Analysis of lipid oxidation during digestion by liquid chromatography–mass spectrometric and nuclear magnetic resonance spectroscopic techniques

Siirretty Doriast

Analysis of lipid oxidation during digestion by liquid chromatography–mass spectrometric and nuclear magnetic resonance spectroscopic techniques

Lipid autoxidation is an unwanted process that affects the quality of food and has impact on human health. Lipid oxidation has been studied extensively, but oxidation during digestion has largely been ignored. Formation of oxidized lipids increases rapidly when protective antioxidants are exhausted. On the other hand, the nature of antioxidants can lead to problems when fortifying foods with too much antioxidants. Pro-oxidative effects of several antioxidants have been observed when used in excessive amounts. Methods for studying lipid oxidation are numerous. Among them are unspecific titrimetric methods and highly specialized chromatographic and mass spectrometric methods. Nuclear magnetic resonance (NMR) spectroscopy, especially the proton (1H) NMR, is a promising technique for fast screening of lipid samples as it is non-destructive and because of the large dynamic scale of the technique. Drawbacks of NMR are that relatively large amount of sample is required for the analysis and that specific molecular structures may be difficult to identify from complex spectrum. This thesis focuses on the study of in vitro lipid oxidation by different chromatographic, mass spectrometric and nuclear magnetic resonance spectroscopic methods. The most significant findings of the studies in this thesis centre around oxidation, hydrolysis, and behaviour of lipids in an artificial digestion model used in the studies. The model simulates the digestion processes of human and can be used to study lipid oxidation in vitro. Also of importance, are the lipid analysis techniques developed for the experiments, as the techniques can be adopted to other fields of scientific studies as well for industrial uses. Four major studies were conducted in this thesis: first an in vitro digestion model was adopted to study the behaviour of differently oxidized rapeseed oils. Simultaneously, a novel HPLC&ndash; evaporative light scattering detector&ndash;MS analysis technique was developed, which enabled the analysis of native and oxidized free fatty acids, monoacylglycerols, diacylglycerols, and triacylglycerols in the chyme produced by the digestion model. The main findings of the study were that thermally oxidized rapeseed oil, chemically oxidized rapeseed oil and unoxidized rapeseed oil were hydrolyzed in a similar manner. No hydroperoxides were detected in the digested samples, even though they were present in the undigested oils. Also, the finding of Abstract vii large amounts of sn-1(3) monoacylglycerols was surprising, questioning the long believed mechanism of triacylglycerol digestion and absorption. In the second study, an ultra-high performance liquid chromatography (UHPLC) analysis technique was developed to replace the previous HPLC method. Analysis time was reduced by a factor of 5.5 without the loss of chromatographic resolution or detection sensitivity. Over 150 compounds were detected from digested and undigested oxidized rapeseed oils with the method. Most significant finding was that toxic core aldehydes present in the undigested oxidized oils were not detected in the extracted chyme. This implies that the aldehydic functions were either lost during the hydrolysis of lipids or that the compounds formed various complexes with other components of the chyme and were not detectable by the analysis technique used. In the third study, a series of antioxidants were assessed for the effects in the artificial digestion model. An improved UHPLC&ndash;ESI&ndash;MS analysis method was developed, which used lithium salt to greatly enhance the ionization and therefore the detection limits of the low level analytes in electrospray ionization&ndash;mass spectrometry. The main findings were that native (unoxidized) rapeseed oil can be oxidized during the digestion processes and that none of the used antioxidants could completely prevent this oxidation. L-ascorbic acid, 6-palmitoyl-O-Lascorbic acid, 3,5-di-tert-butyl-4-hydroxytoluene (BHT), DL-&alpha;-tocopherol, and DL-&alpha;- tocopheryl acetate had different kinds of effects against this oxidation, as measured by the concentration of oxidized lipids in the samples. The findings of our second study were supported by the fourth study in where 1H NMR spectroscopy was used along UHPLC&ndash;ESI&ndash;MS analyses to study the behaviour of core aldehyde-rich oils in the artificial digestion model. Again, no compounds with aldehydic functions were detected by UHPLC&ndash;ESI&ndash;MS analyses of the digested oils even when high amounts of core aldehydes were present in the original oil. However, 1H NMR analyses of several samples revealed that there were some remaining carbonyl functions in the digested samples. The combined results of these analyses techniques strongly hinted that Schiff bases and Michael addition products were formed in the digestion mixture. Overall, the scientific studies conducted in this thesis have increased the knowledge of lipid oxidation and especially provided more detailed information on possible oxidation during lipid digestion. The findings merit for more research in the fieLipid autoxidation is an unwanted process that affects the quality of food and has impact on human health. Lipid oxidation has been studied extensively, but oxidation during digestion has largely been ignored. Formation of oxidized lipids increases rapidly when protective antioxidants are exhausted. On the other hand, the nature of antioxidants can lead to problems when fortifying foods with too much antioxidants. Pro-oxidative effects of several antioxidants have been observed when used in excessive amounts. Methods for studying lipid oxidation are numerous. Among them are unspecific titrimetric methods and highly specialized chromatographic and mass spectrometric methods. Nuclear magnetic resonance (NMR) spectroscopy, especially the proton (1H) NMR, is a promising technique for fast screening of lipid samples as it is non-destructive and because of the large dynamic scale of the technique. Drawbacks of NMR are that relatively large amount of sample is required for the analysis and that specific molecular structures may be difficult to identify from complex spectrum. This thesis focuses on the study of in vitro lipid oxidation by different chromatographic, mass spectrometric and nuclear magnetic resonance spectroscopic methods. The most significant findings of the studies in this thesis centre around oxidation, hydrolysis, and behaviour of lipids in an artificial digestion model used in the studies. The model simulates the digestion processes of human and can be used to study lipid oxidation in vitro. Also of importance, are the lipid analysis techniques developed for the experiments, as the techniques can be adopted to other fields of scientific studies as well for industrial uses. Four major studies were conducted in this thesis: first an in vitro digestion model was adopted to study the behaviour of differently oxidized rapeseed oils. Simultaneously, a novel HPLC&ndash;evaporative light scattering detector&ndash;MS analysis technique was developed, which enabled the analysis of native and oxidized free fatty acids, monoacylglycerols, diacylglycerols, and triacylglycerols in the chyme produced by the digestion model. The main findings of the study were that thermally oxidized rapeseed oil, chemically oxidized rapeseed oil and unoxidized rapeseed oil were hydrolyzed in a similar manner. No hydroperoxides were detected in the digested samples, even though they were present in the undigested oils. Also, the finding of large amounts of sn-1(3) monoacylglycerols was surprising, questioning the long believed mechanism of triacylglycerol digestion and absorption. In the second study, an ultra-high performance liquid chromatography (UHPLC) analysis technique was developed to replace the previous HPLC method. Analysis time was reduced by a factor of 5.5 without the loss of chromatographic resolution or detection sensitivity. Over 150 compounds were detected from digested and undigested oxidized rapeseed oils with the method. Most significant finding was that toxic core aldehydes present in the undigested oxidized oils were not detected in the extracted chyme. This implies that the aldehydic functions were either lost during the hydrolysis of lipids or that the compounds formed various complexes with other components of the chyme and were not detectable by the analysis technique used. In the third study, a series of antioxidants were assessed for the effects in the artificial digestion model. An improved UHPLC&ndash;ESI&ndash;MS analysis method was developed, which used lithium salt to greatly enhance the ionization and therefore the detection limits of the low level analytes in electrospray ionization&ndash;mass spectrometry. The main findings were that native (unoxidized) rapeseed oil can be oxidized during the digestion processes and that none of the used antioxidants could completely prevent this oxidation. L-ascorbic acid, 6-palmitoyl-O-Lascorbic acid, 3,5-di-tert-butyl-4-hydroxytoluene (BHT), DL-&alpha;-tocopherol, and DL-&alpha;-tocopheryl acetate had different kinds of effects against this oxidation, as measured by the concentration of oxidized lipids in the samples. The findings of our second study were supported by the fourth study in where 1H NMR spectroscopy was used along UHPLC&ndash;ESI&ndash;MS analyses to study the behaviour of core aldehyde-rich oils in the artificial digestion model. Again, no compounds with aldehydic functions were detected by UHPLC&ndash;ESI&ndash;MS analyses of the digested oils even when high amounts of core aldehydes were present in the original oil. However, 1H NMR analyses of several samples revealed that there were some remaining carbonyl functions in the digested samples. The combined results of these analyses techniques strongly hinted that Schiff bases and Michael addition products were formed in the digestion mixture. Overall, the scientific studies conducted in this thesis have increased the knowledge of lipid oxidation and especially provided more detailed information on possible oxidation during lipid digestion. The findings merit for more research in the field. &nbsp;</p

Profile and content of residual alkaloids in ten ecotypes of Lupinus mutabilis Sweet after aqueous debittering process

Universidad Nacional Agraria La Molina. Facultad de Industrias AlimentariasThe evaluation of the level of alkaloids in edible Lupinus species is crucial from a food safety point of view. Debittering of lupin seeds has a long history; however, the control of the level of alkaloids after processing the seeds is typically only evaluated by changes in the bitter taste. The aim of this study was to evaluate the profile and residual levels of quinolizidine alkaloids (QA) in (Lupinus mutabilis Sweet) after aqueous debittering process. Samples from 10 ecotypes from different areas of Peru were analyzed before and after the process. Based on results obtained by gas chromatography and mass spectrometry, from eight alkaloids identified before the debittering process, only small amounts of lupanine (avg. 0.0012 g/100 g DM) and sparteine (avg. 0.0014 g/100 g DM) remained in the seeds after the debittering process, and no other alkaloids were identified. The aqueous debittering process reduced the content of alkaloids to levels far below the maximal level allowed by international regulations (≤ 0.2 g/kg DM)

Supercritical CO2 Extraction of Triterpenoids from Chaga Sterile Conk of Inonotus obliquus

Triterpenoids are among the bioactive components of Chaga, the sterile conk of the medicinal fungus Inonotus obliquus. Supercritical fluid extraction of Chaga triterpenoids was carried out with supercritical CO2, while a modified Folch method was used as a comparison. Three temperature-pressure combinations were tested varying between 314-324 K (40-50 degrees C) and 281-350 bars, using time- and volume-limited extractions. Six triterpenoids were identified with GC-MS and quantified with GC-FID: ergosterol, lanosterol, beta-sitosterol, stigmastanol, betulin, and inotodiol. The Folch extraction resulted in recovery of trametenolic acid, which was not extracted by supercritical CO2. Inotodiol was the major triterpenoid of all the extracts, with a yield of 87-101 mg/100 g and 139 mg/100 g, for SFEs and the Folch method, respectively. The contents of other major triterpenoids, lanosterol and ergosterol, varied in the ranges 59-63 mg/100 g and 17-18 mg/100 g by SFE, respectively. With the Folch method, the yields were 81 mg/100 g and 40 mg/100 g, respectively. The highest recovery of triterpenoids with SFE in relation to Folch was 56% and it was obtained at 324 K (50 degrees C) and 350 bar, regardless of extraction time or volume of CO2. The recoveries of lanosterol and stigmastanol were unaffected by SFE conditions. Despite the lower yield, SFE showed several advantages including shorter extraction time and less impact on the environment. This work could be a starting point for further studies on green extraction methods of bioactive triterpenoids from Chaga

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

Direct inlet negative ion chemical ionization tandem mass spectrometric analysis of triacylglycerol regioisomers in human milk and infant formulas

A previously developed direct inlet tandem mass spectrometric method for analysis of triacylglycerol (TAG) regioisomers was updated and validated for operation with current instrumentation with improved sensitivity and throughput. TAG regioisomers in pooled Chinese and Finnish human milk samples, two bovine milk samples and 11 infant formulas were identified and quantified. A total of 241 TAG regioisomers were identified and quantified, consisting of over 60 mol% of all TAGs in the human milk samples. The infant formulas deviated largely from human milk in regioisomeric composition of TAGs. In the Finnish and Chinese human milks, the most abundant ones were 1,3-dioleoyl-2-palmitoylglycerol (OPO; 7.4 and 8.8 mol% of all TAGs) and 1(3)-linoleoyl-2-palmitoyl-3(1)-oleoylglycerol (LPO; 4.7 and 8.3 mol% of all TAGs). In the infant formulas 1,2(2,3)-dioleoyl-3(1)-palmitoylglycerol (OOP) and 1(3)-linoleoyl-2-oleoyl-3(1)-palmitoylglycerol/1(3)-palmitoyl-2-linoleoyl-3(1)-oleoylglycerol (LOP/PLO) were more abundant than OPO and LPO. The differences between human milk and infant formula prompt for further development of current formulas

Quality of Protein Isolates and Hydrolysates from Baltic Herring (Clupea harengus membras) and Roach (Rutilus rutilus) Produced by pH-Shift Processes and Enzymatic Hydrolysis

Fractionation is a potential way to valorize under-utilized fishes, but the quality of the resulting fractions is crucial in terms of their applicability. The aim of this work was to study the quality of protein isolates and hydrolysates extracted from roach (Rutilus rutilus) and Baltic herring (Clupea harengus membras) using either pH shift or enzymatic hydrolysis. The amino acid composition of protein isolates and hydrolysates mostly complied with the nutritional requirements for adults, but protein isolates produced using pH shift showed higher essential to non-essential amino acid ratios compared with enzymatically produced hydrolysates, 0.84-0.85 vs. 0.65-0.70, respectively. Enzymatically produced protein hydrolysates had a lower total lipid content, lower proportion of phospholipids, and exhibited lower degrees of protein and lipid oxidation compared with pH-shift-produced isolates. These findings suggest enzymatic hydrolysis to be more promising from a lipid oxidation perspective while the pH-shift method ranked higher from a nutrient perspective. However, due to the different applications of protein isolates and hydrolysates produced using pH shift or enzymatic hydrolysis, respectively, the further optimization of both studied methods is recommended

Profile and Content of Residual Alkaloids in Ten Ecotypes of Lupinus mutabilis Sweet after Aqueous Debittering Process

The evaluation of the level of alkaloids in edible Lupinus species is crucial from a food safety point of view. Debittering of lupin seeds has a long history; however, the control of the level of alkaloids after processing the seeds is typically only evaluated by changes in the bitter taste. The aim of this study was to evaluate the profile and residual levels of quinolizidine alkaloids (QA) in (Lupinus mutabilis Sweet) after aqueous debittering process. Samples from 10 ecotypes from different areas of Peru were analyzed before and after the process. Based on results obtained by gas chromatography and mass spectrometry, from eight alkaloids identified before the debittering process, only small amounts of lupanine (avg. 0.0012 g/100 g DM) and sparteine (avg. 0.0014 g/100 g DM) remained in the seeds after the debittering process, and no other alkaloids were identified. The aqueous debittering process reduced the content of alkaloids to levels far below the maximal level allowed by international regulations (<= 0.2 g/kg DM)

A novel UHPLC-ESI-MS/MS method and automatic calculation software for regiospecific analysis of triacylglycerols in natural fats and oils

Regioisomeric analysis of triacylglycerols (TAGs) in natural oils and fats is a highly challenging task in analytical chemistry. Here we present a software (TAG Analyzer) for automatic calculation of regioisomeric composition of TAGs based on the mass spectral data from recently reported ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry (UHPLC−ESI−MS/MS) method for analyzing TAG regioisomers. The software enables fast and accurate processing of complex product ion spectra containing structurally informative diacylglycerol [M+NH4−RCO2H–NH3]+ and fatty acid ketene [RCO]+ fragment ions. Compared to manual processing, the developed software offers higher throughput with faster calculation as well as more accurate interpretation of chromatographically overlapping isobaric TAGs. The software determines results by constructing a synthetic spectrum to match the measured fragment ion spectrum, and by reporting the optimal concentrations of TAGs used to create the synthetic spectrum. This type of calculation is often extremely challenging for manual interpretation of the fragment ion spectra of isobaric TAGs with shared fragments, hence the need for automated data processing. The developed software was validated by analyzing a wide range of mixtures of regiopure TAG reference compounds of known composition and a commercial olive oil sample. Additionally, the method was also applied for regiospecific analysis of TAGs in human milk as an example of natural fats and oils with a highly complex TAG profile. The results indicate that the software is capable of resolving regioisomeric composition of natural TAGs even of the most complex composition. This novel calculation software combined with our existing UHPLC-ESI-MS/MS method form a highly efficient tool for regioisomeric analysis of TAGs in natural fats and oils.</p

Työnjohtajana projektinjohtourakoinnissa

Tässä portfolio-opinnäytetyössä käsitellään aihetta työnjohtajana projektinjohtourakoinnissa. Työn tein yhteistyössä työnantajani SRV Rakennus Oy:n kanssa. Työn tavoitteena on avata lukijalle, millaista on projektinjohtourakointi käytännössä etenkin työnjohtajan näkökulmasta työmaalla sekä myös millaisia toimia sen ohjaaminen vaatii. Työn tarkoitus oli myös toimia itseni kehittäjänä ja lisätä tietouttani projektinjohtourakoinnista ja siihen liittyvistä tiedoista ja toimista. Työ toimi myös mittarina sille, miten olen omaksunut rakennusmestarikoulutuksessa tulleita asioita ja osannut niitä käytännössä soveltaa. Opinnäytetyössä käytin hyväkseni käytännössä rakennustyömaalla oppimaani tietoutta sekä rakennusmestariopintojen aikana saatuja tietoja ja niiden soveltamista käytännön työelämässä. Työn teoriaosuuteen hankin lähteiksi projektinjohtourakoinnista kertovaa kirjallisuutta ja poimin niistä tärkeimmät tiedot työni tueksi. Opinnäytetyötä tehdessä tietouteni projektinjohtourakoinnista lisääntyi niin käytännön kuin teoriatiedon osalta. Opintojen päätyttyä vahvuuksiani on työmaan toimintaan liittyvät asiat ja käytännön työn ohjaaminen ja tehtäviensuunnittelu. Opinnot yhdessä työharjoittelun kanssa ovat kehittänyt minua työnjohtajana toimimisessa ja siihen liittyvien käytäntöjen omaksumisessa. Kehitettävää minulla on vielä aikataulusuunnittelussa sekä työmaan hallinnollisten asioiden kanssa. Kehitettävää on myös rakennusteknisten töiden ja eri työvaiheiden johtamisen osalta, koska kokemusta ei ole vielä ehtinyt karttua kovin paljoa. Työni loppuluvussa käyn läpi, mitä olen työni aikana oppinut ja mitä kehitettävää olen aiheesta löytänyt. Käyn myös lävitse, miten olen opintojeni aikana yhdessä työelämässä toimimisen lisäksi kehittynyt työnjohtajana ja mitä valmiuksia minulla nyt jo on sekä miten haluan kehittyä tulevaisuudessa.The subject of the Portfolio Thesis is the project leader in project management contracting. The thesis work was done in cooperation with my employer SRV building company. The thesis describes how project management contracting is made in practice. The thesis also describes the project management theory. In my thesis work I use the things I have learned in the working life and also the things I learned during my studies. When doing the thesis I learned a lot of new information about project management contracting, and I got more information about the theory. The thesis was, in my opinion, devoted to my career. I got the benefit of doing the thesis work for myself. The last chapter of the dissertation undergoes what I learned during the thesis and what I was developing on the subject. I will also tell you how I succeeded in my work and if my goals were set in my work