Identification of active-site residues of sheep liver serine hydroxymethyltransferase

Chemical modification of amino acid residues with phenylglyoxal, N-ethylmaleimide and diethyl pyrocarbonate indicated that at least one residue each of arginine, cysteine and histidine were essential for the activity of sheep liver serine hydroxymethyltransferase. The second-order rate constants for inactivation were calculated to be 0.016 mM-1 X min-1 for phenylglyoxal, 0.52 mM-1 X min-1 for N-ethylmaleimide and 0.06 mM-1 X min-1 for diethyl pyrocarbonate. Different rates of modification of these residues in the presence and in the absence of substrates and the cofactor pyridoxal 5'-phosphate as well as the spectra of the modified protein suggested that these residues might occur at the active site of the enzyme.

Common variants in the trichohyalin gene are associated with straight hair in Europeans

Hair morphology is highly differentiated between populations and among people of European ancestry. Whereas hair morphology in East Asian populations has been studied extensively, relatively little is known about the genetics of this trait in Europeans. We performed a genome-wide association scan for hair morphology (straight, wavy, curly) in three Australian samples of European descent. All three samples showed evidence of association implicating the Trichohyalin gene (TCHH), which is expressed in the developing inner root sheath of the hair follicle, and explaining approximately 6% of variance (p=1.5x10(-31)). These variants are at their highest frequency in Northern Europeans, paralleling the distribution of the straight-hair EDAR variant in Asian populations.

Molecular genetic studies on nemaline myopathy and related disorders

Nemaline myopathy (NM) is a rare muscle disorder characterised by muscle weakness and nemaline bodies in striated muscle tissue. Nemaline bodies are derived from sarcomeric Z discs and may be detected by light microscopy. The disease can be divided into six subclasses varying from very severe, in some cases lethal forms to milder forms. NM is usually the consequence of a gene mutation and the mode of inheritance varies between NM subclasses and different families. Mutations in six genes are known to cause NM; nebulin (NEB), alpha-actin, alpha-tropomyosin (TPM3), troponin T1, beta-tropomyosin (TPM2) and cofilin 2, of which nebulin and -actin are the most common. One of the main interests of my research is NEB. Nebulin is a giant muscle protein (600-900 kDa) expressed mainly in the thin filaments of striated muscle. Mutations in NEB are the main cause of autosomal recessive NM. The gene consists of 183 exons. Thus being gigantic, NEB is very challenging to investigate. NEB was screened for mutations using denaturing High Performance Liquid Chromatography (dHPLC) and sequencing. DNA samples from 44 families were included in this study, and we found and published 45 different mutations in them. To date, we have identified 115 mutations in NEB in a total of 96 families. In addition, we determined the occurrence in a world-wide sample cohort of a 2.5 kb deletion containing NEB exon 55 identified in the Ashkenazi Jewish population. In order to find the seventh putative NM gene a genome-wide linkage study was performed in a series of Turkish families. In two of these families, we identified a homozygous mutation disrupting the termination signal of the TPM3 gene, a previously known NM-causing gene. This mutation is likely a founder mutation in the Turkish population. In addition, we described a novel recessively inherited distal myopathy, named distal nebulin myopathy, caused by two different homozygous missense mutations in NEB in six Finnish patients. Both mutations, when combined in compound heterozygous form with a more disruptive mutation, are known to cause NM. This study consisted of molecular genetic mutation analyses, light and electron microscopic studies of muscle biopsies, muscle imaging and clinical examination of patients. In these patients the distribution of muscle weakness was different from NM. Nemaline bodies were not detectable with routine light microscopy, and they were inconspicuous or absent even using electron microscopy. No genetic cause was known to underlie cap myopathy, a congenital myopathy characterised by cap-like structures in the muscle fibres, until we identified a deletion of one codon of the TPM2 gene, in a 30-year-old cap myopathy patient. This mutation does not change the reading frame of the gene, but a deletion of one amino acid does affect the conformation of the protein produced. In summary, this thesis describes a novel distal myopathy caused by mutations in the nebulin gene, several novel nebulin mutations associated with nemaline myopathy, the first molecular genetic cause of cap myopathy, i.e. a mutation in the beta-tropomyosin gene, and a founder mutation in the alpha-tropomyosin gene underlying autosomal recessive nemaline myopathy in the Turkish population.

Proline-containing β-turns in peptides and proteins: Analysis of structural data on globular proteins

Tetrapeptide sequences of the type Z-Pro-Y-X were obtained from the crystal structure data on 34 globular proteins, and used in an analysis of the positional preferences of the individual amino acid residues in the β-turn conformation. The effect of fixing proline as the second position residue in the tetrapeptide sequence was studied by comparing the data obtained on the positional preferences with the corresponding data obtained by Chou and Fasman using the Z-R-Y-X sequence, where no particular residue was fixed in any of the four positions. While, in general, several amino acid residues having relatively very high or very low preferences for specific positions were found to be common to both the Z-Pro-Y-X and Z-R-Y-X sequences, many significant differences were found between the two sets of data, which are to be attributed to specific interactions arising from the presence of the proline residue.

Potato virus A as a heterologous protein expression tool in plants

Viruksien käyttö tuotekehityksen ja tutkimuksen vaatimien proteiinien tuottamiseen, syötävien rokotteiden kehittämiseen ja geeniterapiaan edustavat kasvavia biotekniikan sovellusalueita. Perunan A-virus (PVA) kuuluu potyviruksiin, joiden proteiinit tuotetaan aluksi yhtenä suurena molekyylinä, joka pilkotaan yksittäisiksi proteiineiksi viruksen itsensä tuottamilla entsyymeillä. Siten virusgenomiin lisätty vieras geeni käännetään proteiiniksi virusproteiinien mukana. Lopputuloksena kaikkia proteiineja tuotetaan kasvisoluissa samansuuruinen määrä. Lisäksi, viruksen proteiinikuoren koontimekanismi sallii perintöaineksen merkittävän lisäyksen ilman että viruksen tartutuskyky merkittävästi heikkenee. Koska virus monistuu ja leviää koko kasviin, jo melko pieni määrä kasveja riittää huomattavan proteiinimäärän tuottamiseen esimerkiksi säännösten mukaisessa kasvihuoneessa. Tämän työn tarkoituksena oli muuntaa PVA:n genomia siten, että virus soveltuisi yhden vieraan proteiinin tai useiden erilaisten proteiinien samanaikaiseen tuottamiseen kasveissa. Aluksi kokeiltiin viruksen replikaasia ja kuoriproteiinia koodaavien genomialueiden välistä kohtaa ja ihmisestä peräisi olevaa geeniä, joka tuotti S-COMT-entsyymiä (katekoli-O-metyylitransferaasi). Sen aktiivisuuden rajoittaminen auttaa Parkinsonintaudin hoidossa. Kasvissa tuotettua S-COMT:ia voitaisiin käyttää lääkekehityksessä estolääkkeiden testaukseen. Kahden viikon kuluttua tartutuksesta tupakan lehdissä oli entsymaattisesti aktiivista S-COMT:ia n. 1 % lehden liukoisista proteiineista. PVA:n P1-proteiinia koodaavalta alueelta oli paikannettu kohta, johon ehkä voitaisiin siirtää vieras geeni. Asia varmistettiin siirtämällä tähän kohtaan meduusan geeni, joka tuottaa UV-valossa vihreänä fluoresoivaa proteiinia (GFP). GFP-geeniä kantava PVA levisi kasvissa ja lisääntyi n. 30-50 %:iin viruksen normaalista pitoisuudesta. Koko kasvi fluoresoi vihreänä UV-valossa. Vieras geeni voidaan sijoittaa myös potyviruksen P1- ja HCpro-proteiineja koodaavien alueiden väliin. Samaan PVA-genomiin siirrettiin kolme geeniä, yksi kuhunkin kolmesta kloonauskohdasta: GFP-geeni P1:n sisälle, merivuokon lusiferaasigeeni P1/HCpro-kohtaan ja bakteerin beta-glukuronidaasigeeni (GUS) replikaasi/kuoriproteiini-kohtaan. Virusgenomin ja itse viruksen pituudet kasvoivat 38 %, mutta virus säilytti tartutuskykynsä. Se levisi kasveissa saavuttaen n. 15 % viruksen normaalista pitoisuudesta. Kaikki kolme vierasta proteiinia esiintyivät lehdissä aktiivisina.

Separate and interacting effects within the catechol-O-methyltransferase (COMT) are associated with schizophrenia

Several lines of evidence have implicated the catechol-O-methyltransferase (COMT) gene as a candidate for schizophrenia (SZ) susceptibility, not only because it encodes a key dopamine catabolic enzyme but also because it maps to the velocardiofacial syndrome region of chromosome 22q11 which has long been associated with SZ predisposition. The interest in COMT as a candidate SZ risk factor has led to numerous case-control and family-based studies, with the majority placing emphasis on examining a functional Val/Met polymorphism within this enzyme. Unfortunately, these studies have continually produced conflicting results. To assess the genetic contribution of other COMT variants to SZ susceptibility, we investigated three single-nucleotide polymorphisms (SNPs) (rs737865, rs4633, rs165599) in addition to the Val/Met variant (rs4680) in a highly selected sample of Australian Caucasian families containing 107 patients with SZ. The Val/Met and rs4633 variants showed nominally significant associations with SZ (P<0.05), although neither of the individual SNPs remained significant after adjusting for multiple testing (most significant P=0.1174). However, haplotype analyses showed strong evidence of an association; the most significant being the three-marker haplotype rs737865-rs4680-rs165599 (global P=0.0022), which spans more than 26 kb. Importantly, conditional analyses indicated the presence of two separate and interacting effects within this haplotype, irrespective of gender. In addition, our results indicate the Val/Met polymorphism is not disease-causing and is simply in strong linkage disequilibrium with a causative effect, which interacts with another as yet unidentified variant approximately 20 kb away. These results may help explain the inconsistent results reported on the Val/Met polymorphism and have important implications for future investigations into the role of COMT in SZ susceptibility.

Comparative analysis of Tritrichomonas foetus (Riedmuller, 1928) cat genotype, T. foetus (Riedmuller, 1928) cattle genotype and Tritrichomonas suis (Davaine, 1875) at 10 DNA loci

The parasitic protists in the genus Tritrichomonas cause significant disease in domestic cattle and cats. To assess the genetic diversity of feline and bovine isolates of Tritrichomonas foetus (Riedmuller, 1928) Wenrich and Emmerson, 1933, we used 10 different genetic regions, namely the protein coding genes of cysteine proteases 1,2 and 4-9 (CP1, 2, 4-9) involved in the pathogenesis of the disease caused by the parasite. The cytosolic malate dehydrogenase 1 (MDH1) and internal transcribed spacer region 2 of the rDNA unit (ITS2) were included as additional markers. The gene sequences were compared with those of Tritrichomonas suis (Davaine. 1875) Morgan and Hawkins, 1948 and Tritrichomonas mobilensis Culberson et al., 1986. The study revealed 100% identity for all 10 genes among all feline isolates (=T. foetus cat genotype), 100% identity among all bovine isolates (=T. foetus cattle genotype) and a genetic distinctness of 1% between the cat and cattle genotypes of T. foetus. The cattle genotype of T. foetus was 100% identical to T. suis at nine loci (CP1, 2,4-8, ITS2, MDH1). At CP9, three out of four T. suis isolates were identical to the T. foetus cattle genotype, while the T. suis isolate SUI-H3B sequence contained a single unique nucleotide substitution. Tritrichomonas mobilensis was 0.4% and 0.7% distinct from the cat and cattle genotypes of T. foetus, respectively. The genetic differences resulted in amino acid changes in the CP genes, most pronouncedly in CP2, potentially providing a platform for elucidation of genotype-specific host-pathogen interactions of T. foetus. On the basis of this data we judge T. suis and T. foetus to be subjective synonyms. For the first time, on objective nomenclatural grounds, the authority of T. suis is given to Davaine, 1875, rather than the commonly cited Gruby and Delafond, 1843. To maintain prevailing usage of T. foetus, we are suppressing the senior synomym T. suis Davaine, 1875 according to Article 23.9, because it has never been used as a valid name after 1899 and T. foetus is widely discussed as the cause of bovine trichomonosis. Thus bovine, feline and porcine isolates should all be given the name T. foetus. This promotes the stability of T. foetus for the veterinary and economically significant venereal parasite causing bovine trichomonosis. (C) 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Identification of a new Polerovirus (family Luteoviridae) associated with cotton bunchy top disease in Australia

Cotton bunchy top (CBT) disease has caused significant yield losses in Australia and is now managed by control of its vector, the cotton aphid (Aphis gossypii). Its mode of transmission and similarities in symptoms to cotton Blue Disease suggested it may also be caused by a luteovirus or related virus. Degenerate primers to conserved regions of the genomes of the family Luteoviridae were used to amplify viral cDNAs from CBT-affected cotton leaf tissue that were not present in healthy plants. Partial genome sequence of a new virus (Cotton bunchy top virus, CBTV) was obtained spanning part of the RNA-dependent-RNA-polymerase (RdRP), all of the coat protein and part of the aphid-transmission protein. CBTV sequences could be detected in viruliferous aphids able to transmit CBT, but not aphids from non-symptomatic plants, indicating that it is associated with the disease and may be the causal agent. All CBTV open-reading frames had their closest similarity to viruses of the genus Polerovirus. The partial RdRP had 90 % amino acid identity to the RdRP of Cotton leafroll dwarf virus (CLRDV) that causes cotton blue disease, while other parts of the genome were more similar to other poleroviruses. The sequence similarity and genome organization of CBTV suggest that it should be considered a new member of the genus Polerovirus. This partial genome sequence of CBTV opens up the possibility for developing diagnostic tests for detection of the virus in cotton plants, aphids and weeds as well as alternative strategies for engineering CBT resistance in cotton plants through biotechnology. © 2012 Australasian Plant Pathology Society Inc.

Northern challanges for profitable production of quality naked oat

Naked oat (Avena sativa f.sp. nuda L.) is the highest quality cereal in northern growing conditions. However the cultivation area of naked oat is remarkably small. Major challenges for naked oat production are to observe its nakedness. The caryopsis of naked oat is sensitive to mechanical damage at harvest, especially at high grain moisture content. The greater the grain moisture content of naked oat at harvest, the more loses of germination capacity was caused by threshing. For producing high quality naked oat seed, it is recommended that harvesting be done at as low grain moisture content as possible. However, if this is not possible, better germination can be ensure with gentle harvest by reducing the cylinder speed. In spite of conventional oat s excellent fat and amino acid composition in animal feed use, as far as nutritional value is concerned, the total energy yield of oat is weaker than other cereals because of the hulls. Also with naked oat the dehulling is not complete, while hull content on different cultivars mostly varied between one to six percent. In addition to genotype, environmental conditions markedly control the expression of nakedness. Thresher settings had only limited effects on hull content. The function of hulls is to protect the groat, but this was confirmed only for Finnish, small grain, cultivar Lisbeth. The oat kernel is generally covered with fine silky hairs termed trichomes. The trichomes of naked oat are partly lost during threshing and handling of grains. Trichomes can cause itchiness in those handling the grains and also accumulate and form fine dust and can block-up machinery. The cultivars differed considerably in pubescence. Some thresher settings, including increased cylinder speed, slightly increased grain polishing such that grains had some areas completely free of trichomes. Adjusting thresher settings was generally not an efficient means of solving the problems associated with naked oat trichomes. The main differences in cultivation costs between naked and conventional oat lie in the amount of seeds required and the drying costs. The main differences affecting the economic result lie in market prices, yield level and feed value. The results indicate that naked oat is financially more profitable than conventional oat, when the crop is sold at a specific price at all yield levels and when the crop is used as feed at highest yield level. At lower yield levels, conventional oat is, in spite of its lower feed value, the more profitable option for feed use. Dehulled oat did not achieve the same economic result as naked oat, as the cost of dehulling, including the hull waste, was considerable. According to this study naked oat can be cultivated successfully under northern conditions, when taking into consideration the soft, naked grain through cultivation chain.

Influence of harvesting strategy on nutrient supply and production of dairy cows consuming diets based on grass and red clover silage

The main objective of this thesis was to elucidate the effects of regrowth grass silage and red clover silage on nutrient supply and milk production of dairy cows as compared with primary growth grass silages. In the first experiment (publication I), two primary growth and four regrowth grass silages were harvested at two stages of growth. These six silages were fed to 24 lactating dairy cows with two levels of concentrate allowance. Silage intake and energy corrected milk yield (ECM) responses, and the range in these response variables between the diets, were smaller when regrowth silages rather than primary growth silages were fed. Milk production of dairy cows reflected the intake of metabolizable energy (ME), and no differences in the ME utilization were found between the diets based on silages harvested from primary growth and regrowth. The ECM response to increased concentrate allowance was, on average, greater when regrowth rather than primary growth silages were fed. In the second experiment (publication II), two silages from primary growth and two from regrowth used in I were fed to rumen cannulated lactating dairy cows. Cows consumed less feed dry matter (DM), energy and protein, and produced less milk, when fed diets based on regrowth silages rather than primary growth silages. Lower milk production responses of regrowth grass silage diets were mainly due to the lower silage DM intake, and could not be accounted for by differences in energy or protein utilization. Regrowth grass silage intake was not limited due to neutral detergent fibre (NDF) digestion or rumen fill or passage kinetics. However, lower intake may be at least partly attributable to plant diseases such as leaf spot infections, dead deteriorating material or abundance of weeds, which are all higher in regrowth compared with primary growth, and increase with advancing regrowth. In the third experiment (publications III and IV), red clover silages and grass silages harvested at two stages of growth, and a mixed diet of red clover and grass silages, were fed to five rumen cannulated lactating dairy cows. In spite of the lower average ME intake for red clover diets, the ECM production remained unchanged suggesting more efficient utilisation of ME for red clover diets compared with grass diets. Intake of N, and omasal canal flows of total non-ammonia N (NAN), microbial and non-microbial NAN were higher for red clover than for grass silage diets, but were not affected by forage maturity. Delaying the harvest tended to decrease DM intake of grass silage and increase that of red clover silage. The digestion rate of potentially digestible NDF was faster for red clover diets than for grass silage diets. Delaying the harvest decreased the digestion rate for grass but increased it for red clover silage diets. The low intake of early-cut red clover silage could not be explained by silage digestibility, fermentation quality, or rumen fill but was most likely related to the nutritionally suboptimal diet composition because inclusion of moderate quality grass silage in mixed diet increased silage DM intake. Despite the higher total amino acid supply of cows fed red clover versus grass silage diets, further milk production responses on red clover diets were possibly compromised by an inadequate supply of methionine as evidenced by lower methionine concentration in the amino acid profile of omasal digesta and plasma. Increasing the maturity of ensiled red clover does not seem to affect silage DM intake as consistently as that of grasses. The efficiency of N utilization for milk protein synthesis was lower for red clover diets than for grass diets. It was negatively related to diet crude protein concentration similarly to grass silage diets.

The Significance of Wild Plants in the Evolutionary Ecology of Three Major Viruses Infecting Cultivated Sweetpotato in Uganda

The studies presented in this thesis contribute to the understanding of evolutionary ecology of three major viruses threatening cultivated sweetpotato (Ipomoea batatas Lam) in East Africa: Sweet potato feathery mottle virus (SPFMV; genus Potyvirus; Potyviridae), Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus; Closteroviridae) and Sweet potato mild mottle virus (SPMMV; genus Ipomovirus; Potyviridae). The viruses were serologically detected and the positive results confirmed by RT-PCR and sequencing. SPFMV was detected in 24 wild plant species of family Convolvulacea (genera Ipomoea, Lepistemon and Hewittia), of which 19 species were new natural hosts for SPFMV. SPMMV and SPCSV were detected in wild plants belonging to 21 and 12 species (genera Ipomoea, Lepistemon and Hewittia), respectively, all of which were previously unknown to be natural hosts of these viruses. SPFMV was the most abundant virus being detected in 17% of the plants, while SPMMV and SPCSV were detected in 9.8% and 5.4% of the assessed plants, respectively. Wild plants in Uganda were infected with the East African (EA), common (C), and the ordinary (O) strains, or co-infected with the EA and the C strain of SPFMV. The viruses and virus-like diseases were more frequent in the eastern agro-ecological zone than the western and central zones, which contrasted with known incidences of these viruses in sweetpotato crops, except for northern zone where incidences were lowest in wild plants as in sweetpotato. The NIb/CP junction in SPMMV was determined experimentally which facilitated CP-based phylogenetic and evolutionary analyses of SPMMV. Isolates of all the three viruses from wild plants were genetically similar to those found in cultivated sweetpotatoes in East Africa. There was no evidence of host-driven population genetic structures suggesting frequent transmission of these viruses between their wild and cultivated hosts. The p22 RNA silencing suppressor-encoding sequence was absent in a few SPCSV isolates, but regardless of this, SPCSV isolates incited sweet potato virus disease (SPVD) in sweetpotato plants co-infected with SPFMV, indicating that p22 is redundant for synergism between SCSV and SPFMV. Molecular evolutionary analysis revealed that isolates of strain EA of SPFMV that is largely restricted geographically in East Africa experience frequent recombination in comparison to isolates of strain C that is globally distributed. Moreover, non-homologous recombination events between strains EA and C were rare, despite frequent co-infections of these strains in wild plants, suggesting purifying selection against non-homologous recombinants between these strains or that such recombinants are mostly not infectious. Recombination was detected also in the 5 - and 3 -proximal regions of the SPMMV genome providing the first evidence of recombination in genus Ipomovirus, but no recombination events were detected in the characterized genomic regions of SPCSV. Strong purifying selection was implicated on evolution of majority of amino acids of the proteins encoded by the analyzed genomic regions of SPFMV, SPMMV and SPCSV. However, positive selection was predicted on 17 amino acids distributed over the whole the coat protein (CP) in the globally distributed strain C, as compared to only 4 amino acids in the multifunctional CP N-terminus (CP-NT) of strain EA largely restricted geographically to East Africa. A few amino acid sites in the N-terminus of SPMMV P1, the p7 protein and RNA silencing suppressor proteins p22 and RNase3 of SPCSV were also submitted to positive selection. Positively selected amino acids may constitute ligand-binding domains that determine interactions with plant host and/or insect vector factors. The P1 proteinase of SPMMV (genus Ipomovirus) seems to respond to needs of adaptation, which was not observed with the helper component proteinase (HC-Pro) of SPMMV, although the HC-Pro is responsible for many important molecular interactions in genus Potyvirus. Because the centre of origin of cultivated sweetpotato is in the Americas from where the crop was dispersed to other continents in recent history (except for the Australasia and South Pacific region), it would be expected that identical viruses and their strains occur worldwide, presuming virus dispersal with the host. Apparently, this seems not to be the case with SPMMV, the strain EA of SPFMV and the strain EA of SPCSV that are largely geographically confined in East Africa where they are predominant and occur both in natural and agro-ecosystems. The geographical distribution of plant viruses is constrained more by virus-vector relations than by virus-host interactions, which in accordance of the wide range of natural host species and the geographical confinement to East Africa suggest that these viruses existed in East African wild plants before the introduction of sweetpotato. Subsequently, these studies provide compelling evidence that East Africa constitutes a cradle of SPFMV strain EA, SPCSV strain EA, and SPMMV. Therefore, sweet potato virus disease (SPVD) in East Africa may be one of the examples of damaging virus diseases resulting from exchange of viruses between introduced crops and indigenous wild plant species. Keywords: Convolvulaceae, East Africa, epidemiology, evolution, genetic variability, Ipomoea, recombination, SPCSV, SPFMV, SPMMV, selection pressure, sweetpotato, wild plant species Author s Address: Arthur K. Tugume, Department of Agricultural Sciences, Faculty of Agriculture and Forestry, University of Helsinki, Latokartanonkaari 7, P.O Box 27, FIN-00014, Helsinki, Finland. Email: tugume.arthur@helsinki.fi Author s Present Address: Arthur K. Tugume, Department of Botany, Faculty of Science, Makerere University, P.O. Box 7062, Kampala, Uganda. Email: aktugume@botany.mak.ac.ug, tugumeka@yahoo.com

Digestion capasity, nutrient digestibilities and physico-chemical conditions in the intestine influenced by the age of growing turkeys

Six experiments have been conducted to examine digestibility and feeding value of domestic Finnish fibre-rich cereals (barley and oats as compared to maize and wheat) and protein sources (rapeseed meal and cake, peas, faba beans, lupin seeds) for growing turkeys and to investigate effects of age of the birds (from 3 to 12 weeks of age) on digestion process and estimated nutrient digestibility and energy values. Besides, an objective of the study was to test applications of digestibility research methodology for turkeys. Total tract digestibility and apparent metabolizable energy (AME) was assayed in experimental cages using excreta collection, and a slaughter method was applied to sample small intestinal digesta for determination of apparent ileal crude protein digestibility (AICPD), jejuno-duodenal digesta viscosity and caecal volatile fatty acid (VFA) concentration. Digesta viscosity decreased and caecal VFA production increased with age of growing turkeys. Digesta retention times in the small intestine were generally longer in the older birds than in the younger ones. Crude fat digestibility and AME increased with age of growing turkeys, especially with viscous diets. AICPD seemed to decrease with age in most cases. Supplementation with β-gucanase-xylanase decreased viscosity, improved crude fat digestibility and metabolizable energy value and increased VFA production especially in barley-fed turkeys and especially in the young birds. Poor protein digestibility and low energy value of rapeseed meal and rapeseed cake decreased their feeding value for turkeys. In addition, a typical goitrogenic effect of rapeseed feeding was detected. Use of legume seeds as feed for growing turkeys is limited mostly by the low energy value in lupin seeds and the low ileal protein and amino acid digestibility in faba beans. Digestibility of fibre-rich protein sources was not improved with age of the turkeys. Euthanizing the turkeys for AICPD determination by carbon dioxide and bleeding led to lower digestibility values than mechanical stunning and cervical dislocation, suggesting inferiority of carbon dioxide stunning in experimental use. Comparison of AICPD and AME results obtained using different markers showed that considerable differences may occur, especially on total tract level, when acid-insoluble ash gave considerably lower AME values than titanium dioxide and chromic oxide.

Effect of phenolic-rich plant materials on protein and lipid oxidation reactions

The antioxidant activity of natural plant materials rich in phenolic compounds is being widely investigated for protection of food products sensitive to oxidative reactions. In this thesis plant materials rich in phenolic compounds were studied as possible antioxidants to prevent protein and lipid oxidation reactions in different food matrixes such as pork meat patties and corn oil-in water emulsions. Loss of anthocyanins was also measured during oxidation in corn oil-in-water emulsions. In addition, the impact of plant phenolics on amino acid level was studied using tryptophan as a model compound to elucidate their role in preventing the formation of tryptophan oxidation products. A high-performance liquid chromatography (HPLC) method with ultraviolet and fluorescence detection (UV-FL) was developed that enabled fast investigation of formation of tryptophan derived oxidation products. Byproducts of oilseed processes such as rapeseed (Brassica rapa L.), camelina (Camelina sativa) and soy meal (Glycine max L.) as well as Scots pine bark (Pinus sylvestris) and several reference compounds were shown to act as antioxidants toward both protein and lipid oxidation in cooked pork meat patties. In meat, the antioxidant activity of camelina, rapeseed and soy meal were more pronounced when used in combination with a commercial rosemary extract (Rosmarinus officinalis). Berry phenolics such as black currant (Ribes nigrum) anthocyanins and raspberry (Rubus idaeus) ellagitannins showed potent antioxidant activity in corn oil-in-water emulsions toward lipid oxidation with and without β-lactoglobulin. The antioxidant effect was more pronounced in the presence of β-lactoglobulin. The berry phenolics also inhibited the oxidation of tryptophan and cysteine side chains of β-lactoglobulin. The results show that the amino acid side chains were oxidized prior the propagation of lipid oxidation, thereby inhibiting fatty acid scission. In addition, the concentration and color of black currant anthocyanins decreased during the oxidation. Oxidation of tryptophan was investigated in two different oxidation models with hydrogen peroxide (H2O2) and hexanal/FeCl2. Oxidation of tryptophan in both models resulted in oxidation products such as 3a-hydroxypyrroloindole-2-carboxylic acid, dioxindolylalanine, 5-hydroxy-tryptophan, kynurenine, N-formylkynurenine and β-oxindolylalanine. However, formation of tryptamine was only observed in tryptophan oxidized in the presence of H2O2. Pine bark phenolics, black currant anthocyanins, camelina meal phenolics as well as cranberry proanthocyanidins (Vaccinium oxycoccus) provided the best antioxidant effect toward tryptophan and its oxidation products when oxidized with H2O2. The tryptophan modifications formed upon hexanal/FeCl2 treatment were efficiently inhibited by camelina meal followed by rapeseed and soy meal. In contrast, phenolics from raspberry, black currant, and rowanberry (Sorbus aucuparia) acted as weak prooxidants. This thesis contributes to elucidating the effects of natural phenolic compounds as potential antioxidants in order to control and prevent protein and lipid oxidation reactions. Understanding the relationship between phenolic compounds and proteins as well as lipids could lead to the development of new, effective, and multifunctional antioxidant strategies that could be used in food, cosmetic and pharmaceutical applications.

Microfiltration in cheese and whey processing

Milk microfiltration (0.05-0.2 um) is a membrane separation technique which divides milk components into casein-enriched and native whey fractions. Hitherto the effect of intensive microfiltration including a diafiltration step for both cheese and whey processing has not been studied. The microfiltration performance of skimmed milk was studied with polymeric and ceramic MF membranes. The changes caused by decreased concentration of milk lactose, whey protein and ash content for cheese milk quality and ripening were studied. The effects of cheese milk modification on the milk coagulation properties, cheese recovery yield, cheese composition, ripening and sensory quality as well as on the whey recovery yield and composition by microfiltration were studied. The functional properties of whey protein concentrate from native whey were studied and the detailed composition of whey protein concentrate powders made from cheese wheys after cheese milk pretreatments such as high temperature heat treatment (HH), microfiltration (MF) and ultrafiltration (UF) were compared. The studied polymeric spiral wound microfiltration membranes had 38.5% lower energy consumption, 30.1% higher retention of whey proteins to milk retentate and 81.9% lower permeate flux values compared to ceramic membranes. All studied microfiltration membranes were able to separate main whey proteins from skimmed milk. The optimal lactose content of Emmental cheese milk exceeded 3.2% and reduction of whey proteins and ash content of cheese milk with high concentration factor (CF) values increased the rate of cheese ripening. Reduction of whey protein content in cheese milk increased the concentration of caseinomacropeptide (CMP) of total proteins in cheese whey. Reduction of milk whey protein, lactose and ash content reduces milk rennet clotting time and increased the firmness of the coagulum. Cheese yield calculated from raw milk to cheese was lower with microfiltrated milks due to native whey production. Amounts of a-lactalbumin (a-LA) and b-lactoglobulin (b-LG) were significantly higher in the reference whey, indicating that HH, MF and UF milk pretreatments decrease the amounts of these valuable whey proteins in whey. Even low CF values in milk microfiltration (CF 1.4) reduced nutritional value of cheese whey. From the point of view of utilization of milk components it would be beneficial if the amount of native whey and the CMP content of cheese whey could be maximized. Whey protein concentrate powders made of native whey had excellent functional properties and their detailed amino acid composition differed from those of cheese whey protein concentrate powders.

Protein cross-linking with oxidative enzymes and transglutaminase : Effects in meat protein systems

The effects of tyrosinase, laccase and transglutaminase (TG) were studied in different meat protein systems. The study was focused on the effects of the enzymes on the gel formation properties of myofibrils, and on the textural and water-holding properties of the heated meat systems. The cross-linking efficiency of a novel Trichoderma reesei tyrosinase was compared to that of the commercial Agaricus bisporus tyrosinase. Trichoderma tyrosinase was found to be superior compared to the Agaricus enzyme in its protein cross-linking efficiency and in the incorporation of a small molecule into a complex proteinaceous substrate. Tyrosinase, laccase and TG all polymerised myofibrillar proteins, but laccase was also found to cause protein fragmentation. A positive connection between covalent cross-link and gel formation was observed with tyrosinase and TG. Laccase was able to increase the gel formation only slightly. With an excessive laccase dosage the gel formation declined due to protein fragmentation. Tyrosinase, laccase and TG had different effects on the texture and water-holding of the heated chicken breast meat homogenates. Tyrosinase improved the firmness of the homogenate gels free of phosphate and with a low amount of meat. TG improved the firmness of all studied homogenates. Laccase weakened the gel firmness of the low-meat, low-salt and low-salt/phosphate homogenates and maintained the firmness on the control level in the homogenate free of phosphate. Tyrosinase was the only enzyme capable of reducing the weight loss in the homogenates containing a low amount of meat and a low amount of NaCl. TG was the only enzyme that could positively affect the firmness of the homogenate gel containing both low NaCl and phosphate amounts. In pilot scale the test products were made of coarsely ground chicken breast fillet with a moderate amount of salt. Increasining the amount of meat, salt and TG contents favoured the development of firmness of the test products. The evaporation loss decreased slightly along with increasing TG and NaCl amounts in the experimental conditions used, indicating a positive interaction between these two factors. In this work it was shown that tyrosinase, laccase and TG affected the same myofibrillar proteins, i.e. myosin and troponin T. However, these enzymes had distinguishable effects on the gel formation of a myofibril system as well as on the textural and water-holding properties of the finely ground meat homogenates, reflecting distinctions at least in the reaction mechanisms and target amino acid availability in the protein substrates for these enzymes.