Browsing by department "Elintarvike- ja ympäristötieteiden laitos"

The literature review deals with the structure of arabinoxylan and the principles of mass spectrometric methods used in analyzing them. The fragmentation of carbohydrate structures, the structural information gained from it, and naming of the resulting neutral fragments and product ions was also discussed. The aim of the experimental work was to analyze linear xylo-oligosaccharides and branched or linear arabinoxylans with known structures using tandem mass spectrometry. Of the arabinoxylo-oligosaccharides analyzed, three were branched while the others had an arabinose substituent at terminal xylose. The oligosaccharides were analyzed as underivatized, alditols, and methylated alditols with both positive and negative ionization. Derivatization was done in order to produce different product ions and to gain information about the effect of reduction and methylation on fragmentation mechanisms. Underivatized oligosaccharides provided most information with negative mode ionization. Low intensity of C-type fragment ions prevented their isolation in MS3-step when reduced oligosaccharides were analyzed. Methylation changed the weights of fragmentation products, which made it possible to detect branching in the oligosaccharide structure. Methylation also changed the fragmentation mechanism which caused differences in the product ion spectra compared to underivatized oligosaccharides. B-ions were fragmented to gain information about linkage positions when analyzing methylated alditols. The mechanism of B-ion fragmentation differs from that of C-ions, and produced information rich neutral fragments. However, low signal intensity made branch site analysis difficult, especially when arabinose substituent was located at terminal xylose.

Aronia berry consist of possible health promoting anthocyanins of which antioxidative character may have a positive impact to reduce risk factors of cardiovascular diseases. When ingested, anthocyanins decompose to several metabolites of which phenolic acids were the main focus of this research. Phenolic acids can be measured in urine which must be pretreated before further analysis. The aim of the study was to optimize a micro scale solid phase (μSPE) purification method for analysing phenolic metabolites in urine. The method was validated by recovery tests (%). The influence of two acids, formic acid and trifluoroacetic acid, were tested for the recovery (%) of six phenolic compounds: ferulic acid, protochatechuic acid, 3,4-dihydroxyphenylacetic acid, hippuric acid, homovanillic acid and cyanidine-3-glucoside. The method was applied to selected spot urine samples of a clinical research with subjects who had ingested for four weeks 100 ml of aronia juice which contained 1 g of phenolic compounds. Three unknown compounds (A, B and C) were identified as possible derivatives of phenolic acids by UHPLC-MS. The research was part of an EU supported BACCHUSproject. μSPE had a purifying effect with the two acids used having a different influence to the recovery of test compounds. However, there was no clear evidence that either of the acids would be better suited for the purification of the phenolic acids used in this study. Two of the three unknown compounds in randomly selected urine samples were identified as possible derivatives of phenolic acids based on their chromatographic and spectrophotometric characters. Compound A was identified as protocatecuiq acid derivative and compound C was tentatively identified as a derivative of ferulic acid. Compound B instead was not identified because it did not have any typical attributes of phenolic acids.

Several unhealthy lifestyle factors are associated with increased sickness absence (SA) and their cost. Diet and physical activity are lifestyle factors that can be altered and thus, they may include potential to effect on employer’s cost of SA. This study aimed to estimate the associations of changes in diet and leisure-time physical activity (LTPA) with employer’s direct cost of subsequent short-term (< 10 working days) SA spells. This study is a part of the Helsinki Health Study which is a longitudinal cohort of 40–60-year-old employees of the City of Helsinki in Finland (n = 8960, response rate 67 %). The participants received a phase 1 questionnaire during 2000–02 and a phase 2 questionnaire in 2007. Data from the phase 1 to the phase 2 were used to examine changes in diet and LTPA. Dietary habits were inquired by using a short food frequency questionnaire. Consumption of fruits (F) and vegetables (V) was used as an indicator of healthiness of participants’ diet. Participants’ F&V consumption was classified into three categories both in the phase 1 and 2: those who consumed neither F nor V daily, those who consumed either F or V daily and those who consumed both of them daily. In the question about LTPA, the participants estimated the intensity and the amount of their weekly leisure-time exercise, and based on these, the average weekly MET-hours were calculated. Participants were classified into three categories both in the phase 1 and 2: inactive (< 14 MET-hours/week), moderately active (≥ 14 MET-hours including LTPA with moderate intensity) and active (≥ 14 MET-hours/week including LTPA with vigorous intensity). Data of short-term SA and salaries were received from the employers’ registers between 2008 and 2012. A two-part model was used to analyze the associations between changes in diet and LTPA with the cost of SA. Employees who improved their F&V consumption from non-daily to daily and persevered physically active got 620 € (95 % CI 1194 €, -47 €) lower cost for the employer than those remaining non-daily F&V consumers and physically inactive through the 5-year follow-up, which means 21 % decrease in cost attributable to F&V consumption and LTPA. When examining changes in diet solely, improving or maintaining a greater F&V consumption tended to get lower cost, whereas the highest cost were among those who decreased their F&V consumption from daily to non-daily. No statistically significant results were found, however. Instead, those who persevered physically active (-546 €, 95 % CI -955 €, -137 €) or improved from moderately active to active (-542 €, 95 % CI -1005 €, -78 €) got 19% less cost for the employer than those remaining inactive through the follow-up. Employees who were inactive either in the phase 1 or 2 or continuously inactive got the highest cost for the employer. Improving employees’ diet and LTPA may reduce employer’s direct cost of short-term SA. Though diet had individually no significant associations with cost, improvements in diet may contribute the beneficial associations of LTPA with employer’s cost. To estimate the total cost savings that improvements in diet and LTPA may produce for the employer, all other direct and indirect cost attributable diet and LTPA, such as presenteeism and medical care cost, should also be evaluated.

The objectives of this study was to investigate the cause of the local whiteness in AURAÒ blue cheese and to find the way which reduces the white cheeses. It was studied if white cheeses have a compact or open structure. White cheeses were classified as compact and open. Scanning electron microscopy was performed on compact, open and mouldy cheeses. Factors which cause compact cheese were also studied. At the beginning of blue cheese manufacturing, citrate was analysed at different time points in different vats. The aim was to make compact cheese in one of the trials. Cheese curd was broken before and after moulding. Alternative factors including the effect of addition of salt to the vat on reducing the amount of white cheeses was also studied. Other variables were the speed of the drying belt, heating of vat, the amount of vat whey and the different moulds were also studied. In addition it was studied how separation of whey affects to the amount of compact cheeses in the drying belt. Microbiological and chemical analyses as well as density and texture measurement were performed on the trials. Most of the white cheeses were compact and the small amount of white cheeses had an open structure. The compact cheeses had less mould and yeast than the mouldy cheeses. The pH was lower and the amount of free amino acids was less in the compact cheeses than the mouldy cheeses. The compact cheeses were more dense than the mouldy cheeses. Measurements from texture analyser showed that the hardness of compact cheese were greater and the fracturability of compact cheese was lower than the hardness or fracturability of mouldy cheese. There were no differences in the amount of citrates between days or vats. Broken cheese curd before moulding caused a more compact structure. The addition of salt in the vat did not affect the white cheeses. The compact cheeses decreased when the speed of the drying belt was reduced. With the use of the new mould, less compact cheeses were produced than with older mould. The removal of water from cheese curds is logarithmic the longer they are on an open drying belt. The compact cheese also decreased logarithmically when cheese curds were on drying belt from zero to ten seconds. There was a greater amount of compact cheeses after moulding in the vat.

Bakteereita ja eukaryootteja esiintyy erittäin runsaasti jätevedessä, jossa ne voivat muodostaa fyysisiä vuorovaikutuksia toistensa kanssa. Fyysisiä vuorovaikutuksia bakteerien ja eukaryoottien välille voi muodostua symbioosien tai saalis-saalistaja suhteen välityksellä. Bakteerit ja eukaryootit osallistuvat myös jäteveden puhdistusprosesseihin, joten näillä molemmilla on iso merkitys puhdistusprosessissa. EpicPCR-menetelmän avulla voidaan tutkia isoja mikrobipopulaatioita sekvensoimalla genomin alueita suuresta määrästä yksittäisiä soluja. Aiemmin menetelmää on käytetty selvittämään sulfaatin pelkistämiseen osallistuvien bakteerien fylogeneettista diversiteettiä järven sedimenteissä. Tämän työn tarkoituksena oli tutkia bakteerien ja eukaryoottien fyysisiä vuorovaikutuksia jätevedessä käyttäen apuna epicPCR-menetelmää. Tutkimuksessa yhdistettiin tiiviissä fyysisessä yhteydessä olleen bakteerin 16S rRNA- ja eukaryootin 18S rRNA -geeni. Samalla selvitettiin miten epicPCR-menetelmä soveltuu käytettäväksi kahden fylogeneettisen 16S rRNA- ja 18S rRNA -geenin yhdistämiseen ja miten jätevesi soveltuu näytteeksi. Tulosten perusteella pohdittiin millaisia voisivat olla bakteerien ja eukaryoottien fyysiset vuorovaikutukset ja mitä ne merkitsevät kummallekin osapuolelle. Fyysisiä yhteisvaikutuksia tutkittiin käyttämällä yksinkertaiseen teknologiaan perustuvaa epicPCR-menetelmää ja sekvensointi suoritettiin Illumina MiSeq -menetelmällä. Sekvenssien käsittely ja analyysi tehtiin käyttäen Unixin Bash -komentorivikieltä ja R-ohjelmointikieltä CSC:n Taito-laskentaklusterilla käyttäen apuna Qiime-työkalupakettia. Tulosten perusteella bakteerien ja eukaryoottien välillä havaittiin fyysisiä yhteyksiä, jotka jakaantuivat kahdella eri tavalla. Jätevedestä löytyi eukaryoottiryhmiä, jotka muodostivat yhteyden useamman bakteeriryhmän kanssa ja eukaryoottiryhmiä, jotka muodostivat yhteyden vain tietyn bakteeriryhmän kanssa. Saalistukseen pystyvät eukaryoottiryhmät muodostivat yhteyksiä useamman bakteeriryhmän kanssa kuten myös määrällisesti enemmän kuin fotosynteettiset eukaryoottiryhmät.

The literature review deals with coeliac disease and wheat, rye and barley prolamins. The characteristics and analysis methods of the prolamins were discussed. Furthermore, the ELISA types, antibodies and reference material in ELISA were introduced in literature review. The aim of this master thesis study was to determine the feasibility of barley C-hordein as reference material in the quantification of wheat gluten in a sandwich ELISA method based on R5 antibody. RP-HPLC was used to determine the compositions of wheat prolamins from 27 wheat cultivars. SDS-PAGE was used for wheat prolamin subgroups identification. The R5 antibody reactivity of prolamins and the same prolamin group from different cultivars were tested. By comparing the R5 reactivity of total gluten of the 27 wheat cultivars, 10% barley C-hordein was used to calibrate the gluten content in spiking test of oat flour and oat biscuits. Omega 1,2-gliadin (Km 13) and γ-gliadin (Km 21) showed rather strong R5 reactivity while ω 5-gliadin (Km 203) and LMW glutenin subunits (Km 523) showed almost no reactivity against R5 antibody. However, the subgroup R5 reactivity differences between cultivars were not significant. The reactivity of total gluten from 27 cultivar varied from Km 14 to 192, with a logarithmic average Km 53. Thus, 10% C-hordein (Km 49) had similar reactivity of the average of all cultivars. In flour spiking test, the recoveries calibrated with PWG gliadin were 57-187%, comparing to 30-115% with 10% C-hordein calibration. In biscuit spiking test, the recoveries calibrated with PWG gliadin and 10% C-hordein were 84-145% and 44-76%.

Syanobakteerit tuottavat bioaktiivisia sekundääri- eli erikoismetabolian tuotteita. Syanobakteerien bioaktiivisia yhdisteitä on rakenteiltaan lukuisia erilaisia ja ne voidaan jakaa pääryhmiin peptidit, polyketidit, alkaloidit ja lipopolysakkaridit. Yhdisteistä on löydetty muun muassa antibakteerisia, antifungaalisia ja antiviraalisia ominaisuuksia. Lisäksi näihin bioaktiivisiin aineisiin kuuluu terveysriskin aiheuttavia toksisia yhdisteitä. Planktiset syanobakteerit ovat saaneet runsaasti huomiota niiden muodostamien massaesiintymien vuoksi, mutta myös benttiset eli sedimentissä tai erilaisilla pinnoilla elävät syanobakteerit voivat aiheuttaa terveysriskin. Benttisillä syanobakteereilla on myös potentiaalia uusien bioaktiivisten yhdisteiden löytymisessä biotekniikan tai lääketeollisuuden tarkoituksiin. Tutkimuksessa seulottiin Itämeren ja järven benttisten syanobakteerien tuottamia bioaktiivisia yhdisteitä. Syanobakteerikannat tunnistettiin 16S rRNA geenin avulla ja niiden bioaktiivisuutta tarkasteltiin maljadiffuusiomenetelmän avulla, PCR:n avulla etsittiin tunnettuja biosynteesigeenejä. Bioaktiivisia yhdisteitä pyrittiin tunnistamaan LC/MS-menetelmällä. Tunnistetut syanobakteerit muodostivat monipuolisen benttisille syanobakteereille tyypillisen lajikirjon, jossa bioaktiivisia ominaisuuksia havaittiin 24 % tutkituista kannoista. Bioaktiivisia ominaisuuksia löydettiin niin meri- kuin järviympäristöistä eristetyiltä kannoilta. Useita bioaktiivisuuksia esiintyi muun muassa Nostoc- ja Anabaena-syanobakteereilla. Benttiset syanobakteerit tuottivat antifungaalisia yhdisteitä, kuten hassallidiinia ja skytofysiiniä sekä sytotoksista anabaenolysiiniä. Lisäksi löydettiin useita syanobaktiinien biosynteesigeenejä. Benttisiltä syanobakteereilta ei havaittu mikrokystiinin tai anatoksiini-a:n biosynteesigeenejä. Antimikrobisia ominaisuuksia löydettiin kahdesta syanobakteerikannasta, joiden tuottamia bioaktiivisia yhdisteitä ei vielä tunnistettu. Benttiset syanobakteerit tuottavat siis useita tunnettuja bioaktiivisia yhdisteitä ja ovat myös potentiaalinen uusien bioaktiivisten yhdisteiden lähde.

Clostridium difficile infection (CDI) is a microbiota-related disease. Typically, antibiotic-induced perturbation of gut microbiota precedes the infection, while a healthy gut microbiota provides protection, i.e. colonization resistance, against it. Furthermore, in the case of recurrent CDI that is not resolved by antibiotics, restoring the gut microbiota with a fecal microbiota transplantation (FMT) is among the few treatment options that really work. Although FMT is effective in the treatment of CDI, the factors behind treatment success remain unclear. Both, the key species and the functions that are necessary to restore the healthy microbiota and eradicate C. difficile, are a matter of speculation. This study was based on the hypothesis that the adherence of some commensal bacteria to the gut epithelial cells could play a role in eradicating C. difficile by competing for epithelial binding with it. Furthermore, the isolation of those bacteria from the donor feces would enable more detailed mechanistic studies and development of a bacterial product for the treatment of CDI in the future. As a pre-selection step, bacterial adhesion to Caco-2 cells was utilized to isolate and cultivate epithelium-adherent bacteria from the donor feces. Microbiota composition of fecal sample, and the adhered and cultured sub-populations thereof, was determined by partial 16S rDNA amplicon sequencing using MiSeq method. The pre-selection approach was successful, since the obtained populations were different, both after the adhesion and cultivation, as compared to the original fecal sample. In addition, most obtained pure isolates adhered well to enterocytes. The ability of fecal bacteria to compete with C. difficile for binding to gut epithelial cells in vitro was also studied. Isolated bacteria from Caco-2-adhered populations were applied in competition and exclusion assays with C. difficile as purified or multi-species cultures, and reduction in C. difficile binding was observed due to the certain bacteria or bacterial populations. These assays still need developing and the results must be confirmed with more repetitions. However, the results are promising and a useful ground for future work in developing bacteriotherapeutic formulations for the treatment of CDI.

Sustainable development and fluctuating petroleum prices encourage manufacturing packaging materials in a more natural and cost-effective way. Green economy utilizes renewable raw materials from the nature which can be used in larger scale applications such as packaging industry. Barrier properties can be enhanced by manufacturing multilayer structures with a coating made of organic materials such as cellulose nanofibrils (CNF). With CNF it is possible to provide barrier and mechanical improvement to packaging material at low added cost. Together with hydrophobic films (e.g., HDPE, LDPE) also the low water vapor permeability and heat sealing properties can be enhanced. The aim of this thesis was to utilize CNF as a barrier layer for bio-based flexible food packaging pouches. A study consisted of examining 1-layer bio-HDPE film, 2-layer bio-HDPE/CNF film and 3-layer HDPE/CNF/LDPE film and testing their permeability (for oxygen, water vapor and aroma), sealability, tensile strength, influence of irradiation and aroma compounds (clove), and suitability for modified atmosphere packaging, MAP (with hazelnuts). The 2- and 3-layer multilayer films provided very good oxygen barrier under dry conditions, although aroma exposure increased the oxygen permeability rates. Pouches made of the 2-layer film were the most feasible for MAP, even though the 3-layer film would also have been an alternative if the film quality had been optimal. The oxidation of fatty acids in hazelnuts could be slowed down by using multilayer structure including CNF barrier under modified atmospheric conditions. This study helps to verify the feasibility of the bio-based multilayer films as a new food packaging material, and demonstrates the use of CNF as a high-barrier layer in a laminate. Bio-HDPE/CNF/bio-LDPE multilayer has potential for utilizing it in flexible food packaging pouches if the manufacture process is standardized leading into homogeneous film quality.

Nykyinen jälkiteollinen ihmiskunta tuottaa energiansa pääasiassa fossiilisilla polttoaineilla. Pariisin ilmastosopimuksen osapuolet sitoutuivat pitämään ilmaston lämpenemisen alle kahdessa celsiusasteessa verrattuna esiteolliseen aikaan ja muuttamaan energiantuotantotapojaan. Yhtenä ratkaisuna nähdään uusiutuvat energianlähteet, joita kuuluvat muun muassa biopolttoaineet. Bioetanoli on maailmanlaajuisesti käytetyin liikenteen biopolttoaine, jonka tuotanto on huomattavasti kasvanut 1980-luvulta lähtien. Tällä hetkellä bioetanolia tuotetaan etupäässä ruoantuotannon kanssa kilpailevista sokeri- ja tärkkelyspitoisista viljelykasveista, mutta niin kutsuttujen toisen sukupolven biopolttoaineiden tuotanto olisi ihmiskunnan ja maapallon kannalta kestävämmällä pohjalla. Toisen sukupolven bioetanolia valmistetaan kasvi- ja puupohjaisesta jätemateriaalista, kuten maa- ja metsätalouden sekä elintarviketeollisuuden jätteistä. Aikaisemmissa tutkimuksissa hyväksi lignoselluloosan ja puuperäisten jätteiden hajottajaksi ja monipuoliseksi entsyymien tuottajaksi tunnistettu, valkolahottajiin kuuluva kääpämäinen kantasieni rusorypykkä Phlebia radiata kykenee samanaikaiseen kiinteän kasvualustan hajotukseen ja etanolifermentaatioon. Tämän vuoksi rusorypykän etanolin tuottoa ja siihen liittyviä perusaineenvaihduntareittejä haluttiin tutkia tarkemmin. Sienen rihmastoa kasvatettiin jätelignoselluloosamateriaaleilla hapettomiksi muuttuvissa fermentaatio-olosuhteissa. Etanolin tuoton ja kasvualustan pilkkoutumisen lisäksi tutkittiin käänteistranskription kvantitatiivisella PCR-reaktiolla lignoselluloosan hajotukseen ja etanolifermentaatioon liittyviä entsyymejä koodaavia geenejä ja näiden geenien ilmentymistä eri kasvuaikapisteissä. Tulosten perusteella havaittiin rusorypykän tuottavan noin 13,5 massa-% etanolia kasvualustan hiilihydraattien sokeripitoisuuden perusteella lasketusta teoreettisesta maksimiarvosta. Lignoselluloosan hajotukseen ja etanolin fermentointiin oleellisesti liittyvien entsyymien geenit ilmentyivät kasvatusten aikana jätelignoselluloosalla tilastollisesti merkitsevin eroin eri aikapisteissä, mikä osoitti hajotustoiminnan, rihmaston kasvun ja etanolin tuoton olevan muuttuvia ja toisiinsa vaikuttavia prosesseja. Sieni pystyi hyödyntämään kiinteää kasvualustaansa irrottaen siitä käyttöönsä sokereita. Rihmaston kasvusta ja elävästä sienibiomassasta viitteitä antavaa ergosterolia havaittiin koko kuukauden pituisen kasvatuksen ajan. Lisäksi kaasukehässä havaittiin odotettuja fermentaatiomuutoksia, kuten hiilidioksidin kertymistä, ja happikaasu oli neljässä viikossa lähes kulutettu loppuun. Tulosten perusteella rusorypykkä osoittautui tehokkaaksi jätelignoselluloosan pilkkojaksi ja kestäväksi etanolin tuottajaksi. RNA-eristys kiinteältä kasvualustalta sekä aineenvaihduntageenien ilmentymisen analysointi RT-qPCR-menetelmällä mahdollistivat rusorypykän transkriptomin jatkotutkimuksen geenien säätelyn selvittämiseksi fermentoivissa ja lähes hapettomissa olosuhteissa.