NMR Metabolomics of Foods – Investigating the Influence of Origin on Sea Buckthorn Berries, Brassica Oilseeds and Honey

The origin of foods plays an important role in their metabolome (the set of compounds present as products of metabolic events). The compositions of food plants are inevitably determined by a number of inherent and external factors – most importantly by the genotype (species, subspecies, cultivar, variety) and the prevailing conditions and weather parameters at each growth environment. The declaration of food origin can be defined and protected by law. The constantly increasing consumer awareness towards food origin, authenticity and quality has set the need for efficient tools for their verification. Metabolomics based on nuclear magnetic resonance (NMR) spectroscopy is increasingly being applied in analysing food composition and quality and in detecting food frauds and adulterations. The aim of the current work was to determine the influence of origin-related variables in food composition and quality by using 1H NMR metabolomics. The model foods – sea buckthorn (Hippophaë rhamnoides) berries, oilseeds of Brassica spp. and varietal honey – represent different foods with special sensory, nutritional, bioactive, commercial and national significance. The sea buckthorn berry metabolites were investigated in respect to the genotype (subspecies, cultivar) and geographical origin, with special emphasis on the influence of northern latitudes and related conditions. In the oilseeds, the inter-species variation and the influence of environmental and developmental stage on the seed composition was investigated. NMR profiling was applied in characterising the marker compounds for different honey types for botanical authentication. Multivariate analysis methods such as principal component (PCA) and discriminant analyses (PLS-DA, OPLS-DA) were applied in every sub-study to determine the key metabolites and origin-related factors characterising the food samples. The sea buckthorn subspecies were mainly distinguished by the relatively high content of ethyl-β-D-glucopyranoside (ssp. rhamnoides) and malic acid and vitamin C (ssp. sinensis). The northern latitude and respective conditions (the length of growth season, temperature, radiation and precipitation) was shown to alter the chemical composition of berries of the same genetic origin. In subarctic latitudes, the berries formed more ascorbic acid while the levels of ethyl glucose remained relatively low. The berries of cultivar 'Tytti' contained more ethyl glucoside while the berries of 'Terhi' contained more quinic acid in comparison. Calculated from the start of the growing season until harvest, the effective temperature sum (degree days) and the radiation sum correlated positively with ethyl glucoside that accumulated up to six-fold in overripe berries in southern Finland. The sea buckthorn berries (ssp. sinensis) grown at over 2000 m altitude contained typically more ascorbic and malic acids. The seeds of turnip rape was characterised by a relatively higher sucrose and polyunsaturated fatty acid content over oilseed rape that had a higher content of sinapine and oil in general. Growth conditions with reduced temperature added to the level of unsaturation in the oilseed lipids and delayed the seed development. The varietal honeys were classified with the aid of NMR profiling, as the typical sugar composition and other botanical markers were characterised. Also, previously unreported markers were designated for dandelion honeys. The correlations between complex food metabolomes and the origin-related variables were easily accomplished with NMR metabolomics. Especially, the effect of northern conditions on the growth place-dependent compositional flexibility (phenotypic plasticity) of the plant foods was deemed considerable. The results of this thesis can be further used to determine food quality, origin and authenticity and as an aid in plant breeding operations.Alkuperällä on suuri vaikutus elintarvikkeen metabolomiin eli aineenvaihduntatuotteiden kokonaisuuteen. Erityisesti kasviperäisten elintarvikkeiden koostumukseen vaikuttavat lukuisat sisäiset ja ulkoiset alkuperään liittyvät tekijät, kuten perimä (laji, alalaji, lajike) ja kasvupaikalle tyypilliset ympäristö- ja sääolosuhteet. Elintarvikkeen alkuperä voidaan määritellä ja suojata lainsäädännöllisin perustein. Kuluttajien kasvanut kiinnostus ja tietämys elintarvikkeiden alkuperää, aitoutta ja laatua kohtaan on lisännyt tehokkaiden ja luotettavien laadunvarmistusmenetelmien tarvetta. Varsinkin ydinmagneettista resonanssispektroskopiaan (NMR) perustuvaa metabolomiikkatutkimusta hyödynnetään yhä useammin elintarvikkeiden koostumuksen, laadun ja aitouden analysoinnissa. Tämän tutkimuksen tarkoituksena oli selvittää alkuperän vaikutusta tyrnimarjojen (Hippophaë rhamnoides), rypsin- ja rapsinsiementen (Brassica spp.) sekä lajihunajan koostumukseen 1H-NMR-metabolomiikan avulla. Nämä elintarvikkeet ovat kansallisesti ja kaupallisesti arvokkaita ja mielenkiintoisia niille tyypillisten aistittavien, ravitsemuksellisten ja bioaktiivisten ominaisuuksien ansiosta. Tyrnimarjojen koostumusta vertailtiin eri alalajien (ssp. rhamnoides ja ssp. sinensis) ja lajikkeiden ('Terhi' ja 'Tytti') sekä kasvupaikkojen (Suomi, Kiina, Kanada) välillä. Tavoitteena oli erityisesti selvittää, miten erityisesti pohjoisille leveysasteille tyypilliset olosuhteet vaikuttavat marjojen aineenvaihduntatuotteisiin. Öljysiementen kohdalla tutkittiin myös miten lajikohtainen perimä sekä kasvupaikan/-olosuhteiden ja siemenen kehittymisvaihe vaikuttavat siementen kemialliseen koostumukseen ja laatuun. Hunajien tapauksessa NMR-metabolomiikkaa hyödynnettiin kasvialkuperäkohtaisten sormenjälkiyhdisteiden tunnistamiseen ja kotimaisten lajihunajien kasvialkuperän varmentamiseen. Kaikissa osatutkimuksissa sovellettiin pääkomponentti-(PCA) ja diskriminanttianalyysiin (PLS-DA, OPLS-DA) perustuvia monimuuttujamenetelmiä tärkeimpien näyteryhmiä erottavien ja määrittävien yhdisteiden ja taustatekijöiden selvittämiseksi. Tyrnin alalajit erottuivat pääasiassa suhteellisesti korkean etyyli-β-D-glukopyranosidin (ssp. rhamnoides) sekä omenahappo- ja C-vitamiini-pitoisuuden (ssp. sinensis) perusteella. Pohjoisen leveysasteen ja sille tyypillisten olosuhteiden (kasvukauden pituus, lämpötila, säteily, sademäärä) todettiin muokanneen samaa geneettistä alkuperää olevien marjojen kemiallista koostumusta. Subarktisilla leveyksillä tyrnimarjaan muodostui enemmän askorbiinihappoa ja etyyliglukosidin määrä oli alhainen. 'Tytti'-lajikkeen marjat sisälsivät enemmän etyyliglukosidia, kun taas 'Terhi' sisälsi vastaavasti enemmän kviinihappoa. Kasvukauden tehoisa lämpösumma ja säteilysumma korreloivat positiivisesti etyyliglukosidin kanssa, jota kertyi ylikypsiin marjoihin Etelä-Suomessa jopa kuusinkertainen määrä kypsiin verrattuna. Yli 2000 metrin korkeudessa kasvaneissa tyrnimarjoissa (ssp. sinensis) oli tyypillisesti korkeampi omena- ja askorbiinihappopitoisuus. Suhteellisesti korkeampi sakkaroosipitoisuus ja monityydyttymättömien rasvahappojen osuus oli tyypillisempää rypsille, kun taas rapsi erottui rypsistä korkeamman öljy- ja sinapiinipitoisuuden perusteella. Kylmempi kasvupaikka lisäsi monityydyttymättömien rasvahappojen osuutta öljysiemenissä ja hidasti siemenen kehittymistä. NMR-profiloinnin avulla lajihunajat pystyttiin luokittelemaan kullekin hunajalle ominaisen sokerikoostumuksen ja muiden kasvialkuperästä kertovien merkkiyhdisteiden perusteella. Voikukkahunajalle tunnistettiin myös aiemmin raportoimattomia merkkiyhdisteitä. NMR-metabolomiikan avulla pystyttiin helposti selvittämään monimutkaisten aineenvaihduntatuotteiden kokonaisuuksien ja elintarvikkeen alkuperään liittyvien muuttujien välisiä yhteyksiä. Varsinkin pohjoisten kasvuolosuhteiden vaikutus kasviperäisten elintarvikkeiden koostumukselliseen vaihteluun oli huomattava. Väitöskirjan tuloksia voidaan hyödyntää elintarvikkeiden laadun, alkuperän ja aitouden varmistamisessa sekä kasvinjalostuksen apuna.Siirretty Doriast

1H NMR Metabolomics of Chinese Human Milk at Different Stages of Lactation among Secretors and Non-Secretors

Human milk is an intricate, bioactive food promoting infant health. We studied the composition of human milk samples collected over an 8-month lactation using 1H NMR metabolomics. A total of 72 human breast milk samples were collected from ten Chinese mothers at eight different time points. The concentrations of ten human milk oligosaccharides (HMOs), fucose and lactose were quantified. Six of the mothers were classified as Lewis-positive secretors (Se+Le+) and four as Lewis-positive non-secretors (Se−Le+) based on the levels of 2′-fucosyllactose (2′-FL), lacto-N-fucopentaose (LNFP) II, lactodifucotetraose (LDFT) and lacto-N-neotetraose (LNnT). Acetate, citrate, short/medium-chain fatty acids, glutamine and urea showed a time-dependent trend in relation to the stage of lactation. The concentrations of 2′-FL, 3-FL (3-fucosyllactose), 3′-SL (3′-sialyllactose), LDFT, LNFP I, LNFP II, LNFP III, LNnT, LNT (lacto-N-tetraose), and fucose were statistically different between secretors and non-secretors. A temporal difference of approximately 1–2 months between the development of non-secretor and secretor HMO profiles was shown. The results highlighted the importance of long-term breastfeeding, especially among non-secretors.</p

Untargeted Metabolic Fingerprinting Reveals Impact of Growth Stage and Location on Composition of Sea Buckthorn (Hippophaë Rhamnoides) Leaves

Sea buckthorn (Hippophaë rhamnoides) is increasingly cultivated to produce raw materials for food and nutraceuticals. There is little knowledge on composition of sea buckthorn leaves (SBLs) and the key factors influencing the composition. This research aims to unravel the metabolic profile of SBLs and the effects of cultivar, location and stage of growth, and climatic conditions on the metabolic profile of SBLs. Leaves of two sea buckthorn cultivars grown in the south and north of Finland during two consecutive growth seasons were studied using untargeted nuclear magnetic resonance (NMR) metabolomics. The highest variance in the metabolic profile was linked to the growth stage, wherein leaves from the first 7 weeks of harvest were characterized with higher abundance of polyphenols, while relatively higher abundance of carbohydrates and sugars was observed in the later weeks. The growth location attributed for the second highest variation, wherein the north–south comparison identified fatty acids and sugars as discriminatory metabolites, and the potential association of metabolome to natural abiotic stressors was revealed. An inverse correlation between carbohydrate/sugar content as well as fatty acids of higher carbon chain length with the temperature variables was evident. The supervised chemometric models with high sensitivity and specificity classified and predicted the samples based on growth stage and location, and cultivar. Nontargeted NMR-metabolomics revealed the metabolic profile of SBLs and their variation associated with various biotic and abiotic factors. Cultivar and growth stage are key factors to consider when harvesting SBLs for use in food and nutraceuticals.Practical ApplicationGlobally, sea buckthorn cultivation has been rapidly increasing due to the known health-promoting benefits of the berries and leaves of the plant. The current research obtained new comprehensive information on the compositional profile of sea buckthorn leaves as well as the impact of major contributory factors, such as cultivars, the advancement of growth stage, geographical location, and weather parameters. The findings of this research provide new knowledge and guidance for plant breeding, cultivation and commercial utilization of sea buckthorn leaves as raw materials for food, feed, and nutraceuticals.</p

Characterization and Quantification of Nonanthocyanin Phenolic Compounds in White and Blue Bilberry (Vaccinium myrtillus) Juices and Wines Using UHPLC-DAD−ESI-QTOF-MS and UHPLC-DAD

The nonanthocyanin phenolic compounds in juice and wine produced from fruits of white bilberry, a nonpigmented mutant of Vaccinium myrtillus, and blue bilberry (pigmented variety) were analyzed using liquid chromatography with a diode array detector (LC-DAD) and LC-DAD−electrospray ionization-quadrapole/time of flight hybrid mass spectrometry (ESI-QTOF-MS). On the basis of elution order, UV−vis spectra, accurate mass data, and fragmentation pattern and standards, 42 compounds including 22 phenolic acids, 15 flavonols, and 5 flavan-3-ols, were identified in juices and wines prepared from the two bilberry varieties. The levels of most individual nonanthocyanin phenolic compounds in white bilberry products were significantly lower than those in pigmented ones. In bilberry juices, phenolic acids were the most predominant, accounting for approximately 80% of total phenolic content, with p-coumaroyl monotropeins and caffeic acid hexoside being the major phenolic acids. After fermentation, the total contents of phenolic acids, flavonols, and nonanthocyanin phenolic compounds significantly increased, while the content of total flavan-3-ols decreased significantly. p-Coumaroyl monotropeins still dominated in the wine products, while caffeic acid content showed dramatic elevation with the significant drop of caffeic acid hexoside.</p

Effect of Acylated and Nonacylated Anthocyanins on Urine Metabolic Profile during the Development of Type 2 Diabetes in Zucker Diabetic Fatty Rats

The effect of nonacylated and acylated anthocyanins on urinary metabolites in diabetic rats was investigated. Nonacylated anthocyanins extract from bilberries (NAAB) or acylated anthocyanins extract from purple potatoes (AAPP) was given to Zucker diabetic fatty (ZDF) rats for 8 weeks at daily doses of 25 and 50 mg/kg body weight. 1H NMR metabolomics was applied to study alterations in urinary metabolites from three time points (weeks 1, 4, and 8). Both types of anthocyanins modulated the metabolites associated with the tricarboxylic acid cycle, gut microbiota metabolism, and renal function at weeks 1 and 4, such as 2-oxoglutarate, fumarate, alanine, trigonelline, and hippurate. In addition, only a high dose of AAPP decreased monosaccharides, formate, lactate, and glucose levels at week 4, suggesting improvement in energy production in mitochondria, glucose homeostasis, and oxidative stress. This study suggested different impacts of AAPP and NAAB on the metabolic profile of urine in diabetes. </p

NMR-based metabolomics approach on optimization of malolactic fermentation of sea buckthorn juice with Lactiplantibacillus plantarum

This work investigated the impact of malolactic fermentation on the metabolomic profile of sea buckthorn juice to optimize the fermentation process for flavor modification. Six strains of L. plantarum were used with varied pH of the juice, cell acclimation, and fermentation time. 1H-NOESY spectra were acquired from fresh and fermented juices with a total of 46 metabolites identified. Less sugars and quinic acid were metabolized at pH 2.7 while oxidation of ascorbic acid was reduced at pH 3.5. l-Malic acid, essential amino acids, and nucleosides were consumed early during fermentation while sugars in general were consumed later in the fermentation. If deacidification is the main target of fermentation, strains that produce less acids and ferment less sugars, shorter fermentation time, and lower starter pH should be used. Higher starter pH and longer fermentation time promote formation of antimicrobial compounds and potentially increase antioxidant stability. </p

Oxidative stability, oxidation pattern and α-tocopherol response of docosahexaenoic acid (DHA, 22:6n-3)-containing triacylglycerols and ethyl esters

DHA is most often used in supplements either in its triacylglycerol or ethyl ester form. Currently, there is only little published data on the differences in the oxidative stability and α-tocopherol response between the two lipid structures, as well as on the oxidation patterns of pure DHA. This study investigated the oxidative stability, α-tocopherol response and oxidation pattern of DHA incorporated in triacylglycerols and as ethyl esters with an untargeted approach after oxidation at 50 °C in the dark. Liquid and gas chromatographic methods with mass spectrometric detection and nuclear magnetic resonance spectroscopy were applied. DHA was more stable in triacylglycerols than as ethyl esters without α-tocopherol addition. With α-tocopherol added the opposite was observed. The oxidation products formed during triacylglycerol and ethyl ester oil oxidation were mostly similar, but also some structure-related differences were detected in both volatile and non-volatile oxidation products

Aromatic Characteristics of Passion Fruit Wines Measured by E-Nose, GC-Quadrupole MS, GC-Orbitrap-MS and Sensory Evaluation

This study investigated the volatile composition and aromatic features of passion fruit wines using a combination of gas chromatography–quadrupole mass spectrometry (GC-qMS), gas chromatography–Orbitrap–mass spectrometry (GC-Orbitrap-MS), electronic nose (E-nose) and sensory evaluation. The results showed that these passion fruit wines possessed different aromatic features confirmed by E-nose. Seventeen sulfur compounds and seventy-eight volatiles were detected in these passion fruit wines using GC-Orbitrap-MS and GC-qMS, respectively. Forty-four volatiles significantly contributed to the overall wine aroma. These wines possessed passion fruit, mango, green apple, lemon and floral aromas confirmed by sensory evaluation. The partial least squares regression analysis indicated that sulfides, esters and terpenes, and terpenes mainly correlated to the passion fruit, mango and green apple aroma, respectively. Sulfur compounds significantly affected the aroma of passion fruit wine. The findings in this study could provide useful insight toward the quality control of passion fruit wine.</p

Profiles of Volatile Compounds in Blackcurrant (Ribes nigrum) Cultivars with a Special Focus on the Influence of Growth Latitude and Weather Conditions

The volatile profiles of three blackcurrant ( Ribes nigrum L.) cultivars grown in Finland and their responses to growth latitude and weather conditions were studied over an 8 year period by headspace solid-phase microextraction (HS-SPME) followed by gas-chromatographic-mass-spectrometric (GC-MS) analysis. Monoterpene hydrocarbons and oxygenated monoterpenes were the major classes of volatiles. The cultivar 'Melalahti' presented lower contents of volatiles compared with 'Ola' and 'Mortti', which showed very similar compositions. Higher contents of volatiles were found in berries cultivated at the higher latitude (66 degrees 34' N) than in those from the southern location (60 degrees 23' N). Among the meteorological variables, radiation and temperature during the last month before harvest were negatively linked with the volatile content. Storage time had a negative impact on the amount of blackcurrant volatiles

Evaluation of the composition and oxidative status of omega-3 fatty acid supplements on the Finnish market using NMR and SPME-GC–MS in comparison with conventional methods

Previous studies disagree on the oxidative status of omega-3 supplements. The great deviation raises concerns about quality and the methods used to monitor it. This study investigated 49 omega-3 products for their fatty acid content, lipid class and oxidative status using official methods, gas and liquid chromatography with mass spectrometry and nuclear magnetic resonance spectroscopy. With minor deviations, omega-3 fatty acid content and lipid class of all products were as declared. 24% of studied products exceeded thresholds set by The Global Organization for EPA and DHA Omega-3s for peroxide and/or p-anisidine value suggesting a compromised oxidative status. However, peroxide and/or p-anisidine value were only suitable for detection of lipid oxidation in 90% or 73%, respectively, of the products. Analysis of volatile oxidation compounds can be an alternative method for p-anisidine value. Nuclear magnetic resonance spectroscopy was shown to be a rapid method for determination of oil type and lipid class.</p