Browsing Opinnäytteet ja väitöskirjat by organization "Doctoral Programme in Food Chain and Health"

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  • Ahlberg, Sara (Helsingin yliopisto, 2020)
    Aflatoxins continue to be a food safety problem globally, especially in developing regions. Prevalent food contaminating aflatoxins are B1 (AFB1) and M1 (AFM1). These are human carcinogens and have potentially severe health impacts. Almost all (99.5 %) milk samples from Nairobi were contaminated with AFM1, highlighting the urgent need to create functional solutions to improve food safety. Based on the aflatoxin levels and milk consumption, risks were calculated: cancer risk caused by AFM1 was lower among consumers purchasing from formal markets (0.003 cases per 100,000) than for low-income consumers (0.006 cases per 100,000) purchasing from informal markets. Overall cancer risk (0.004 cases per 100,000) from AFM1 alone was low. Because of AFM1 in milk, 2.1 % of children below three years in middle-income families, and 2.4 % in low-income families, could be stunted. Overall, 2.7 % of children could hypothetically be stunted due to AFM1 exposure from milk. Based on these results AFM1 levels found in milk could contribute to an average of -0.340 height for age z-score reduction in growth. The exposure to AFM1 from milk is 46 ng/day on average, but children bear higher exposure of 3.5 ng/kg bodyweight (bw)/day compared with adults, at 0.8 ng/kg bw/day. Aflatoxins are produced by Aspergillus flavus fungus, which is prevalent in soils. Certain strains of lactic acid bacteria (LAB) have been reported inhibiting fungal growth. 171 LAB strains were tested against aflatoxin producing A. flavus fungi. The three LAB strains showing the highest antifungal activity were identified as Lactobacillus plantarum. None of the strains was able to completely inhibit fungal growth under conditions favorable for fungi and suboptimal for LAB. The three indigenous LAB Lactobacillus strains and one Lactococcus strains were tested for their AFM1 binding abilities in different conditions and after different treatments along with two reference Lactobacillus strains. The binding of AFM1 by LAB strains varied between 11 to 100 % in the biocontrol solution analysis, being approximately at the level of 40 % throughout the analysis sets. A significant amount of effort and resources have been invested in an attempt to control aflatoxins. However, these efforts have not substantially decreased the prevalence nor dietary exposure to aflatoxins in developing countries. The growth reduction of aflatoxin producing fungi with LAB could be one potential option, but there are still major issues to solve prior to any practical applications. A different approach to control aflatoxins suggesting the usage of binding agents in foods and lactic acid bacteria (LAB) have been studied extensively for this purpose. However, when assessing the results comprehensively and reviewing the practicality and ethics of use, risks are evident, and concerns arise. In conclusion, there are too many issues with using LAB for aflatoxin binding for it to be safely promoted. Arguably, using binders in human food might even worsen food safety in the longer term. A more comprehensive food safety approach has to be taken to solve this ongoing crisis.
  • Tolonen, Sanna (Helsingin yliopisto, 2019)
    Previous findings suggest that bone mineral density and bone loss are highly genetically determined but the further details of bone genetics remain partly unknown. In thesis, it was studied for the first time whether the single nucleotide polymorphisms of lactase and apolipoprotein E (APOE) genes are associated with the peripheral quantitative computed tomography (pQCT) bone traits of radius and tibia. In addition to the gene polymorphisms, the associations of physical activity in childhood and adulthood with various bone traits as outcomes were examined. This is also a current topic since despite the fact that bone-loading physical activities are essential in building healthy bones; it seems that modern communities do not encourage most of us to move and exercise enough. Study subjects of the present thesis are part of the Cardiovascular Risk in Young Finns Study which was initiated in 1980 with 3596 persons aged 3, 6, 9, 12, 15 and 18. The findings presented are additionally based on the follow-ups conducted in 1986, 2001 and 2007. The pQCT and quantitative ultrasound bone measurements and fracture data were collected in 2008 when the participants were 31-46 years old. Lactase C/T-13910 genotyping (rs4988235) was performed using a 5’ nuclease assay and the APOE genotypes (rs429358 and rs7412) and the APOE promoter polymorphisms -219 and +113 (rs405509 and rs440446) using TaqMan SNP genotyping assays. Dietary intakes were collected through 48-hour dietary recall interviews and later in 2007 with a 131-item food frequency questionnaire. Physical activity in childhood was assessed using a questionnaire including items on leisure-time physical activity, organised exercise, participation in competitions, and intensity level. In adulthood, pedometer-determined steps were used to quantify the amount of physical activity undertaken in 2007. In Study I, small differences in trabecular densities at the distal sites of radius and tibia were found in men between the lactase genotypes. Men with the T/T genotype had ~3% higher values than the carriers of T/C and C/C genotypes. They also had higher calcium intake than men carrying the C allele. In Study II, the carriers of the APOE ԑ4 allele had higher total-cholesterol and LDL-cholesterol serum concentrations than the non-carriers. In addition, the ԑ4 allele was associated with some lower cortical density but greater bone mineral content (BMC) at the tibial diaphysis. Women with the APOE promoter -219T/T allele had on average 7-8% lower cortical and compressive strengths at the distal sites of radius and tibia than the female G/G carriers. At the tibial shaft, the mean values of cortical strength were 2-2.5% lower in subjects with the -219T/T allele compared to the G/G carriers. Men with the -219T/T allele also had 1-2% lower cortical density and cortical strength at the radial shaft but 4-5% greater total areas at the radius than men with the G/G allele. In addition, women with the -219T/T allele whose longitudinal saturated fat intake was the highest (≥35.5 g/day) had the lowest total area and torsional strength at the radial shaft. Almost identical results were found in women and men with the +113G/C genotypes (G/G, G/C and C/C) as reported for the -219G/T genotypes (G/G, G/T and T/T). In Study III, there were no statistically significant differences in the risk of low-energy fractures between the different physical activity groups of 3-18-year-old children and adolescents. In addition, no differences were found in adult tibial traits between the physical activity groups of 3-6-year-olds. In females, the highest level of physical activity at the age of 9-18 years was significantly associated with a lower likelihood of below median values of compressive strength at the distal tibia, and total and cortical areas, and BMC and strength indices at the tibial shaft compared to the lowest activity level in adolescence (ORs 0.33-0.53). Similarly in males, total area at the distal site and cortical area and strength at the tibial shaft were less likely to be below the median values in the highest physical activity group compared to the lowest activity group of adolescents (ORs 0.48-0.53). In Study IV, women within the highest tertile of daily steps had 3.8% and 0.5% greater broadband ultrasound attenuation and speed of sound at the calcaneus compared to women in the lowest tertile. In tibia, women in the highest tertile (> 8765 daily steps) had on average 1-5.4% greater bone cross-sectional area, BMC, trabecular density and compressive strength at the distal site and 1.6-2.7% greater total and cortical bone areas, BMC and torsional strength at the shaft compared to their study peers. Similarly, BMC and BSI at the distal radius and bone areas, BMC and torsional strength at radial shaft were 1.7-3.4% greater in women within the highest tertile of daily steps compared to the women with a lower number of daily steps. In men, the differences in studied bone traits were mainly non-significant across the tertiles of daily steps. Statistically significant results presented have p-values of ≤0.05. In conclusion, the genetic heterogeneity in lactase gene or the APOE ԑ2/ԑ3/ԑ4 genetic variation seemed to have little effect on the studied bone traits at the radius and tibia. Instead, the APOE promoter -219T/T and +113C/C alleles were associated with lower cortical bone phenotypes in both genders compared to the G/G carriers. In women, these supposed risk alleles of APOE promoter polymorphisms were also associated with lower total area and torsional strength at the radial shaft when the intake of saturated fat was high. In addition, a high level of physical activity at the age of 9-18 years, but not in younger children, was associated with wider and stronger weight-bearing tibia in both females and males. In adulthood, a higher amount of physical activity measured as daily steps was associated with greater bone cross-sectional area, mineral mass and strength at the calcaneus, tibia and radius in women.
  • Bhattarai, Mamata (Helsingin yliopisto, 2020)
    Galactoglucomannans (GGMs) are principal hemicelluloses in softwood tissues. Spruce GGM obtained from different recovery approaches are currently studied for applications as emulsifiers and stabilizers of dispersed systems. Natural polysaccharides, owing to their origin from a complex matrix, have an intrinsic affinity for the association. Their associative properties are affected by the recovery approach since it influences purity and intrinsic characteristics (e.g., molar mass, degree of substitution). Understanding the impact of the GGM recovery approach on its associative behavior, currently limited, is essential to comprehend the stabilization mechanism of GGM in dispersed systems as well in the expansion of its functional applications. This doctoral project investigated the associative behavior of GGM at a semi-dilute concentration of 1% in an aqueous medium and its effect in an oil-in-water emulsion system, with GGM recovered from spruce sawdust/wood chips using pressurized hot water extraction, BLN process (modified pressurized hot water extraction process), and effluent of the thermo-mechanical pulping process. All studied GGM samples existed in the form of polysaccharide molecules and supramolecular fraction. It was observed that the recovery approach and sample purity affected the molar mass of the polysaccharide fraction as well as the share and structural properties of the supramolecular fraction. The supramolecular fraction was observed to be either colloidal aggregates, agglomerates, solid particles, or a combination in varying proportions. Next, these samples were studied in emulsions. Differences in the macromolecular state of samples were found to influence interfacial morphology and stability of emulsions. Following, the associative behavior of purified GGM obtained from the pressurized hot water extraction process was studied at acidic, neutral, and alkaline pH and upon addition of sodium chloride. Associative behavior of the sample displayed a positive correlation with acidic pH condition and time, improving emulsion stability. The sample exhibited an upper limit of GGM to oil ratio for efficient emulsification and stabilization ability, implying the presence of a limited amount of active emulsifying component. Currently, novel biomaterials are being developed from wood biomass. The findings of this study contributed to the characterization of colloidal properties of GGM at a nanometric scale, thereby enhancing its scope of future applications. These findings regarding the solubility of GGM would also be relevant in existing operations of paper and pulping industries, as well as for aspiring biorefineries in identifying optimal GGM recovery approach.
  • Arte, Elisa (Helsingin yliopisto, 2019)
    Wheat is the world’s most important staple food, providing one-fifth of the daily protein consumed globally. However, the majority of wheat is used as refined flour, in which the nutritionally superior bran layers and germ are removed during milling, thus producing yearly a massive amount of underutilised food side streams. Better exploitation of the side streams and development of new plant-based protein ingredients are required to ensure the future global demand for food protein. This study aimed to examine hydrolytic enzymes and lactic acid fermentation as tools to improve the bioavailability, nutritional quality and technological properties of wheat bran proteins for food applications. The study showed that proteolytic activity, either by endogenous or exogenous enzymes, was crucial for increasing protein liberation and solubilisation from wheat bran, whereas microbial activity was required for improving the nutritional quality of the proteins. The application of commercial carbohydrases or proteases was able to either solubilise the bran cell walls or the proteins from the residues of endosperm in bran but was not effective in liberating proteins within aleurone cells. The endogenous enzymes of wheat bran, activated by chemical acidification, increased the protein solubilisation up to 75% with a simultaneous increase in in vitro protein digestibility (from 14% to 20%). However, bioprocessing by lactic acid bacteria (LAB), yeast and cell wall-degrading enzymes (Depol 761P and Viscoferm) was found as the most beneficial and microbiologically safe method to improve the solubilisation and nutritional quality of bran proteins. This bioprocessing meth-od resulted in a protein solubilisation of 52% and significantly improved the in vitro protein digestibility to 39%. In this work, the bioprocessing of wheat bran by LAB and yeast, with and without cell wall-degrading enzymes and phytase prior to the production of protein isolates, was found to influence the biochemical and technological properties of the bran proteins. The bioprocessed protein isolates had significantly higher protein content (80%), presumably due to the degradation of starch and soluble arabinoxylans during the bioprocessing. In general, the bioprocessing of bran resulted in a lower protein solubilisation of the protein isolates and had no influence on the emulsifying properties of the isolates in oil-in-water emulsions. However, bioprocessing by lactic acid fermentation together with cell wall-degrading enzymes almost doubled the foaming stability. Furthermore, wheat breads were made by substituting 20% of the total energy by proteins from the isolates. Wheat breads enriched with the lactic acid fermented bran protein isolate was found to have the most optimal technological characteristics, showing delayed staling and lower firmness during four days’ storage in comparison to bread enriched with a protein isolate produced without bioprocessing. In conclusion, by utilising lactic acid fermentation in combination with selected hydrolytic enzymes, the aleurone cell walls can be degraded and the proteins liberated for microbial modification, leading to improved protein bioavailability, nutritional quality and technological functionality. This study is the first to show the potential of using bioprocessing for the development of new wheat bran-based protein ingredient for food applications.
  • Pöntinen, Anna (Helsingin yliopisto, 2019)
    Listeria monocytogenes is a remarkable bacterium, as it is able to shift from a capable environmental saprophyte into a severe intracellular pathogen. As a strictly foodborne pathogen, L. monocytogenes poses a notable risk, particularly to those consumers among the risk groups for whom invasive listeriosis is potentially fatal. Furthermore, modern consumption habits and increasingly favoured ready-to-eat foods, often consumed without proper heating, increase the risk of acquiring the foodborne disease. The aim of this study was to investigate the genetic mechanisms conferring wide-ranging stress tolerance in L. monocytogenes. Two-component systems, comprising a sensor histidine kinase and a cognate response regulator, aid bacteria in sensing and adapting to changes in both surrounding environmental as well as intracellular conditions. The histidine kinases, in particular, have lacked comprehensive studies on their roles in the stress tolerance of L. monocytogenes. Thus, histidine kinases were studied by expressional analyses under cold conditions and by mutationally disrupting each histidine kinase-encoding gene in a parental model strain, L. monocytogenes EGD-e. The modified strains were individually challenged at high (42.5 degrees C) and low (3.0 degrees C) temperatures, high (9.4) and low (5.6) pH levels, and high salt (6% NaCl), ethanol (3.5 vol%) and hydrogen peroxide (5 mM) concentrations. Expressional studies and growth experiments on genetically modified strains proved lisK and yycG to respectively play central roles in the acclimation and immediate growth of L. monocytogenes at low temperatures. The most substantial increase in gene expression under cold conditions was that of the chemotaxis gene cheY with 236-fold upregulation at 3 degrees C. The disrupted ΔliaS strain displayed impaired growth in response to all the other stresses, particularly at a high temperature and under osmotic stress. These studies demonstrated the prominent importance of the histidine kinase-encoding genes yycG and lisK to cold tolerance and liaS, with roles in the growth of L. monocytogenes under multiple stresses. To shed light on the accessory genetic mechanisms that cause large strain variation in L. monocytogenes in withstanding heat treatments, heat resistance-conferring traits were further investigated by means of whole-genome sequencing. Comparing the complete genomes of heat-resistant L. monocytogenes AT3E and -sensitive AL4E strains revealed the heat-resistant strain to harbour a novel 58-kb plasmid, pLM58, which was absent in the sensitive strain. Furthermore, curing of the plasmid in AT3E produced a marked decrease in heat resistance from virtually no reduction to a 1.1 cfu/ml log10 reduction at 55.0 degrees C. In pLM58, a 2,155-bp open reading frame annotated as an ATP-dependent ClpL protease-encoding gene was identified. Conjugation of the coding sequence and the putative promoter of the clpL gene into a natively heat-sensitive L. monocytogenes 10403S strain, in turn, enhanced the survival of the strain from a 1.2 cfu/ml log10 reduction to a 0.4 cfu/ml log10 reduction in heat challenge at 55.0 degrees C. In this study, we presented the first evidence of plasmid-mediated heat resistance in L. monocytogenes and identified the protease ClpL to be a novel plasmid-borne heat-resistance mediator. The emerging resistance of L. monocytogenes to benzalkonium chloride, a quaternary ammonium compound widely used as a detergent in food-processing facilities, is a significant concern for food safety and public health. The resistance of 392 L. monocytogenes isolates from Finland (n = 197) and Switzerland (n = 195) to benzalkonium chloride was assessed. A minimal inhibitory concentration of 20 µg/ml was defined. Altogether, 11.5% of the strains proved to be resistant to benzalkonium chloride. Serotype 1/2c harboured the highest prevalence, 32.4% (11/34), of benzalkonium chloride-resistant strains, while in total, most of the resistant strains belonged to serotype 1/2a. Altogether, 68.9% of the resistant strains harboured at least one of the efflux pump system-encoding genes, bcrABC, emrE or qacH, known to confer benzalkonium chloride resistance in L. monocytogenes. We found resistant strains with partially or completely efflux pump-dependent benzalkonium chloride resistance, with the exception of the known resistance-mediating efflux pumps, suggesting the existence of other resistance-contributing efflux pump systems. The lacking of known efflux pump system-encoding genes in addition to efflux pump-independent benzalkonium chloride resistance, in turn, indicates the contribution of completely novel benzalkonium chloride resistance mechanisms. The aim of these studies was to shed light on the genes contributing to the versatile stress tolerance abilities and strain variation of the severe foodborne pathogen, L. monocytogenes. Knowledge of such traits may aid in developing targeted strategies and measures to identify and control the contamination and risks caused, in particular, by stress-tolerant L. monocytogenes strains.
  • Luukkanen, Jenni (Helsingin yliopisto, 2019)
    Official control in slaughterhouses, consisting of meat inspection and food safety inspection, has an important role in ensuring meat safety, animal health and welfare, and prevention of transmissible animal diseases. Meat inspection in the European Union (EU) includes the inspection of food chain information, live animals (ante-mortem inspection), and carcasses and offal (post-mortem inspection). Food safety inspections are performed to verify slaughterhouses’ compliance with food safety legislation and are of the utmost importance, especially if slaughterhouses’ self-checking systems (SCSs) fail. The aim of this study was to investigate the prerequisites for official control such as the functionality of the task distribution in meat inspection and certain meat inspection personnel-related factors. In addition, needs for improvement in slaughterhouses’ SCSs, meat inspection, and food safety inspections, including control measures used by the official veterinarians (OVs) and their efficacy, were examined. In the EU, competent authorities must ensure the quality of official control in slaughterhouses through internal or external audits, and the functionality of these audits was also studied. Based on our results, meat inspection personnel (OVs and official auxiliaries [OAs]), slaughterhouse representatives, and officials in the central authority were mainly satisfied with the functionality of the present task distribution in meat inspection, although redistributing ante-mortem inspection from the OVs to the OAs was supported by some slaughterhouse representatives due to perceived economic benefit. Ante-mortem inspection was assessed as the most important meat inspection task as a whole for meat safety, animal welfare, and prevention of transmissible animal diseases, and most of the respondents considered it important that the OVs perform ante-mortem inspection and whole-carcass condemnation in red meat slaughterhouses. In a considerable number of slaughterhouses, OA or OV resources were not always sufficient and the lack of meat inspection personnel decreased the time used for food safety inspections according to the OVs, also affecting some of the red meat OAs’ post-mortem inspection tasks. The frequency with which OVs observed post-mortem inspection performed by the OAs varied markedly in red meat slaughterhouses. In addition, roughly one-third of the red meat OAs did not consider the guidance and support from the OVs to be adequate in post-mortem inspection. In various parts of this study, OVs’ demand for increased guidance and support from their superiors in control actions of a difficult nature was emphasized. According to our results, the most common non-compliance in slaughterhouses concerned hygiene such as cleanliness of premises and equipment, hygienic working methods, and maintenance of surfaces and equipment. Chief OVs in a few smaller slaughterhouses reported more frequent and severe non-compliances than other slaughterhouses, and in these slaughterhouses the usage of written time limits and enforcement measures by the OVs was more infrequent than in other slaughterhouses. Deficiencies in documentation of food safety inspections and in systematic follow-up of corrections of slaughterhouses’ non-compliance had been observed in a considerable number of slaughterhouses. In meat inspection, deficiencies in inspection of the gastrointestinal tract and adjacent lymph nodes were most common and observed in numerous red meat slaughterhouses. Internal audits performed to evaluate the official control in slaughterhouses were considered necessary, and they induced correction of observed non-conformities. However, a majority of the interviewed OVs considered that the meat inspection should be more thoroughly audited, including differences in the rejections and their reasons between OAs. Auditors, for their part, raised a need for improved follow-up of the audits. Our results do not give any strong incentive to redistribute meat inspection tasks between OVs, OAs, and slaughterhouse employees, although especially from the red meat slaughterhouse representatives’ point of view the cost-efficiency ought to be improved. Sufficient meat inspection resources should be safeguarded in all slaughterhouses, and meat inspection personnel’s guidance and support must be emphasized when developing official control in slaughterhouses. OVs ought to focus on performing follow-up inspections of correction of slaughterhouses’ non-compliance systematically, and also the documentation of the food safety inspections should be developed. Hygiene in slaughterhouses should receive more attention; especially in slaughterhouses with frequent and severe non-compliance, OVs should re-evaluate and intensify their enforcement. The results attest to the importance of internal audits in slaughterhouses, but they could be developed by including auditing of the rejections and their underlying reasons and uniformity in meat inspection.
  • Silventoinen, Pia (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.
  • Xu, Yan (Helsingin yliopisto, 2018)
    Fava bean is a good source of plant protein that is increasingly gaining attention due to its health benefits and sustainability. However, the addition of fava bean flour or protein concentrate at high concentrations to food products may result in an unsatisfying texture. Therefore, texture modification of the fava bean matrix is essential to improve its usability in various food systems. Microbial exopolysaccharides (EPS) are effective texture modifiers, with lactic acid bacteria (LAB) being widely used in the food industry to produce them. In this thesis, EPS were produced in situ from sucrose by LAB during the fermentation of fava bean flour or fava bean protein concentrate (FPC), and their texture modification effects on fava bean matrix were evaluated. Leuconostoc pseudomesenteroides DSM 20193 and Weissella confusa VTT E-143403 were found to be good dextran producers in fava bean matrix. With the same starter, sucrose addition strongly increased paste viscosity after fermentation. By separately hydrolyzing the fermented paste with dextranase and levanase, this increase was demonstrated to be primarily driven by dextran. The gel-strengthening ability of EPS was revealed by dynamic oscillatory rheology analysis, with obvious elasticity increases in sucrose-enriched pastes after fermentation. Dextrans produced by Ln. pseudomesenteroides DSM 20193 and W. cibaria Sj 1b showed considerable gel-strengthening ability. Two mechanisms of degradation for raffinose family oligosaccharides (RFO) were established in this thesis, involving plant-derived α-galactosidase and microbial levansucrase (LSR). In fava bean flour, RFO were preferentially degraded by endogenous α-galactosidase, producing galactose and sucrose that could be further used for EPS synthesis. In the absence of endogenous α-galactosidase, LSR could act on RFO, forming melibiose, manninotriose, and manninotetraose. A joint function of endogenous α-galactosidase and microbial LSR in RFO degradation was also observed. Texture evaluation of fermented FPC pastes revealed higher firmness, consistency, cohesiveness, and index of viscosity in sucrose-enriched pastes. Proteolysis of fava bean protein was very weak after fermentation, thus contributing less to texture modification of FPC pastes than did EPS. Confocal laser scanning microscopy of fermented fava bean protein showed more concentrated protein aggregates in dextran-enriched pastes. The study of fava bean protein-dextran interactions indicates the importance of intermolecular interactions between these two polymers in determining the rheological properties of the system. During acidification, dextran stabilized the network of fava bean protein through intermolecular interactions. Conjugation of dextran to fava bean protein through the Maillard reaction decreased both viscosity and gel elasticity during protein gelation. The molar mass and conformation of dextrans affected their behavior in the protein system, especially during protein gelation. This thesis shows for the first time the feasibility of connecting EPS production by LAB to the fermentation of fava bean matrix, and identifies two promising dextran producers for use in this matrix. Analysis of the microstructure of fava bean protein with dextran and fava bean protein–dextran interactions clearly showed the role of dextran in the protein network. Furthermore, the results indicate that different EPS producers may allow texture tailoring of the fava bean matrix, which may contribute to the development of novel plant protein-based food or meat substitutes.
  • Wang, Yaqin (Helsingin yliopisto, 2020)
    Utilization of grain legumes (such as faba bean) and minor cereals (such as sorghum and millet) is constantly growing since consumers are increasingly interested in sustainable, plant based and health promoting foods. Grain legumes and minor cereals are raw materials with high nutritional quality due to high content of protein, dietary fibre and other bioactive compounds. Utilization of these grains is challenging as they have negative impact on product texture and flavour. Sourdough technology is one of the “clean label” options to improve technological functionality of these grains. The aim of this thesis was to study the influence of faba bean, sorghum and millet on technological and nutritional properties of composite wheat bread. Wheat flour was replaced with faba bean (30%), sorghum or millet flours (50%), which were either native or fermented. Utilization of native flours had detrimental effect on the rheological properties of dough as well as the volume, texture and sensory properties of bread in comparison to 100% wheat control breads. In contrast, mildly acidified and dextran-containing flours improved all properties of composite breads. The functionality of sourdough was based on sufficient production of dextran and mild acidification. Faba bean sourdough fermented with Weissella confusa VTT E-143403 (dextran content of 5.2% dry weight) improved the specific volume (21%) and texture of breads, especially softness (12%). However, faba bean sourdough fermented with Leuconostoc pseudomesenteroides DSM 20193 (dextran content of 3.6% d.w.) decreased bread volume and increased crumb hardness, probably due to the higher acidification. Furthermore, efficacy to improve shelf-life (delay staling rate) was shown to be linked to slower starch retrogradation and improved water retention. Sourdough fermentation also increased the level of free phenolic compounds in millet and sorghum. Fermentation of millet decreased starch in vitro digestibility (lower predicted glycemic index), while improving the in vitro digestibility of proteins. These changes may be attributed to the production of organic acids and concomitant activation of hydrolytic enzymes like glycoside hydrolase, cellulases, esterases and proteases. Utilization of tailored sourdough technology had a significant impact on the sensory properties of sorghum breads. Sourdough fermentation of sorghum without dextran increased unpleasant flavour properties such as acidic, bitter flavour and aftertaste, probably due to increased content of acids and small molecular weight polyphenols (e.g. caffeic acid). This study showed that dextran containing sorghum breads had less intensive acidic and bitter flavour and milder aftertaste even though the actual acidity and polyphenol compositions was the same as in control breads. This revealed the exceptional ability of dextran to mask acidic and bitter flavour notes. This observation was further verified by adding purified dextran (0.12−0.96% bread weight) to white wheat bread together with acid and bitter flavour compounds (lactic/acetic acid and caffeine). When the amount of dextran was sufficient (> 0.43% b.w.), the perceived intensity of acidic and bitter flavours in the bread decreased. This thesis demonstrated efficient production of dextran in situ in faba bean, sorghum and millet flours during sourdough fermentation, which facilitates production of nutritionally high quality composite breads without additives. Additionally, this thesis revealed for the first time the ability of dextran to modify sensation of acidic and bitter flavours, which allows future product innovations in plant based foods.
  • Kytö, Elina (Helsingin yliopisto, 2020)
    Purchase behavior is a frequent but highly complicated human behavior affected by numerous factors. In the food industry, several sensory and consumer research responses are generally used to predict future sales to maximize market success. However, the failure rate is estimated to be extremely high, and new methods and measurements for prediction are needed. This thesis investigates the role of different consumer and sensory research measurements – especially the measure of purchase intention – in food purchase behavior prediction. The aim is to examine the ability of purchase intention, and other explicit and implicit responses, to predict food purchases with varying amounts of product information. The focus is on factors related to the consumer (perception, previous usage, emotions, habits, and beliefs) and the product (expectations, product type, and sensory quality as internal factors, and brand and package design as external factors). The association between the amount of product information, the different measurements, and the actual purchase behavior regarding two types of dairy snack products (a flavored protein quark and a natural yogurt) is studied. The product information phases were 1) expectation based on the brand and packaging pictures that simulated an in-store experience (before trying the product), 2) sensory quality perception based on blind tasting, and 3) actual perception based on tasting with brand and packaging pictures that simulated an in-home experience (after tasting the product). The responses were measured using explicit responses (purchase intention, pleasantness, emotions, recommendation, brand relationship, and willingness to eat again) and implicit responses (event-related potential, reaction time, and pupil size). In addition, various consumer-related factors, such as previous use of and familiarity with the studied products by brand, change seeking, purchase drivers, purchase moment, and purchase decision level, were measured. Finally, purchase behavior was measured using self-reported surveys within a one-month period after the evaluations. Purchase intention and recommendation responses measured in the actual perception condition were found to be the most accurate predictors of food purchases. Overall, explicit responses were associated with purchase behavior more strongly than implicit responses. Furthermore, measurements after only sensory quality perception were poor predictors of purchases. In conclusion, purchase behavior was predicted better when more product information was available.
  • Jaakkonen, Anniina (Helsingin yliopisto, 2020)
    Cattle are commonly asymptomatic carriers of Shiga toxin-producing Escherichia coli (STEC) and Campylobacter jejuni, which cause gastroenteritis in humans. Especially STEC infections may lead to severe or fatal consequences. Both STEC and C. jejuni are intermittently shed in cattle feces and can contaminate bulk tank milk via fecal contamination during milking. These bacteria are effectively eliminated from milk by pasteurization, but the consumption of unpasteurized milk, or raw milk, poses a risk of infection. In recent years, the consumption of raw milk has become more popular, with public demand to relax legislation that restricts sales of raw milk. However, on-farm epidemiology of these pathogens have warranted further investigation to support the development of on-farm risk management practices and pathogen monitoring of dairy farms that sell raw milk to consumers. These studies investigated a milkborne outbreak caused by STEC (Study I) and obtained longitudinal data on the contamination of bulk tank milk by STEC and C. jejuni, and explored on-farm contamination routes of these pathogens (Study II). Furthermore, the studies revealed strain characteristics of C. jejuni that may affect survival and persistence of this pathogen in milk (Study III). The occurrence of STEC and C. jejuni, or C. jejuni alone, was determined in bulk tank milk, in-line milk filters of the milking machine, cattle feces, and the farm environment on four dairy farms (STEC and C. jejuni in Studies I and II) or one dairy farm (C. jejuni in Study III). STEC and C. jejuni isolates from the dairy farms were further subjected to phenotypic characterization and whole-genome sequencing, followed by comparative genomic analyses to explore gene contents and phylogenetic relationships between the isolates. Furthermore, questionnaire data were collected to trace back the outbreak source (Study I) and to determine on-farm risk factors associated with milk contamination using a logistic regression model (Study II). Ultimately, the results contributed to the revision of the Finnish legislation that restrict the sales of raw milk in 2017 (Study II). Study I elucidated the reservoirs and transmission routes of atypical, sorbitol-fermenting (SF) STEC O157, which have largely been unknown. The study presents microbiological and epidemiologic evidence that an outbreak of SF STEC O157 with 11 cases originated from a recreational farm housing dairy cattle and was transmitted via the consumption of raw milk. Thus, these results strongly support bovine origin of SF STEC O157. In longitudinal monitoring (Study II), one clone of STEC O157:H7, which represented a bovine-associated lineage, was simultaneously isolated on each of the three dairy farms. STEC O157:H7 persisted in two herds for up to 12 months, and a similar but distinct clone was reintroduced in one herd 2.5 years after the previous detection. These results support evidence that few STEC O157:H7 clones persist on-farm simultaneously. Unlike STEC, both persistent and numerous sporadic C. jejuni strains appeared simultaneously on dairy farms (Studies II and III). Persistence for 11 months or longer was associated with a few C. jejuni genotypes, especially the host generalist sequence type (ST) ST-883. C. jejuni of ST-883 outperformed other STs in environmental fitness, representing the only ST that could be isolated from bulk tank milk and milk filters and the dominant ST found from environmental samples. Therefore, ST-883 imposes a higher contamination pressure on milk than other STs among the farm isolates and represents a candidate for on-farm risk-based monitoring. In the longitudinal monitoring, STEC was rarely isolated from bulk tank milk and milk filters and only simultaneously with fecal isolation. Higher detection rates were obtained from milk filters than milk by both culture methods and real-time PCR. Therefore, milk filters are more reliable sampling targets for monitoring of STEC than milk. Isolation of C. jejuni from milk and milk filters was associated with C. jejuni clone rather than sample material, but the isolation rates of C. jejuni appeared generally poor. To enhance the isolation rates for monitoring purposes, the sampling regime also warrants further consideration. Reduced milk contamination by STEC was associated with on-farm practices: pasturing and culling of dairy cows and rigorous cleansing in the barn. Higher outdoor temperatures were associated with increased milk contamination. In Study III, C. jejuni of ST-883 persistently contaminated bulk tank milk of a dairy farm for seven months or longer after having caused a milkborne outbreak. Although ST-883 survived in refrigerated raw milk longer than other STs from the same farm, the persistence of ST-883 in bulk tank milk was likely affected by other phenotypic traits such as biofilm formation. Outbreak strain of ST-883 reversibly adapted to survival in bulk tank milk, showing biofilm formation in an on/off manner among replicate cultures and cellular heterogeneity by phase variation in genes related to capsule and oxidative stress response. Furthermore, the outbreak strain harbored a pTet-like genomic element, which may have contributed to higher biofilm quantities. This study identified candidate phenotypic and genotypic mechanisms affecting survival and persistence of C. jejuni in milk. Taken together, STEC and C. jejuni can persist on dairy farms for months or longer and contaminate bulk tank milk despite stringent on-farm hygiene measures. Although these measures cannot totally prevent milk contamination, they likely reduce the contamination pressure on milk. Therefore, cost-effective hygiene measures should be applied on all farms that sell raw drinking milk to consumers. Detection of pathogens from milk may be challenging, and milk may also be contaminated by highly virulent STEC and C. jejuni strains that show atypical phenotype, increasing their environmental endurance or hampering their detection. Therefore, only heat treatment of raw milk before consumption can adequately assure its food safety.
  • Kant, Ravi (Helsingin yliopisto, 2018)
    This thesis details an in silico exploration of the genetic potential of the Gram-positive genus Lactobacillus through the establishment and analysis of a pan-genome dataset. Lactobacilli are an intensively researched and studied group of bacteria, which is in part owed to their exploitation for various man-made food and industrial purposes and their advocated beneficial use as gut probiotics. Bacterial pan-genomics is an outgrowth of the comparative genomics field and by comparing the genomes from many strains of the same species the data obtained serves to catalogue the entire genetic repertoire available to a particular species. In effect, the pan-genome represents the complete assortment of putative genes in a species (or genus) of bacteria. The research presented in this thesis is comprised of three distinct studies (I, II, and III), each of which used the pan-genome approach to give a theoretical account of Lactobacillus genetics. In study I, a pan-genome was assembled at the genus level using 20 fully sequenced genomes from 14 different Lactobacillus species. Here, complete genome sequences were selected for creating a broader framework that would allow more comprehensive genomic comparisons. Varying aspects were apparent among the genome sequences used, including sizes that ranged from ~1.8 to ~3.3 Mbps and a G+C content of between ~33% and ~51%. The assembled pan-genome was sized at 14,000 protein-encoding genes, and out of which a small 383-gene Lactobacillus core genome (LCG) was derived. The genetic content of the LCG was used for reconstructing a molecular phylogeny, which then permitted a taxonomic grouping of the 20 genomes into three main clades. Additional classifications of the LCG involved identifying core group and signature group genes, as well as so-called ORFan genes that were further sorted as either LCG-specific or group-specific. In study II, a pan-genome of the Lactobacillus rhamnosus species was constructed from 13 different genomes. L. rhamnosus is a highly adaptable bacterium that thrives in a variety of hosts and environments. Presumably, there is little doubt that numerous genetic peculiarities are the source of the niche-related phenotypes that enable the inherent ecological adaptability of L. rhamnosus strains. For this, the genetic content of the assembled pan-genome was examined for those geno-phenotypic variations occurring at the cell-surface level and whether these correlate to a particular habitat preference of various L. rhamnosus strains. The L. rhamnosus pan-genome itself had an estimated size of 4,893 protein-encoding genes, which was further partitioned into the 2,095-gene core and 2,798-gene accessory genomes. Pan-genomic comparisons were benchmarked against the gut-adapted L. rhamnosus GG strain and focused primarily on seven functionally characterized surface-exposed proteins. Most notably, the operonic genes for the mucoadhesive SpaCBA pilus were part of the accessory genome and can be regarded as a genomic novelty in L. rhamnosus. Nonetheless, for those L. rhamnosus strains with a functional SpaCBA piliation trait, this would improve niche-specific fitness and presumably prolong transient (allochthonous) colonization of mucosal epithelial surfaces in the gut or elsewhere in the body. In study III, a pan-genomic appraisal was performed on the Lactobacillus ruminis species by compiling the genomes of nine different strains obtained from human, bovine, porcine, and equine digestive tracts. L. ruminis is a piliated and flagellated strict anaerobe and one of the few indigenous (autochthonous) lactobacilli in the gut. The pan-genome was utilized to pinpoint the molecular basis for the intractable colonization behavior of L. ruminis, where the focus was on those geno-phenotypes associated with cellular surface morphology and anaerobic fermentation and respiration. The size of the L. ruminis pan-genome was predicted to contain 4,301 protein-related genes, while the number of genes in the core and accessory genomes was 1,234 and 3,067, respectively. As inferred from the pan-genomic data, the presence of certain surface proteins and a substitute anaerobic energy-yielding metabolism might represent the adaptive phenotypes that help make L. ruminis a gut-autochthonic species.
  • Pesonen, Maiju (Natural Resources Institute Finland (Luke), 2020)
    The decrease in the dairy cattle population observed in recent years threatens to reduce the level of beef production. Although the number of beef cows has increased by 20% over the last 10 years, there is a clear discrepancy between the demand for and supply of domestic beef. Consequently, slaughterhouse pricing favours heavy carcasses and the average carcass weights of slaughtered animals have increased in recent years. There is a paucity of information on the effects of the breed on the performance, carcass characteristics and meat quality of beef breed bulls raised to heavy carcass weights. The first aim of this thesis was to evaluate the growth performance, carcass traits and meat quality of different beef breeds in the Finnish beef cattle population. The second aim was to evaluate the potential for improvement of carcass and meat quality traits through crossbreeding compared to purebred animals. The third objective was to evaluate carcass fat scores in relation to carcass weights in different breed groups. In addition, the effects of the proportion of concentrates and rapeseed meal (RSM) supplementation on animal performance, carcass characteristics and meat quality parameters were determined for Hereford (Hf) and Charolais (Ch) bulls. To achieve these aims five experiments were carried out. The objectives of the first experiment in which Hf and Ch bulls were offered grass silage-based diets, were to determine the effects on the performance, carcass traits and meat quality of the proportion of concentrate in the diet, and the inclusion of RSM in a barley-based concentrate. The objective of the second and third experiments were to study performance and meat quality of purebred Hf, Ch bulls and Hf × Ch crossbred bulls and Angus (Ab) and Limousin (Li) bulls and Ab × Li crossbred bulls which were offered grass silage-grain-based rations and raised to heavy carcass weights. The objective of the fourth experiment was to determine the growth and carcass traits of beef breed bulls and heifers. The data collected from Finnish slaughterhouses. The objective of the fifth experiment was to study the potential for improvement in the gain and carcass traits through Ab × beef breed crossbreeding compared to purebred Ab bulls and through Hereford Hf × beef breed crossbreeding compared to purebred Hf bulls. Continental breeds tended to have carcass traits that suit the Finnish beef production system well under the current Finnish feeding management approach. British breeds produced more intramuscular fat in the meat and have a higher sensory quality compared to Continental breeds. The carcass traits of British breeds can be enhanced for the current market demand by crossbreeding British breed dams with Continental breeds. The grass silage-grain-based diet suited beef breeds well for growing and finishing diets. The concentrate level can be reduced for British breeds. Continental breeds will benefit from increased concentrate levels in the diet. Protein supplementation does not add any substantial advantages to the diet. Using protein supplements will increase the environmental impacts of beef production.
  • Lopez Valladares, Gloria (Helsingin yliopisto, 2019)
    Isolates of Listeria monocytogenes (N=932) collected from human cases of invasive listeriosis in Sweden between 1958 and 2010 were serotyped and characterised with pulsed-field gelelectrophoresis (PFGE) and AscI restriction enzyme. The genotype diversity of L. monocytogenes isolates was investigated and related to genotypic results from epidemiological information on human infection, in order to detect possible clustering of L. monocytogenes genotypes over time, season, location, age, or gender (Paper I). From 1972 to 1995, serovar 4b was the predominant serovar; however, in 1996, serovar 1/2a became the major serovar among human listeriosis cases in Sweden. Based on the number and distribution of all bands in the profile, 63 PFGE types belonging to serovars 1/2b, 3b and 4b and 119 PFGE types belonging to serovars 1/2a and 1/2c were identified (Paper I). The PFGE types were further assembled into PFGE groups, based on the number and distribution of small bands below 145.5 kb (Papers II and III). As the genomic region of small bands is genetically more conservative than in large bands, the distribution of small bands establishes relatedness of strains and defines genetic markers for both lineages. Cold-smoked salmon (Salmo salar) and gravad salmon packed under modified atmosphere or vacuum from three manufacturers were purchased in Sweden and Germany in 2005 and the occurrence and levels of L. monocytogenes were analysed (Paper IV): 56 products were analysed and eleven harboured L. monocytogenes. From the positive samples, 56 isolates were analysed with AscI, and 11 isolates were further analysed with ApaI: five AscI PFGE types were identified, four belonging to serovar 1/2a and one to 4b. Forty-three (n=43: 76.8%) isolates shared serovar 1/2a and 13 (23.2%) shared serovar 4b and all AscI types were identified among human clinical strains in Sweden. Moreover, three gravad salmon samples harboured two PFGE types each from different lineages, serovar 1/2a and serovar 4b. Although, in most of the products, the level of L. monocytogenes was less than 100 cfu/g, the highest level was 1500 cfu/g. The occurrence of L. monocytogenes was 12.9% in gravad salmon, encountered in three manufacturers (A, B, C) and 28% in cold-smoked salmon only from manufacturer A. Although the level of L. monocytogenes in RTE fish products is generally low, these products, should be considered possible sources of listeriosis in Sweden. A patient may harbour more than one L. monocytogenes PFGE type that can be determined through PFGE and AscI restriction enzyme. However, to avoid misleading conclusions, several L. monocytogenes colonies should be isolated and characterised from different sites from the same patient or mother-baby pairs (Paper V).
  • Summa, Maija (Helsingin yliopisto, 2019)
    Human noroviruses (HuNoVs) are yearly responsible for a large number of acute human gastroenteritis cases globally in all age groups. Typically, the virus transmits via the fecal-oral route from person to person, causing strong symptoms such as nausea, vomiting, and diarrhea, which usually disappear in a few days. However, HuNoVs cause also numerous food-related illnesses in developed countries, including Finland, inducing gastroenteritis outbreaks through contaminated water and foodstuffs. According to the reports of the European Commission, both in Europe and in Finland the most common foods causing HuNoV outbreaks are shellfish, berries (especially frozen raspberries), vegetables, and mixed foods, which most likely became contaminated by a sick food handler. Noroviruses belong to the Caliciviridae family and are classified into seven genogroups. HuNoVs belong to genogroups I (GI), II (GII), and IV (GIV). Other genogroups contain only animal noroviruses. Noroviruses are generally regarded as host-species-specific, but the possibility of zoonotic transmission and infections has been discussed for over a decade for several genotypes. The purpose of this study was to develop a simple and rapid method for detection of HuNoVs in food. The potential zoonotic nature of HuNoVs, particularly whether animals can serve as transmitters for these viruses, was also investigated. In the past two decades, numerous methods for detecting HuNoVs in food have been developed. However, many of these are time-consuming and the sensitivity of the methods has been highly variable. In this work, four published extraction methods for detection of HuNoV in food (lettuce, ham, and frozen berries) were compared. The method based on alkaline elution and polyethylene glycol (PEG) precipitation was found to be the most reliable detection method for all three food matrices tested. The recovery efficiency of the method with frozen raspberries was on average 28%. Two rapid methods for detection of HuNoV in frozen raspberries were also presented. The rapid method based on direct RNA extraction yielded the same recovery levels (32%) as the PEG precipitation method. The method proved to be sensitive because it detected HuNoV also with a virus level of 100 genome copies in a 25 g sample. Moreover, the method detected HuNoV in naturally contaminated berry samples that were linked to outbreaks of disease. A treatment with either a chloroform-butanol mixture or dilution of the food samples for the RT-PCR reaction was efficient in reducing the effect of PCR inhibitors. The same effect was achieved with PEG as a supplement in the food samples. Thirty-nine frozen berry samples purchased from local stores in 2010, 2014, and 2017 were screened. All berries tested negative for HuNoVs GI and GII. HuNoV genome was detected in the feces of 31 birds, two rats, and four pet dogs. The genotypes found in six bird samples and all dog samples were the same as those commonly found in human samples at the time of sampling. HuNoVs can be detected in food samples also in small numbers using the rapid method presented in this study. The use of PEG as a supplement was found to reduce inhibition of the RT-PCR reaction in the two rapid methods, and therefore, the commonly used chloroform-butanol treatment, which easily loses viruses during processing, could be omitted. The results of animal samples strongly indicate that wild birds, pet dogs, and possibly also rats may be involved in the transmission of HuNoVs to food, water, and surfaces
  • Xie, Chong (Helsingin yliopisto, 2020)
    Vitamin B12 is a micronutrient that is predominantly present in food of animal origin. Therefore, developing plant-origin food with a nutritionally relevant content of vitamin B12 can increase the dietary intake of this vitamin among people with limited consumption of animal products. Since its chemical synthesis is overly complicated and expensive, the commercial vitamin B12 used for food fortification is exclusively produced via a biotechnological process. As compared to fortification with this commercial form of vitamin B12, in situ fortification via fermentation can be a more cost-effective alternative. As a commonly consumed staple food, grains are excellent vehicles for enrichment with micronutrients. Propionibacterium freudenreichii is the only food-grade microorganism with the ability to produce vitamin B12. Because P. freudenreichii has a low growth rate and is sensitive to acidic conditions, sterilized grain materials have mostly been used so far to produce a high vitamin B12 content. The sterilization process, however, alters the technological properties of grain-based raw materials and decreases the feasibility of the process. The present thesis focuses on in situ fortification of vitamin B12 in native grain materials by fermentation with P. freudenreichii. This study has demonstrated that fermentation of wheat flour, whole-wheat flour and wheat bran with P. freudenreichii resulted in a physiologically significant level of vitamin B12 (up to 155 ng/g dw) after 7 days. Whole-grain wheat flour and wheat bran had a higher content of vitamin B12 than refined wheat flour. However, the propagation of Enterobacteriaceae indicated that monoculture fermentation with P. freudenreichii cannot dominate the microflora, to guarantee microbial safety and control endogenous microbiota present in grain materials. Thus, an effective co-culture of Lactobacillus brevis ATCC 14869 and P. freudenreichii was established through a pre-screening to ensure microbial safety. During co-fermentation in wheat bran, P. freudenreichii produced a high level of vitamin B12 (ca. 183 ng/g dw on day 3). Moreover, controlling pH during fermentation could greatly enhance the vitamin B12 production (up to 332 ng/g dw on day 3). Meanwhile, L. brevis showed a strong inhibition on the propagation of Enterobacteriaceae during fermentation, as expected. The wider applicability of the established co-culture was demonstrated by fermenting 11 types of grain materials, including cereals, pseudocereals and legumes, with P. freudenreichii and L. brevis. P. freudenreichii produced a nutritionally significant level of vitamin B12 in most of the grain materials. The highest production was found in the rice bran (ca. 742 ng/g dw), followed by the buckwheat bran (ca. 631 ng/g dw), after fermentation. Meanwhile, the addition of L. brevis was able to dominate indigenous microbes during fermentation and thus greatly improve microbial safety during the fermentation of different grain materials. Overall, this thesis demonstrates that the fermentation of grain materials with P. freudenreichii and an appropriate co-culture, such as L. brevis, is a promising way to provide vitamin B12 in non-sterilized grain-based materials, without compromising microbial safety. Meanwhile, selecting raw materials that provide optimal conditions for P. freudenreichii can significantly improve the efficacy of vitamin B12 synthesis.
  • Adebayo, Folasade Abiola (Helsingin yliopisto, 2019)
    Diet is an indicator of health and chronic disease. Food consumption patterns of a particular population may define their nutrient intake, including vitamin D, revealing their health profiles. The food consumption patterns among immigrants often differ from those of the host population, and health inequalities exist between these two groups. Vitamin D insufficiency (S-25(OH)D <50 nmol/L), which has been associated with bone disorders, such as osteoporosis, and risk for cancers and other chronic diseases, is a public health problem among populations at northern latitudes, especially during winter because of low ultraviolet B irradiation and reduced skin synthesis of vitamin D. Nevertheless, the risk of vitamin D insufficiency is higher among non-Western immigrants, particularly dark-skinned ones, living in these regions. In the Nordic countries, vitamin D status in the majority of the host populations seems to be better than that of immigrants. This study seeks insights into immigrants’ nutrition with the aims of examining food consumption and vitamin D status among immigrants of Russian, Somali, and Kurdish background. The study also aimed to investigate whether ethnic differences exist in the response of serum 25-hydroxyvitamin D (S-25(OH)D) to vitamin D3 supplementation between Somali and Finnish women. The thesis was based on three datasets: i) a cross-sectional population-based Migrant Health and Wellbeing Study (Maamu Study), ii) the nationally representative Finnish Health 2011 Survey (Health 2011), and iii) the randomized controlled trial Marwo-D intervention study. Study I (n=1372) comprised immigrant participants aged 18-64 years from the Maamu Study. Healthy food consumption frequencies were evaluated among 527 Russian, 337 Somali, and 508 Kurdish men and women, through dietary questions in interviews. Data on socio-demographic factors were obtained from a sampling frame and through interviews. Potential socio-demographic determinants of healthy food consumption were assessed by logistic regression. Immigrants of Russian background more frequently consumed healthy foods, especially rye bread, vegetables, fruits, and berries, than Kurds and Somalis. Female sex, older age, and higher education were positively associated with healthy food consumption. Low consumption of fresh vegetables, fruits and berries was observed among Somali immigrants. In Study II, the S-25(OH)D concentrations of 1310 immigrants (446 Russians, 364 Somalis, and 500 Kurds) aged 18-64 years from the Maamu Study and a Finnish reference group aged 30-64 years from the Health 2011 Survey (n=798) were standardized according to the Vitamin D Standardization Program (VDSP) by liquid chromatography-tandem mass spectrometry. Data on socio-demographic, lifestyle, and dietary habits were obtained from a sampling frame and through structured interviews or through a self-administered health questionnaire. Vitamin D status (S-25(OH)D) of the immigrant groups was analysed relative to the Finnish reference group through linear regression. The consumption of dietary vitamin D sources and the potential socio-demographic, lifestyle, and dietary determinants of low vitamin D status i.e. deficiency (S-25(OH)D <30 nmol/L) and insufficiency (<50 nmol/L), were evaluated with logistic regression analyses. The prevalence of vitamin D deficiency and insufficiency was higher among the immigrants, especially Somalis and Kurds, than among the Finns (p<0.001). Consumption of vitamin D-rich foods differed between the immigrant groups; vitamin D-fortified fat spread was commonly used by a higher proportion of Somalis than Russians and Kurds; fish consumption was less frequent among Kurds than among Russians and Somalis; and higher proportions of Russians and Kurds consumed vitamin D-fortified dairy daily than Somalis (p<0.001 for all). The main determinants of low S-25(OH)D concentration were daily smoking, alcohol consumption, obesity, and winter blood sampling (p≤0.04). Older age, physical activity, consumption of fish, vitamin D-fortified fat spread, and dairy products, and use of vitamin D supplements were associated with reduced odds of low S-25(OH)D concentration (p≤0.04). In Study III, 191 subjects were screened and 147 women (Somali n=72, Finns n=75) aged 21-64 years were randomized to receive placebo or 10 or 20 µg vitamin D3/d in a 5-month trial during winter in the Helsinki area (60oN). S-25(OH)D concentrations were assessed by liquid chromatography-tandem mass spectrometry. Background and dietary data were collected through a detailed questionnaire and a validated semi-quantitative interview-administered food frequency questionnaire. Response of S-25(OH)D to vitamin D3 supplementation was assessed with repeated-measures analysis of covariance. Vitamin D status (S-25(OH)D) and vitamin D intake from diet and supplements were analysed. High prevalence of vitamin D insufficiency was observed among Somali women at screening. Interestingly, total vitamin D intake was higher among Somalis, but their baseline mean S-25(OH)D concentrations were lower than among Finns (p<0.001 and p=0.001, respectively). Moderate vitamin D3 supplementation at doses of 10 µg and 20 µg effectively increased mean S-25(OH)D in both Somali and Finnish women, without ethnic differences in the response to supplementation (p>0.05). In conclusion, food consumption patterns among immigrants with Russian, Somali, and Kurdish background were not similar. Healthy foods, particularly rye bread, vegetables, fruits, and berries, were consumed more by Russian immigrants than by participants with Kurdish and Somali background. Frequent consumption of fresh vegetables and fruits and berries was uncommon among Somalis. Higher consumption frequency of the healthy foods was associated with some socio-demographic factors, namely female sex, older age, and higher education. Likewise, differences existed in the consumption of vitamin D-rich foods between the immigrant groups. Use of vitamin D-fortified fat spread was more frequent among Somalis than among Russians and Kurds; fish consumption frequency was lower among Kurds than among Russians and Somalis; and vitamin D-fortified dairy was more frequently consumed daily by Russians and Kurds than by Somalis. The extent of the risk of low vitamin D status also differed between immigrant groups; higher prevalence of vitamin D deficiency and insufficiency was observed among Somalis and Kurds than among Russians. Hence, the immigrant groups cannot be considered a homogeneous group. In addition, non-fair-skinned immigrants are at higher risk of deficiency/insufficiency than their host populations. This thesis demonstrated that moderate vitamin D3 supplementation was effective in increasing S-25(OH)D in both Somali and Finnish women, and supports previous findings that ethnicity has no effect on the response of S-25(OH)D to vitamin D supplementation. Promotion of healthy food consumption patterns, including fruits, vegetables, whole-grains, and vitamin D-rich foods, is essential among immigrant groups to improve overall health.
  • Juvonen, Minna (Helsingin yliopisto, 2020)
    Oligosaccharides potentially present in cereal matrices have attracted attention because of their health-related properties, such as prebiotic potential. In addition to those that are naturally present, oligosaccharides can be formed during processing by the hydrolysis of cereal polysaccharides, such as arabinoxylan, by endogenous or microbial enzymes. In sourdough fermentation, additional poly- and oligosaccharides may be produced by lactic acid bacteria (LAB). In the work performed for this thesis, mass spectrometry (MS) methods were implemented in the structural analysis of various enzymatically produced oligosaccharides. The research was focused on finding solutions to separating and identifying oligosaccharide isomers. To determine the linkage positions of linear mixed-linked glucooligosaccharides (GLOS), the feasibility of the positive and negative electrospray ionisation multiple-stage MS (ESI-ITMSn) were compared. Next, the ability of ESI-ITMSn to detect the branching points and linkage positions of complex arabinoxylan-oligosaccharides (A)XOS was studied. The MS method was also coupled to hydrophilic interaction liquid chromatography (HILIC) to separate and identify the (A)XOS in mixtures. Finally, the potential of travelling wave ion mobility spectrometry (TWIMS) combined with MS to differentiate the isomeric (A)XOS was evaluated. To study the structures of the isomeric acceptor products formed by dextransucrase from LAB, 13C-labelling was applied with ESI-ITMSn. The ESI-ITMSn method provided structural information about the molecular weights, sequences and linkage positions of the linear mixed-linked GLOS and (A)XOS. Negative ionisation was found to be more informative than the positive ionisation mode in analysing the linkage positions. The stepwise C-ion fragmentation in negative mode from the reducing end towards the non-reducing end allowed to study the linkage diagnostic fragment ions from the middle and the non-reducing end in both linear and branched oligosaccharides. O-2 or/and O-3 arabinofuranosyl substituents of AXOS were determined by the presence or absence of diagnostic ions in the ESI-ITMS3 analysis in the negative mode. The ESI-ITMSn method, combined with 13C-isotope labelling, enabled the analysis of unique trisaccharides produced by dextransucrase as well as the further detailed analysis of the isomeric fragment ions. Coupling tandem mass spectrometry (MS/MS) to HILIC enabled the separation and identification of all AXOS by the combination of retention times and the MS/MS spectra. CID-TWIMS-MS/MS was shown to be a powerful tool in differentiating the isomeric AXOS when both the fragment ions and the precursor ions were analysed.
  • Nathanail, Alexis (Helsingin yliopisto, 2019)
    Fungi are capable of producing an array of heterogenous toxic secondary metabolites, i.e. mycotoxins, which may acutely or chronically impact human and animal health following the consumption of contaminated agricultural commodities. Mycotoxins, like most xenobiotics, are prone to structural alterations via metabolic processes in living organisms but can also undergo changes during food manufacturing. The resulting compounds, defined as “modified mycotoxins”, possess distinct chemical properties, with potentially unique toxicological characteristics, and often coexist with their precursor forms in food- and feedstuffs. The impetus of this Ph.D. thesis largely stems from the dearth of evidence available on these compounds and aspires to contribute evidence for addressing the underlying debate: Are modified mycotoxins relevant to food/feed safety? In this context, liquid chromatography–tandem mass spectrometric methods, employing fit-for-purpose sample preparation approaches, were developed and validated for the simultaneous determination of Fusarium mycotoxins and their modified forms. At first, conventional sample preparation techniques commonly utilised in mycotoxin analyses were evaluated against automated on-line sample clean-up. On-line clean-up and the standard “extract and shoot” approach offered optimal overall performance and achieved compliance with legislative criteria. The natural occurrence of the Fusarium mycotoxins HT-2 toxin, T-2 toxin, deoxynivalenol, nivalenol, zearalenone and derivatives thereof was investigated by conducting a nationwide survey of Finnish barley, oats and wheat grains. Deoxynivalenol was the most abundant mycotoxin (in 93% of the cereal samples), and at unusually high levels compared to adjacent years, followed by the modified mycotoxin deoxynivalenol-3-glucoside (81%). All 10 additional modified mycotoxins included in the method were detectable at widely varying concentrations. The relative proportions of modified/parent mycotoxins were mostly between 15–55%. Furthermore, the metabolism of HT-2 toxin and T-2 toxin was studied in barley and wheat. Specifically, tracing of their metabolism was accomplished by untargeted metabolomics based on stable isotopic labelling and liquid chromatography–high resolution mass spectrometry. Structural elucidation of the detected compounds indicated the presence of several novel modified mycotoxins, including glucoside, malonyl-glucoside, acetyl and feruloyl conjugates of the parent toxins. Time course kinetics of the in planta metabolites revealed the HT-2 toxin-3-glucoside as the primary detoxification product, which was rapidly formed in both crops. The experiments also determined the extent of metabolism of the parent toxins, while highlighting those modified forms present at harvest. Lastly, the metabolic fate of HT-2 toxin, T-2 toxin, deoxynivalenol and deoxynivalenol-3-glucoside was investigated during a four-day beer brewing fermentation with lager yeast. Yeast tolerated high toxin levels and was able to remove 9–34% of dosed toxins from wort by adsorption and/or biotransformation. The original contribution of this work can be summarised as the discovery of several novel modified Fusarium mycotoxins and related metabolic pathways, generation of essential natural occurrence data and gaining of further insight into mycotoxin-plant/fungal interactions, all of which were facilitated by state-of-the-art analytical tools.
  • Zhang, Yuemei (Helsingin yliopisto, 2020)
    The thesis aimed to study the effects and mechanisms of freezing-thawing treatments on myofibrillar protein denaturation and to explore the role of myofibrillar protein denaturation in causing the formation of thaw loss. The freezing-thawing of porcine M. longissimus thoracis et lumborum (LTL) increased the free Ca2+ concentration and the subsequent chilled storage promoted an accelerated decrease of activities of calpain-1 and calpain-2, compared to unfrozen meat. Proteasome activity was observed to be around 40% lower after freezing-thawing. The observed increased purge loss and decreased water-holding capacity (WHC) of myofibrils indicated myofibrillar protein denaturation occurring during the freezing-thawing treatment. In the investigation of freezing-induced denaturation of myofibrillar and sarcoplasmic proteins of LTL in relation to freezing rate, fast frozen samples (cold metal plate/-80°C) had a characteristic freezing time of 12 min, while samples frozen at slow rate (air/-20°C) had a freezing time of around 174 min. Slow freezing led to around 30% larger thaw loss in parallel with reduced WHC and increased surface hydrophobicity of myofibrils, indicating more severe myofibrillar protein denaturation in slow compared to fast freezing. A model is proposed to explain the importance of myofibrillar protein denaturation in relation to the freezing rate in the origin of thaw loss: In slow freezing, protons are accumulated with concentrating solutes in the unfrozen water leading to a decline of pH causing denaturation of structural proteins. In fast freezing small ice crystals might trap protons inducing less decline of pH and thus less myofibrillar protein denaturation and reduced thaw loss when compared with slow freezing. Sarcoplasmic protein denaturation also was shown to occur in freezing-thawing as evaluated by differential scanning calorimetry and tryptophan fluorescence properties of drip, which was, however, independent of freezing rate. The role of decreased pH (from pH 5.5 to 5.2), combined with high ionic strength (2 M KCl), in causing myofibrillar protein denaturation was studied by exposing fresh minced meat to either high ionic strength only or to high ionic strength with decreased pH to mimic conditions estimated to be in the unfrozen water of frozen meat during freezing. Exposure to high ionic strength caused an increase of WHC of the isolated myofibrils, whereas exposure to high ionic strength combined with low pH reduced WHC and increased surface hydrophobicity of the myofibrils. These results suggest that decreased pH combined with increased ionic strength in the unfrozen water of frozen meat largely would explain myofibrillar protein denaturation and thus the thaw loss occurring in frozen-thawed meat. The storage at -3 °C of fast or slow frozen pork prior to final thawing at 2 °C diminished the impact of the freezing rate on myofibrillar protein attributes, and differences between fast and slow freezing were no longer significant for WHC and surface hydrophobicity of the isolated myofibrils when frozen samples subsequently were kept at -3 °C for 7 days. The results suggest that a marked myofibrillar protein denaturation is taking place with extended storage time at -3 °C. In conclusion, freezing-thawing increases water loss in meat and slow freezing causes a higher increase when compared to freezing at fast rate. Myofibrillar and sarcoplasmic protein denaturation occurs in freezing and thawing. The rate of freezing produces a significant effect in the myofibrillar fraction: A slow freezing will develop a more severe protein denaturation than a fast freezing. Consequently, the myofibrillar protein denaturation, related to the freezing rate, is proposed to contribute to the generation of thaw loss. However, a subsequent storage of frozen meat at -3 °C before final thawing seems to diminish the beneficial effects of fast freezing on myofibrillar protein characteristics, compared to slow freezing, due to an additional protein denaturation. Therefore, it is recommended that meat industry adjusts the thawing capacity to minimize the passage time in temperature of -3 °C in meat.