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.

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 °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, ß-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 °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.

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.

Impact of lactic acid fermentation on sensory and chemical quality of dairy analogues prepared from lupine (Lupinus angustifolius L.) seeds.

Lupine (Lupinus sp.) is a valuable source of plant proteins. There is little knowledge on the impact of food processing on composition and sensory properties of lupine products. In this research, we investigated the impact of fermentation with five starters of lactic acid bacteria on the sensory quality and flavor-active compounds in dairy analogues prepared from sweet lupine (Lupinus angustifolius L.). The sensory qualities of unfermented and fermented products were studied with generic descriptive analysis and affective tests. Acids and sugars were analyzed with GC-FID and volatiles with HS-SPME-GC-MS and GC-O. Fermentation increased sourness and 'vinegar' odor and reduced the 'beany' odor and flavor as well as the unpleasantness of flavor. Formation of volatiles during the fermentation was dependent on the starters. However, all fermentations increased the contents of lactic, acetic, and hexanoic acids, while reducing the contents of hexanal, described as 'grassy' in the unfermented lupine sample.

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.

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.

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).

Regiospecific Analysis of Triacylglycerols by Ultrahigh-Performance-Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry.

An ultrahigh-performance-liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) method was developed for the analysis of AAB and ABC type triacylglycerol (TG) regioisomers. Excellent linear regressions were established between the ratio of [RR]+ product ions and the proportion of regioisomers of TGs. The method was further optimized by analysis of 18 regiospecific pairs of AAB type TGs and five triplets of regioisomers of ABC type TGs with acyl carbon number (ACN) ranging from 36 to 54 and the number of double bonds (DB) from 0 to 7. Reverse linear relationships were recognized between the slope of the calibration curve and the number of double bonds of the sn-2 fatty acids. Negative linear regressions were found between the intercepts of the calibration curves and the sum of ACN + DB of sn-2 fatty acids. The method was highly repeatable as shown by the low deviation and high stability of the calibration curves at different concentrations and between different periods of analysis. This is the first time that calibration curves for the ABC type TGs are reported. The results provide crucial and novel information for reliable and quantitative determination of regioisomeric TGs in natural fats and oils.

Determination of vitamin K composition of fermented food.

A rapid ultra-high performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometric (UHPLC-APCI-MS/MS) method was developed for the analysis of vitamin K compounds: phylloquinone (PK) and menaquinones (MK-n). Non-chlorinated mobile phase composition was optimized for separation of eight vitamin K compounds on a reversed phase column in 10 min. Sample treatment with liquid and solid phase extractions and by the use of MK-4 as an internal standard enabled the quantitation of microgram level of vitamin K compounds in food. The method was used to screen and quantitate vitamin K from 17 fermented food products. The highest amount of PK was detected in kimchi (42 µg/100 g), whereas the highest MK-7 content was detected in natto (902 µg/100 g). Some MK-9 was present in kefir (5 µg/100 g). Two Chinese fermented soybean pastes contained significant amount of MK-6 (5-36 µg/100 g), MK-7 (12-86 µg/100 g), and MK-8 (22-44 µg/100 g).

Fish Oil Finishing Diet Maintains Optimal n-3 Long-Chain Fatty Acid Content in European Whitefish (Coregonus lavaretus).

This study examined the effect of substituting vegetable oil for fish oil in feed, with subsequent re-introduction of fish oil-rich feed (finishing feeding) in late stages of growth, on the fatty acids of cultivated European whitefish (Coregonus lavaretus). Restorative finishing feeding with fish oil-rich feed for 15 and 25 weeks was sufficient to change the total content of nutritionally valuable long-chain n-3 fatty acids, eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3), to correspond to that of fish fed the fish oil-rich feed throughout their lifespan. Under natural conditions, 15 and 25 weeks correspond to weight gains of 75% and 100% (i.e. doubling), respectively. Also, the fatty acid profile of the fish was restored after finishing periods of 15 and 25 weeks. Limiting the use of fish oil by lowering the overall fat content of the feed (no vegetable oil added) resulted in a decrease in the long-chain n-3 fatty acids. Based on the results, after receiving a vegetable oil-rich diet, restorative fish oil-rich feeding in the last stages of growth in European whitefish is nutritionally justified in order to balance nutritional gain for consumers with sustainable use of finite marine oils. The results encourage commercial efforts to further utilize and optimize finishing feeding practices.

CO2 Plant Extracts Reduce Cholesterol Oxidation in Fish Patties during Cooking and Storage.

Cholesterol oxidation products (COPs) in foods may pose risks for human health. Suitable antioxidants can reduce the formation of COPs in industrial products. Consumer awareness of food additives has brought a need for more natural alternatives. This is the first study on the effects of supercritical CO2 extracts of rosemary, oregano, and an antimicrobial blend of seven herbs, tested at two levels (1 and 3 g/kg fish), against cholesterol oxidation in patties made of a widely consumed fish species, Atlantic salmon (Salmo salar), during baking and storage. Cholesterol oxidation was reduced by the extracts as indicated by lowered levels of 7α-hydroxycholesterol, 7ß-hydroxycholesterol, and 7-ketocholesterol, which were quantified by GC-MS. The total amount of COPs was smaller in all of the cooked samples containing the plant extracts (<1 µg/g extracted fat) than in the cooked control (14 µg/g). Furthermore, the plant extracts exhibited protective effects also during cold storage for up to 14 days.

Cultivation of Nannochloropsis for eicosapentaenoic acid production in wastewaters of pulp and paper industry.

The eicosapentaenoic acid (EPA) containing marine microalga Nannochloropsis oculata was grown in an effluent from anaerobic digestion of excess activated sludge from a wastewater treatment plant serving a combination of a pulp and a paper mill and a municipality (digester effluent, DE), mixed with the effluent of the same wastewater treatment plant. The maximum specific growth rate and photosynthesis of N. oculata were similar in the DE medium and in artificial sea water medium (ASW) but after 7 days, algae grown in the DE medium contained seven times more triacylglycerols (TAGs) per cell than cells grown in ASW, indicating mild stress in the DE medium. However, the volumetric rate of EPA production was similar in the ASW and DE media. The results suggest that N. oculata could be used to produce EPA, utilizing the nutrients available after anaerobic digestion of excess activated sludge of a pulp and paper mill.

The impact of beef steak thermal processing on lipid oxidation and postprandial inflammation related responses.

Oxidised lipid species, their bioavailability and impact on inflammatory responses from cooked beef steak are poorly characterised. Oxidised lipid species from pan-fried (PF) and sous-vide (SV) thermally processed beef were determined with UHPLC-ESI/MS. Twenty-three lipid oxidation products increased with thermal processing and differences between the PF and SV steaks were measured. Fifteen oxidised lipids were measured in post-meal plasma after a cross-over randomised clinical study. Postprandial plasma inflammatory markers tended to remain lower following the SV meal than the PF meal. High levels of conjugated dienes were measured in the HDL fraction, suggesting that the protective effect of HDL may extend to the reverse-transport of oxidised lipid species. Oxidised lipids in a single meal may influence postprandial oxidative stress and inflammation. Further studies are required to examine the lipid oxidative responses to increased dietary oxidative lipid load, including the reverse transport activity of HDL.

Effects of CO2 plant extracts on triacylglycerol oxidation in Atlantic salmon during cooking and storage.

Increasing concern of consumers on the safety of synthetic food additives has created high interest in natural preservatives in food industry. Plant extracts produced by supercritical CO2 technology from rosemary (R), oregano (O) and an antimicrobial blend (AB) consisting of seven different plants were studied for their effects on lipid oxidation in Atlantic salmon (Salmo salar). Fish pieces were marinated with rapeseed oil containing 0, 1, 2 or 4 g of plant extracts/kg of fish. After cooking the pieces were stored in refrigerator for 26 days. Peroxide values (PVs) were determined and oxidised triacylglycerols (TAGs) measured by UHPLC-ESI/MS at 0, 7, 14 and 26 days of storage. During the first two weeks of storage, AB delayed oxidation by at least one week compared to control samples as shown by PVs (<10 meq. O2) and by the oxidised TAGs. Oregano and rosemary showed also some antioxidative potential.

Effects of antioxidants on rapeseed oil oxidation in an artificial digestion model analyzed by UHPLC-ESI-MS.

A normal diet contains large quantities of oxidized fatty acids, glycerolipids, cholesterol, and their cytotoxic degradation products because many foods in the diet are fried, heated, or otherwise processed and consumed often after long periods of storage. There is also evidence that the acid medium of the stomach promotes lipid peroxidation and that the gastrointestinal tract is a major site of antioxidant action, as demonstrated by various colorimetric methods. The identity and yields of specific products of lipid transformation have seldom been determined. The present study describes the molecular species profiles of all major gastrointestinal lipids formed during digestion of autoxidized rapeseed oil in an artificial digestion model in the presence of L-ascorbic acid, 6-palmitoyl-O-L-ascorbic acid, 3,5-di-tert-butyl-4-hydroxytoluene (BHT), DL-α-tocopherol, and DL-α-tocopheryl acetate. Differences in oxidized lipid profiles were detected in the samples digested in the presence of different antioxidants, but none of them could prevent the formation of oxidized lipids or promote their degradation in a gastric digestion model. The lack of effect is attributed to the inappropriate nature of the gastrointestinal medium for the antioxidant activity of these vitamins and BHT. A fast ultrahigh performance liquid chromatographic-electrospray ionization-mass spectrometric method was developed for the analysis of lipolysis products, including epoxy, hydroperoxy, and hydroxy fatty acids, and acylglycerols, utilizing lithium as ionization enhancer.

Liquid chromatography-light scattering detector-mass spectrometric analysis of digested oxidized rapeseed oil.

Rapeseed oil was oxidized chemically and thermally to produce two distinct oxidized oils. These oils, along with unoxidized oils, were subjected to an artificial digestion model to simulate the digestive processes in humans. Lipid digestion involves lipases that break down the intact triacylglycerol (TAG) molecules first to diacylglycerols, and eventually to sn-2-monoacylglycerols (MAG) and free fatty acids. A high performance liquid chromatography-evaporative light scattering detector-electrospray ionization-mass spectrometric (HPLC-ELSD-ESI-MS) method was developed to monitor the lipolysis and the presence of oxidized lipids. The HPLC-ELSD-ESI-MS analysis enabled the separation and detection of nearly all the lipid species present in the sample after TAG hydrolysis. The HPLC-MS analyses of digestion products revealed that oxidized triacylglycerols are hydrolyzed by the digestive enzymes in a manner similar to that of native, unoxidized molecules. Significant amounts of sn-1(3)-MAG were found in all the samples after lipolysis, however, more of these were found in unoxidized rapeseed oil samples than in the oxidized oils. Several oxidized molecules were identified with the aid of synthesized oxylipids. This novel method is scalable to small-scale preparative fractionation of oxidized lipid molecules from a complex digestion sample. Also, the fingerprint-like, diagnostic, MS profiles of oxidized oils, reference compounds, and digestion products may be a great aid in comprehensive analysis of lipid oxidation and lipolysis.

Use of lipidomics for analyzing glycerolipid and cholesteryl ester oxidation by gas chromatography, HPLC, and on-line MS.

Various analytical techniques have been adopted for the isolation and identification of the oxolipids and for determining their functionality. Gas chromatography in combination with mass spectrometry (MS) has been specifically utilized in analysis of isoprostanes and other low molecular weight oxolipids, although it requires derivatization of the solutes. In contrast, liquid chromatography (LC) in combination with on-line MS has proven to be well suited for analysis of intact oxolipids without (or minimal) derivatization. LC-MS has also been helpful for the identification of lipidomic changes resulting from covalent binding of lipid ester core aldehydes to amino lipids, amino acids, peptides, and proteins. This chapter reviews the use of the above techniques for lipidomic analysis of the autoxidation products of cholesteryl esters and glycerolipids as practiced in the authors' laboratories.

Lipidomic analysis of glycerolipid and cholesteryl ester autooxidation products.

Thin-layer chromatography (TLC), gas chromatography (GC), and liquid chromatography (LC) in combination with mass spectrometry (MS) have been adopted for the isolation and identification of oxolipids and for determining their functionality. TLC provides a rapid separation and access to most oxolipids as intact molecules and has recently been effectively interfaced with time-of-flight (TOF) MS (TOF-MS). GC with flame ionization (FI) (GC/FI) and electron impact (EI) MS (GC/EI-MS) has been extensively utilized in the analysis of isoprostanes and other low-molecular-weight oxolipids, although these methods require derivatization of the analytes. In contrast, LC with ultraviolet (UV) absorption (LC/UV) or evaporate light scattering detection (ELSD) (LC/ELSD) as well as electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) MS (LC/ESI-MS) or LC/APCI-MS has proven to be well suited for the analysis of intact oxolipids and their conjugates without or with minimal derivatization. Nevertheless, kit-based colorimetric and fluorescent procedures continue to serve as sensitive indicators of the presence of hydroperoxides and aldehydes.