Differences in Seed Weight, Amino Acid, Fatty Acid, Oil, and Squalene Content in γ-Irradiation-Developed and Commercial Amaranth Varieties (Amaranthus spp.).

Grain amaranth is known as an alternative crop with exclusive nutritional value and health benefits. The purpose of this study was to investigate the effect of gamma irradiation on quantitative and qualitative amaranth seed traits, including 1000-seed weight, amino acids, fatty acids content, oil, and squalene yield. Two Slovak mutant varieties "Pribina" (A. cruentus) and "Zobor" (A.hypochondriacus x A. hybridus) were evaluated and compared to nonirradiated controls Ficha (A. cruentus L.) and K-433 (A. hypochondriacus x A. hybridus) and commercial varieties, Aztec (A. cruentus L.), Plainsman and Koniz (A. hypochondriacus x A. hybridus). Mutant varieties, "Pribina" and "Zobor", showed superior 1000-seed weight performance compared to all investigated amaranth samples. The change in quantitative seed trait was accompanied by significantly higher oil and squalene content compared to commercial varieties. Moreover, significantly higher content of essential linoleic acid was detected in mutant variety "Zobor". The present findings suggest that seeds of irradiation-derived varieties have high nutritional potential and can be used as a supplementary crop in the human diet.

Simplified determination of the content and average degree of acetylation of chitin in crude black soldier fly larvae samples.

Insects are considered a promising alternative protein source for food and feed, but contain significant amounts of chitin, often undesirable due to indigestibility, disagreeable texture and negative effect on nutrients intake. Fractionation strategies are thus increasingly being applied to isolate and valorize chitin separately. The analysis of chitin generally requires an intensive pretreatment to remove impurities, and derivatization to generate sufficient detector response. In this work, a liquid chromatography method, without pretreatment nor derivatization, was developed for the simultaneous determination of chitin content and degree of acetylation in non-purified samples of black soldier fly (BSF) larvae. The method is found to be more suitable, compared to traditional methods, for assessing high degrees of acetylation. For the first time, the degree of acetylation of BSF chitin (81 ± 2%) is reported. Additionally, the chitin content of BSF soft tissues is estimated at approximately 20% of the total chitin content (8.5 ± 0.1%).

Influence of nitrogen depletion in the growth of N. oleoabundans on the release of cellular components after beadmilling.

Cell disruption by bead milling of Neochloris oleoabundans grown under nitrogen repleted (NR) and nitrogen depleted (ND) conditions was evaluated based on the release of biochemicals and biomass to the supernatant. Additionally, energy consumption for cell disruption was calculated per kg of released component. Bead milling of NR cells resulted on average in 34% (w/w) release of biochemicals into the supernatant, with a similar composition as the untreated microalgal biomass. With ND cells, the release was higher and more selective, i.e. 57%, 59%, 68% and 56% (w/w) of respectively biomass, proteins, carbohydrates and lipids whereof 92%, 57% and 46% (w/w) respectively of phospho-, glyco- and neutral lipids.

Automated analytical standard production with supercritical fluid chromatography for the quantification of bioactive C17-polyacetylenes: a case study on food processing waste.

Food processing enterprises produce enormous amounts of organic waste that contains valuable phytochemicals (e.g. C17-polyacetylenes). Knowledge on the phytochemicals content is a first step towards valorisation. Quantification of C17-polyacetylenes is however often hampered by the lack of commercially available standards or by tedious multistep in-house standard production procedures. In the current study, a new and straightforward supercritical fluid chromatography purification procedure is described for the simultaneous production of 2 analytical C17-polyacetylene standards. Respectively, 5 and 6 mg of falcarinol and falcarindiol were purified in 17 h on analytical scale. After confirming the identity and quality (97% purity) by Nuclear Magnetic Resonance, accurate mass-Mass Spectrometry (am-MS) and Photo Diode Array (PDA) detection the C17-polyacetylene standards were used for the analysis of industrial vegetable waste with Liquid Chromatography coupled to PDA and am-MS detection. Measurements showed varying concentrations of C17-polyacetylenes in the organic waste depending on its nature and origin.

Tackling the challenge of selective analytical clean-up of complex natural extracts: the curious case of chlorophyll removal.

Alkaline saponification is often used to remove interfering chlorophylls and lipids during carotenoids analysis. However, saponification also hydrolyses esterified carotenoids and is known to induce artifacts. To avoid carotenoid artifact formation during saponification, Larsen and Christensen (2005) developed a gentler and simpler analytical clean-up procedure involving the use of a strong basic resin (Ambersep 900 OH). They hypothesised a saponification mechanism based on their Liquid Chromatography-Photodiode Array (LC-PDA) data. In the present study, we show with LC-PDA-accurate mass-Mass Spectrometry that the main chlorophyll removal mechanism is not based on saponification, apolar adsorption or anion exchange, but most probably an adsorption mechanism caused by H-bonds and dipole-dipole interactions. We showed experimentally that esterified carotenoids and glycerolipids were not removed, indicating a much more selective mechanism than initially hypothesised. This opens new research opportunities towards a much wider scope of applications (e.g. the refinement of oils rich in phytochemical content).

Generic characterization of apolar metabolites in red chili peppers (Capsicum frutescens L.) by orbitrap mass spectrometry.

The aim of the present study was to develop a generic analytical method for the identification and quantitation of apolar plant metabolites in biomass using liquid chromatography-photodiode array-accurate mass mass spectrometry (LC-PDA-amMS). During this study, a single generic sample preparation protocol was applied to extract apolar plant metabolites. Compound identification was performed using a single generic screening method for apolar compounds without the need for dedicated fractionation. Such a generic approach renders vast amounts of information and is virtually limited by only the solubility and detector response of the metabolites of interest. Method validation confirmed that this approach is applicable for quantitative purposes. Furthermore, an identification-quantitation strategy based on amMS and molar extinction coefficients was used for carotenoids, eliminating the need for reference standards for each carotenoid. To challenge the validated method, chili peppers (Capsicum frutescens L.) were analyzed to unravel their complex phytochemical composition (carotenoids, glycolipids, glycerolipids, capsaicinoids, lipid-soluble vitamins).

Improving Method Reliability in Carotenoid Analysis through Selective Removal of Glycerolipid Interferences by Lipase Treatment.

Saponification is most often used to hydrolyze glycerolipid interferences during carotenoid analysis. Ester bonds of other plant metabolites such as carotenoids are, however, also hydrolyzed during saponification, thus altering the natural carotenoid composition. A straightforward and selective cleanup procedure was therefore developed involving the enzymatic hydrolysis of matrix glycerolipids. The optimized procedure (100 µL of extracted vegetable or algal oil in 20 mL of 50:50 phosphate buffer/methanol with 25 µL of sodium n-octyl sulfate, 30 mg of bile salts, and 250 µL of NaCl solution (5 mM), magnetic stirring for 2 h at 40 °C with 1 mL of Lipozyme TL 100 L and 1 mL of Lipozyme CALB L) removed the greater part of triglycerides (94.8-100.0%) and diglycerides (88.2-99.8%) while preserving the natural carotenoid composition.

Ultra high performance liquid chromatography versus high performance liquid chromatography: stationary phase selectivity for generic carotenoid screening.

Aim of study was to find the most suitable LC column for generic carotenoid screening. To represent the diversity of carotenoids in nature and to optimize chromatographic separation, a set of carotenoid standards was carefully chosen to account for the various classes of carotenoids. The HPLC C30 column has since long been the 'golden standard' in the chromatographic separation of carotenoids. Since approximately one decade, new UHPLC technology has led to much shorter analysis times, smaller peak widths and higher chromatographic resolution. However, there are currently no UHPLC columns on the market containing the specific stationary phase chemistry of the HPLC C30 column. Therefore during this study, we investigated the separation of carotenoids on a set of UHPLC columns and compared it to their separation on the HPLC C30 column. Comparison of carotenoids separations on the different stationary phases with objective column comparison parameters clearly indicated that the HPLC C30 column is an overall better performer in the separation of carotenoids. This is due to the lack of UHPLC column chemistries that are adapted for carotenoid analysis. However, analysis time on the HPLC C30 column takes about four times longer compared to UHPLC analysis. Therefore, with the range of columns that are commercially available nowadays, a choice has to be made between very high selectivity (HPLC C30 column) and analysis times that are adapted to modern laboratory requirements (UHPLC technology). Therefore, carotenoid separations would be even more performing if an appropriate UHPLC C30 column would be available.

Unravelling ionization and fragmentation pathways of carotenoids using orbitrap technology: a first step towards identification of unknowns.

Vegetables are a major source of carotenoids and carotenoids are identified as potentially important natural antioxidants that may aid in the prevention of several human chronic degenerative diseases. Characterization of carotenoids in organic biological matrices is a crucial step in any research valorization trajectory. This study reports for the first time the use of high mass resolution and exact mass orbitrap technology for the elucidation of carotenoid fragmentation pathways. This contributes to the generation of new tools for identifying unknown carotenoids based on fragmentation patterns. Two different chromatographic methods making use of different mobile phases resulted in the generation of different ion species because of the large influence of the mobile phase solvent composition on ionization. It was shown that depending on the molecular ion species that are generated (protonated ions or radical molecular ions), different fragments are formed when applying higher energy collisional dissociation. Fragmentation and the abundance of fragments provide valuable structural information on the type of functional groups, the polyene backbone and the location of double bonds in ring structures of carotenoids. Furthermore, coherence between specific substructures in the molecules and characteristic fragmentation patterns was observed allowing the assignment of fragmentation patterns for carotenoid substructures that can theoretically be extrapolated to carotenoids with similar (sub)structures. Differentiation between isomeric carotenoids by compound specific fragments could however not be made for all the isomeric groups under study. As a wide variety of isomeric forms of carotenoids exist in nature, the combination of good chromatographic separation with high resolution mass spectrometry and other complementary qualitative structure elucidation techniques such as a photo diode array detector and/or nuclear magnetic resonance spectroscopy are indispensable for unambiguous identification of unknown carotenoids.

Catalytic glycerol conversion into 1,2-propanediol in absence of added hydrogen.

Conversion of glycerol into high yields of 1,2-propanediol in absence of added hydrogen is possible with Pt impregnated NaY zeolite characterized by extra-zeolitic metal particles combined with zeolite Brønsted acidity.

The chicken pituitary expresses an ovoinhibitor-like protein in subpopulations of some, but not all, hormone-producing cell types.

Ovoinhibitor is a serine protease-inhibiting protein that was originally purified from egg whites. It is secreted by the oviduct under the control of estrogen and progesterone and it specifically inhibits serine proteinases such as trypsin and chymotrypsin. During recent attempts to raise monoclonal antibodies (Mabs) against chicken bursa of Fabricius proteins, one Mab was produced that specifically recognized chicken ovoinhibitor. This was the first demonstration of ovoinhibitor in an avian immune organ. We presently report on the expression of an ovoinhibitor-like molecule by the pituitary of the chicken as revealed by immunocytochemistry and RT-PCR. Immunofluorescent dual staining experiments using the mouse anti-ovoinhibitor Mab in conjunction with polyclonal antibodies against various hypophysial hormones revealed partial co-localization of an ovoinhibitor-like molecule with growth hormone (GH), luteinizing hormone (LH), and pro-opiomelanocortin (POMC), in a subset of the respective hormone producing cells. By contrast, no co-localization with prolactin (PRL) could be reliably demonstrated. RT-PCR of hypophysial mRNA using ovoinhibitor gene-specific primers yielded an amplicon that was 20% shorter than predicted on the basis of the published ovoinhibitor sequence. Sequencing revealed that of the represented exons only the central portion was expressed in the pituitary and that both 5' and 3' ends of each exon had been truncated. While expression of ovalbumin-like serine protease inhibitors (serpins) has been previously reported in the rat pituitary, to our knowledge, this is the first report of a Kazal-type serine protease inhibitor in the vertebrate neuroendocrine system.

Identification of cGnRH-II in the median eminence of Japanese quail (Coturnix coturnix japonica).

In a previous paper, we described the presence of cGnRH-II in the quail (Coturnix coturnix japonica) and chicken (Gallus gallus) median eminence using highly specific antibodies directed against a polypeptide corresponding to the C-terminal portion of cGnRH-II (van Gils et al., 1993). This finding remained very controversial, since no other study, with any other antibody, had ever reported the presence of cGnRH-II immunoreactive fibers in the median eminence of birds. In this study, the cGnRH-II immunoreactive substances in quail median eminence were isolated by RP-HPLC and identified by RIA. To eliminate the possibility that the cGnRH-II-like immunoreactivity in the median eminence was due to a cross-reaction of our anti-cGnRH-II antiserum with an unknown peptide, the cGnRH-II immunoreactive substances, present in a quail median eminence extract, were isolated by immunoaffinity chromatography using immunoaffinity-purified antibodies. In the eluate of the immunoaffinity column only one peptide could be detected by mass spectrometry. This peptide had a mass of 1235.56 Da, which is the same as synthetic cGnRH-II. In addition, MS/MS fragmentation generated an amino acid sequence corresponding to the sequence of cGnRH-II. The present study therefore identified indisputably cGnRH-II in the median eminence of the quail.