Folates in rye: Determination and enhancement by food processing

The average daily intake of folate, one of the B vitamins, falls below recommendations among the Finnish population. Bread and cereals are the main sources of folate, rye being the most significant single source. Processing is a prerequisite for the consumption of whole grain rye; however, little is known about the effect of processing on folates. Moreover, data on the bioavailability of endogenous cereal folates are scarce. The aim of this study was to examine the variation in as well as the effect of fermentation, germination, and thermal processes on folate contents in rye. Bioavailability of endogenous rye folates was investigated in a four-week human intervention study. One of the objectives throughout the work was to optimise and evaluate analytical methods for determining folate contents in cereals. Affinity chromatographic purification followed by high-performance liquid chromatography (HPLC) was a suitable method for analysing cereal products for folate vitamers, and microbiological assay with Lactobacillus rhamnosus reliably quantified the total folate. However, HPLC gave approximately 30% lower results than the microbiological assay. The folate content of rye was high and could be further increased by targeted processing. The vitamer distribution of whole grain rye was characterised by a large proportion of formylated vitamers followed by 5-methyltetrahydrofolate. In sourdough fermentation of rye, the studied yeasts synthesized and lactic acid bacteria mainly depleted folate. Two endogenous bacteria isolated from rye flour were found to produce folate during fermentation. Inclusion of baker s yeast in sourdough fermentation raised the folate level so that the bread could contain more folate than the flour it was made of. Germination markedly increased the folate content of rye, with particularly high folate concentrations in hypocotylar roots. Thermal treatments caused significant folate losses but the preceding germination compensated well for the losses. In the bioavailability study, moderate amounts of endogenous folates in the form of different rye products and orange juice incorporated in the diet improved the folate status among healthy adults. Endogenous folates from rye and orange juice showed similar bioavailability to folic acid from fortified white bread. In brief, it was shown that the folate content of rye can be enhanced manifold by optimising and combining food processing techniques. This offers some practical means to increase the daily intake of folate in a bioavailable form.

Cereal alkylresorcinols as dietary biomarkers-absorption and occurrence in biological membranes

Several studies link the consumption of whole-grain products to a lowered risk of chronic diseases, such as certain types of cancer, type II diabetes, and cardiovascular diseases. However, the final conclusions of the exact protective mechanisms remain unclear, partly due to a lack of a suitable biomarker for the whole-grain cereals intake. Alkylresorcinols (AR) are phenolic lipids abundant in the outer parts of wheat and rye grains usually with homologues of C15:0- C25:0 alkyl chains, and are suggested to function as whole-grain biomarkers. Mammalian lignan enterolactone has also previously been studied as a potential whole-grain biomarker. In the present work a quantified gas chromatography-mass spectrometry method for the analysis of AR in plasma, erythrocytes, and lipoproteins was developed. The method was used to determine human and pig plasma AR concentrations after the intake of whole-grain wheat and rye products compared to low-fibre wheat bread diets to assess the usability of AR as biomarkers of whole-grain intake. AR plasma concentrations were compared to serum ENL concentrations. AR absorption and elimination kinetics were investigated in a pig model. AR occurrence in human erythrocyte membranes and plasma lipoproteins were determined, and the distribution of AR in blood was evaluated. Plasma AR seem to be absorbed via the lymphatic system from the small intestine, like many other lipophilic compounds. Their apparent elimination half-life is relatively short and is similar to that of tocopherols, which have a similar chemical structure. Plasma AR concentrations increased significantly after a one- to eight-week intake of whole-grain wheat and further on with whole-grain rye bread. The concentrations were also higher after habitual Finnish diet compared to diet with low-fibre bread. Inter-individual variation after a one-week intake of the same amount of bread was high, but the mean plasma AR concentrations increased with increasing AR intake. AR are incorporated into erythrocyte membranes and plasma lipoproteins, and VLDL and HDL were the main AR carriers in human plasma. Based on these studies, plasma AR could function as specific biomarkers of dietary whole-grain products. AR are exclusively found in whole-grains and are more suitable as specific biomarkers of whole-grain intake than previously investigated mammalian lignan enterolactone, that is formed from several plants other than cereals in the diet. Plasma AR C17:0/C21:0 -ratio could distinguish whether whole-grain products in the diet are mainly wheat or rye. AR could be used in epidemiological studies to determine whole-grain intake and to better assess the role of whole-grains in disease prevention.

Interactions of natural products with β-lactoglobulins, members of the lipocalin family

Natural products constitute an important source of new drugs. The bioavailability of the drugs depends on their absorption, distribution, metabolism and elimination. To achieve good bioavailability, the drug must be soluble in water, stable in the gastrointestinal tract and palatable. Binding proteins may improve the solubility of drug compounds, masking unwanted properties, such as bad taste, bitterness or toxicity, transporting or protecting these compounds during processing and storage. The focus of this thesis was to study the interactions, including ligand binding and the effect of pH and temperature, of bovine and reindeer β-lactoglobulin (βLG) with such compounds as retinoids, phenolic compounds as well as with compounds from plant extracts, and to investigate the transport properties of the βLG-ligand complex. To examine the binding interactions of different ligands to βLG, new methods were developed. The fluorescence binding method for the evaluation of ligand binding to βLG was miniaturized from a quartz cell to a 96-well plate. A method of ultrafiltration sampling combined with high-performance liquid chromatography was developed to assess the binding of compounds from extracts. The interactions of phenolic compounds or retinoids and βLG were investigated using the 96-well plate method. The majority of flavones, flavonols, flavanones and isoflavones and all of the retinoids included were shown to bind to bovine and reindeer βLG. Phenolic compounds, contrary to retinol, were not released at acidic pH. Those results suggest that βLG may have more binding sites, probably also on the surface of βLG. An extract from Camellia sinensis (L.) O. Kunze (black tea), Urtica dioica L. (nettle) and Piper nigrum (black pepper) were used to evaluate whether βLG could bind compounds from plant extracts. Piperine from P. nigrum was found to bind tightly and rutin from U. dioica weakly to βLG. No components from C. sinensis bound to βLG in our experiment. The uptake and membrane permeation of bovine and reindeer βLG, free and bound with retinol, palmitic acid and cholesterol, were investigated using Caco-2 cell monolayers. Both bovine and reindeer βLG were able to cross the Caco-2 cell membrane. Free and βLG-bound retinol and palmitic acid were transported equally, whereas cholesterol could not cross the Caco-2 cell monolayer free or bound to βLG. Our results showed that βLG can bind different natural product compounds, but cannot enhance transport of retinol, palmitic acid or cholesterol through Caco-2 cells. Despite this, βLG, as a water-soluble binding protein, may improve the solubility of natural compounds, possibly protecting them from early degradation and transporting some of them through the stomach. Furthermore, it may decrease their bad or bitter taste during oral administration of drugs or in food preparations. βLG can also enhance or decrease the health benefits of herbal teas and food preparations by binding compounds from extracts.

Young people's perceptions of the wood products industry : a relational view

The basis for this study was in poor attractiveness of the wood products industry among young people as a field to study and work in. The purpose was to produce new information of how to improve the relationship between young people and the wood products industry in order to better attract young people with different relational orientation. A survey was conducted among students of comprehensive schools and students of wood industry at vocational schools selected by systematic cluster sampling. The final sample consisted of 613 students. The study combined the theories and concepts of relationships, communication and trust of several disciplines. In addition, it applied theories of relationship marketing, stakeholders, publics, involvement and concepts of reputation and values. It studied the central relational elements in the form of antecedents, relationship state and its consequences. The study examined, how young people with different background and level of interest perceive wood industry as a field to study and work in from relational point of view, what are the central deficiencies in perceived relational elements and what are the public relations activities enhancing the relationship between wood industry and young people with less and high interest in the sector. The results indicate poor visibility of the wood industry among young people: unfamiliarity with the industry and unawareness of the opportunities to study in the field. It appeared that instead of increasing only information sharing, interactive communication in different forms is needed. The study also suggests that behaviors of the industry sector advancing perceived trustworthiness are of crucial importance. Moreover, the wood industry needs to pay attention to its behaviors and communication also among other stakeholder groups, especially the media, as reputation plays an important role in building up trust and satisfaction between young people and the sector. Finally, the less and highly interested young people were found to assess the relationship partly through different relational elements. In order to develop the relationship with highly interested young people they should be regarded clearly as future employees of the wood industry through activities affirming that they are desired and valued employees in the sector. Further, openness of information disclosure, whether concerning current situation or future prospects, seems to increase credibility and attractiveness of the wood industry. Highly interested young people were also found to appreciate socially responsible activities. The less interested young people seem to be insecure about the reliability of the wood industry as an employer, as well as, its ability and interest to invest in young people s skills. In addition,involvement in issues relevant for young people was found crucial in enhancing the relationship with the less interested young people.The conclusions of the study provide tools for enhancing the attractiveness of the wood industry among young people not only to the industry itself, but also to its advocates, teachers and student counselors of comprehensive and vocational schools, authorities and policy makers.

Undersowing in a northern climate: effects on spring cereal yield and risk of nitrate leaching

Disadvantages of invariable cereal cropping, concern of nutrient leaching and prices of nitrogen (N) fertilizer have all increased during last decades. An undersown crop, which grows together with a main crop and after harvest, could mitigate all those questions. The aim of this study was to develop undersowing in Finnish conditions, so that it suits for spring cereal farming as well as possible and enhances taking care of soil and environment, especially when control of N is concerned. In total, 17 plant species were undersown in spring cereals during the field experiments between 1991-1999 at four sites in South and Central Finland, but after selection, eight of them were studied more thoroughly. Two legumes, one grass species and one mixture of them were included in long-term trials in order to study annually repeated undersowing. Further, simultaneous broadcasting of seeds instead of separate undersowing was studied. Grain yield response and the capacity of the undersown crop to absorb soil N or fix N from atmosphere, and the release of N were of greatest interest. Seeding rates of undersown crops and N fertilization rates during annually repeated undersowing were also studied. Italian ryegrass (Lolium multiflorum Lam., IR) absorbed soil nitrate N (NO3-N) most efficiently in autumn and timothy (Phleum pratense L.) in spring. The capacity of other grass species to absorb N was low, or it was insufficient considering the negative effect on grain yield. Red clover (Trifolium pratense L.) and white clover (Trifolium repens L.) suited well in annually repeated undersowing, supplying fixed N for cereals without markedly increased risk of N leaching. Autumn oriented growth rhythm of the studied legumes was optimal for undersowing, whereas the growth rhythm of grasses was less suited but varied between species. A model of adaptive undersowing system was outlined in order to emphasize allocation of measures according needs. After defining the goal of undersowing, many decisions are to be done. When diminishing N leaching is primarily sought, a mixture of IR and timothy is advantageous. Clovers suit for replacing N fertilization, as the positive residual effect is greater than the negative effect caused by competition. A mixture of legume and non legume is a good choice when increased diversity is the main target. Seeding rate is an efficient means for adjusting competition and N effects. Broadcasting with soil covering equipment can be used to establish an undersown crop. In addition, timing and method of cover crop termination have an important role in the outcome. Continuous observing of the system is needed as for instance conditions significantly affect growth of undersown crop and on the other hand N release from crop residues may increase in long run.

Tracking and identifying wastewater toxicants and assessing their biodegradation products

Screening of wastewater effluents from municipal and industrial wastewater treatment plants with biotests showed that the treated wastewater effluents possess only minor acute toxic properties towards whole organisms (e.g. bacteria, algae, daphnia), if any. In vitro tests (sub-mitochondrial membranes and fish hepatocytes) were generally more susceptible to the effluents. Most of the effluents indicated the presence of hormonally active compounds, as the production of vitellogenin, an egg yolk precursor protein, was induced in fish hepatocytes exposed to wastewater. In addition, indications of slight genotoxic potential was found in one effluent concentrate with a recombinant bacteria test. Reverse electron transport (RET) of mitochondrial membranes was used as a model test to conduct effluent assessment followed by toxicant characterisations and identifications. Using a modified U.S. EPA Toxicity Identification Evaluation Phase I scheme and additional case-specific methods, the main compound in a pulp and paper mill effluent causing RET inhibition was characterised to be an organic, relatively hydrophilic high molecular weight (HMW) compound. The toxicant could be verified as HMW lignin by structural analyses using nuclear magnetic resonance. In the confirmation step commercial and in-house extracted lignin products were used. The possible toxicity related structures were characterised by statistical analysis of the chemical breakdown structures of laboratory-scale pulping and bleaching effluents and the toxicities of these effluents. Finally, the biological degradation of the identified toxicant and other wastewater constituents was evaluated using bioassays in combination with chemical analyses. Biological methods have not been used routinely in establishing effluent discharge limits in Finland. However, the biological effects observed in this study could not have been predicted using only routine physical and chemical effluent monitoring parameters. Therefore chemical parameters cannot be considered to be sufficient in controlling effluent discharges especially in case of unknown, possibly bioaccumulative, compounds that may be present in small concentrations and may cause chronic effects.

Developments and Microbiological applications in African foods: Emphasis on Nigerian Wara cheese

African indigenous foods have received limited research. Most of these indigenous foods are fermented and they form part of the rich nutritional culture of many groups in African countries. The industrialization and commercialisation of these indigenous African fermented foods should be preceded by a thorough scientific knowledge of their processing which can be vital in the elimination of hunger and poverty. This study highlighted emerging developments and the microbiology of cereal-based and cassava-based food products that constitute a major part of the human diet in most African countries. In addition, investigations were also carried out on the coagulant of the Calotropis procera plant used in traditional production of Nigerian Wara cheese and on the effects of adding a nisin producing Lactococcus lactis strain originating from human milk to Nigerian Wara cheese. Fermented cereal-based food such as ogi utilize popular African and readily available grains maize, millet or sorghum as substrates and is popular as a weaning diet in infants. In this study, the bulkiness caused by starch gelatinization was solved by amylase treatments in the investigation on cooked and fermented oat bran porridge. A similar treatment could reduce the viscosity of any cereal porridge. The properties of the Sodom apple leaves (Calotropis procera) extract in cheesemaking were studied. C. procera was affected by monovalent (K+ and Na+) and divalent (Mg2+ and Ca2+) cations during coagulation. The rennet strength of this coagulant was found to be 7 % compared to animal rennet at 35 °C. Increasing the incubation temperature to 70 °C increased the rennet strength 28-fold. The molecular weight of the partially purified protease was determined by SDS-PAGE and was confirmed by Zymography to be approximately 60 kilodaltons. The high proteolytic activity at 70 °C supported the suitability of the protease enzyme as a coagulant in future commercial production of Nigerian Wara cheese. It was also possible to extend the shelf life of Wara cheese by a nisin producing lactic acid bacteria Lactococcus lactis LAC309. The levels of nisin in both whey and curd fractions of Wara were investigated, results showed a 3 log reduction of toxicogenic Bacillus licheniformis spiked on Wara after 3 days. These studies are the first in Finland to promote the advancement of scientific knowledge in African foods. Recognizing these indigenous food products and an efficient transfer of technology from the developed countries to industrialize them are necessary towards a successful realization of the United Nations Millenium Development Program.

HPLC analysis of plant sterol oxidation products

Increased interest in the cholesterol-lowering effect of plant sterols has led to development of plant sterol-enriched foods. When products are enriched, the safety of the added components must be evaluated. In the case of plant sterols, oxidation is the reaction of main concern. In vitro studies have indicated that cholesterol oxides may have harmful effects. Due their structural similarity, plant sterol oxidation products may have similar health implications. This study concentrated on developing high-performance liquid chromatography (HPLC) methods that enable the investigation of formation of both primary and secondary oxidation products and thus can be used for oxidation mechanism studies of plant sterols. The applicability of the methods for following the oxidation reactions of plant sterols was evaluated by using oxidized stigmasterol and sterol mixture as model samples. An HPLC method with ultraviolet and fluorescence detection (HPLC-UV-FL) was developed. It allowed the specific detection of hydroperoxides with FL detection after post-column reagent addition. The formation of primary and secondary oxidation products and amount of unoxidized sterol could be followed by using UV detection. With the HPLC-UV-FL method, separation between oxides was essential and oxides of only one plant sterol could be quantified in one run. Quantification with UV can lead to inaccuracy of the results since the number of double bonds had effect on the UV absorbance. In the case of liquid chromatography-mass spectrometry (LC-MS), separation of oxides with different functionalities was important because some oxides of the same sterol have similar molecular weight and moreover epimers have similar fragmentation behaviour. On the other hand, coelution of different plant sterol oxides with the same functional group was acceptable since they differ in molecular weights. Results revealed that all studied plant sterols and cholesterol seem to have similar fragmentation behaviour, with only relative ion abundances being slightly different. The major advantage of MS detection coupled with LC separation is the capability to analyse totally or partly coeluting analytes if these have different molecular weights. The HPLC-UV-FL and LC-MS methods were demonstrated to be suitable for studying the photo-oxidation and thermo-oxidation reactions of plant sterols. The HPLC-UV-FL method was able to show different formation rates of hydroperoxides during photo-oxidation. The method also confirmed that plant sterols have similar photo-oxidation behaviour to cholesterol. When thermo-oxidation of plant sterols was investigated by HPLC-UV-FL and LC-MS, the results revealed that the formation and decomposition of individual hydroperoxides and secondary oxidation products could be studied. The methods used revealed that all of the plant sterols had similar thermo-oxidation behaviour when compared with each other, and the predominant reactions and oxidation rates were temperature dependent. Overall, these findings showed that with these LC methods the oxidation mechanisms of plant sterols can be examined in detail, including the formation and degradation of individual hydroperoxides and secondary oxidation products, with less sample pretreatment and without derivatization.

Prolamin degradation in sourdoughs

This thesis examines protein behaviours that occur during cereal fermentations. The focus is on the prolamin degradation in sourdoughs. The thesis also looks at what happens to the oat globulins during an oat bran acidification process. The cereal prolamins are unique proteins in many respects. The wheat prolamins (glutenins and gliadins) are responsible for the formation of the gluten that provides the viscoelastic properties to wheat doughs whereas the rye prolamins (secalins) are unable to develop gluten-like structures. In addition, many baking technological features, such as flavour, shelf-life and dough properties are affected by the protein degradation that might occur during processing. On the other hand, the prolamins contain protein structures that are harmful to gluten sensitive people. It is thus evident that the degradation of the prolamins in sourdough processes may be approached from various aspects. This thesis describes some of these approaches. Four different cereal fermentations were carried out. Wheat sourdough (WSD) and rye sourdough (RSD) fermentations represented traditional sourdoughs. A germinated-wheat sourdough (GWSD) was a novel sourdough type that was prepared using germinated wheat grains that had high and diverse proteolytic activities. The oat bran fermentation (OBF) represented a fermentation system that lacked functional cereal proteases. The high molecular weight glutenins and rye secalins were degraded during the WSD and RSD fermentations, respectively. It was noteworthy that in WSD only a very limited degradation of the gliadins occurred. The gliadins were, however, hydrolysed very extensively during the GWSD fermentation. No protein degradation was observable in the OBF system. Instead the acidification altered the solubility of the oat globulins and this finally led to their aggregation. This thesis confirms that the endogenous proteases of cereals hydrolyse cereal prolamins in sourdoughs. The thesis also shows that the proteolytic activity of the used cereal raw material determines the extent of proteolysis that occurs in sourdough. This means that bakers may adjust the protein degradation in their sourdoughs by selecting the raw material based on its proteolytic activity. The thesis also demonstrates that by using germinated grains, with high and diverse proteolytic activity in sourdough preparations, the prolamins can be extensively degraded. Whether such highly proteolytic food technology could be used to manufacture new gluten-free cereal-based products for gluten sensitive people remains to be solved.