Yannick Weesepoel

Carotenoid composition of berries and leaves from six Romanian sea buckthorn (Hippophae rhamnoides L.) varieties

Berries and leaves from six varieties of Carpathians sea buckthorn (Hippophae rhamnoides L., ssp. Carpatica) were analysed for their carotenoid composition (free and esterified) using a combination of HPLC-PAD, GC-MS and UHPLC-PAD-ESI-MS techniques. GC-MS techniques revealed the fatty acid profile specific for each berry variety, while targeted UHPLC-MS analysis identified the fatty acids involved in carotenoids esterification: palmitic (C16:0), myristic (C14:0) and stearic (C18:0). Total carotenoid content varied between 53 and 97 mg/100g dry weight in berries, and between 3.5 and 4.2mg/100g DW in leaves. The carotenoid di-esters represented the main fraction among berry varieties having zeaxanthin di-palmitate as major compound, while leaves contained only free carotenoids like lutein, β-carotene, violaxanthin and neoxanthin. Principal component analysis identified the suitable carotenoid biomarkers characteristic for the Carpathians sea buckthorn from Romania with contribution to their taxonomic classification and authenticity recognition.

Nitrogen-depleted Chlorella zofingiensis produces astaxanthin, ketolutein and their fatty acid esters: a carotenoid metabolism study

Natural carotenoids such as astaxanthin, β,β-carotene and lutein are pigments with a high market value. We studied the effects of nitrogen depletion on the carotenoid metabolism of Chlorella zofingiensis (Chlorophyta) and the subsequent treatment with diphenylamine (DPA), an inhibitor of the biosynthesis of secondary ketocarotenoids. Pigments were identified and quantified based on reversed phase ultra-high performance liquid chromatography photodiode array tandem mass spectrometry (RP-UHPLC-PDA-MSn). Nitrogen depletion (without DPA) resulted in a degradation of chlorophylls and primary carotenoids and an accumulation of astaxanthin, ketolutein, canthaxanthin, adonixanthin and β,β-carotene. The DPA treatment decreased the overall production of β,β-carotene derivatives (sum of astaxanthin, canthaxanthin, echinenone and adonixanthin); however, the production of ketolutein and degradation of primary carotenoids were not modified. This suggests that the regulatory mechanisms controlling the flux towards ketolutein and primary carotenoids were not affected by the decreased levels of β,β-carotene derivatives. In addition, DPA increased production of the individual carotenoids, adonixanthin and echinenone. Insight into the regulation of microalgal carotenoid biosynthesis as demonstrated in this paper is essential when a large-scale carotenoid production process is to be optimised or a recombinant C. zofingiensis strain is to be designed with the intention of excessively producing primary or secondary carotenoids.

Growth and pigment accumulation in nutrient-depleted Isochrysis aff. galbana T-ISO

The effect of three different nutrient depletions (nitrogen, sulphur and magnesium) on the growth and pigment accumulation of the haptophyte Isochrysis aff. galbana (clone T-ISO) has been studied. Pigments were quantified based on RP-UHPLC-PDA-MSn analysis. All nutrient depletions led to reduced maximal biomass concentrations. Besides, all nutrient-depleted cultures accumulated 3-hydroxyechinenone. To our knowledge, this is the first time that 3-hydroxyechinenone has been found in I. aff. galbana T-ISO. Most 3-hydroxyechinenone, as well as the most echinenone and diatoxanthin, were found in the nitrogen-limited culture in which a more severe limitation resulted in higher cellular contents. Similar to accumulation of diatoxanthin, accumulation of 3-hydroxyechinenone and echinenone may be part of a global (stress) response mechanism to oversaturating light conditions.

Food Fraud and Authenticity : Emerging issues and future trends

Abstract Food fraud is a significant and growing problem, driven by globalization, economic opportunity, and the low probability and severity of punishment. Although food fraud is economically motivated, it may result in serious health consequences. Therefore, emerging food fraud issues are described in this chapter, including the usage of food fraud databases. Analytical verification of food fraud and food authentication is needed to support proper food safety management systems. However, due to time and money constraints, only a restricted number of samples can be analyzed in a laboratory. For analysis outside the laboratory, rapid, nondestructive, nontargeted methods are needed. This can be either handheld equipment for food safety inspectors or in-line equipment for the food manufacturers.

Sodiation as a tool for enhancing the diagnostic value of MALDI-TOF/TOF-MS spectra of complex astaxanthin ester mixtures from Haematococcus pluvialis

The microalga Haematococcus pluvialis produces the pigment astaxanthin mainly in esterified form with a multitude of fatty acids, which results in a complex mixture of carotenol mono- and diesters. For rapid fingerprinting of these esters, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS) might be an alternative to traditional chromatographic separation combined with MS. Investigation of ionization and fragmentation of astaxanthin mono- and diester palmitate standards in MALDI-TOF/TOF-MS showed that sodium adduct parent masses [Mu2009+u2009Na](+) gave much simpler MS(2) spectra than radical / protonated [M](+●) / [Mu2009+u2009H](+) parents. [Mu2009+u2009Na](+) fragments yielded diagnostic polyene-specific eliminations and fatty acid neutral losses, whereas [M](+●) / [Mu2009+u2009H](+) fragmentation resulted in a multitude of non-diagnostic daughters. For diesters, a benzonium fragment, formed by polyene elimination, was required for identification of the second fatty acid attached to the astaxanthin backbone. Parents were forced into [Mu2009+u2009Na](+) ionization by addition of sodium acetate, and best signal-to-noise ratios were obtained in the 0.1 to 1.0u2009mM range. This method was applied to fingerprinting astaxanthin esters in a crude H. pluvialis extract. Prior to MALDI-TOF/TOF-MS, the extract was fractionated by normal phase Flash chromatography to obtain fractions enriched in mono- and diesters and to remove pheophytin a, which compromised monoester signals. All 12 types of all-trans esterified esters found in LC were identified with MALDI-TOF/TOF-MS, with the exception of two minor monoesters.

Fatty acids attached to all-trans-astaxanthin alter its cis-trans equilibrium, and consequently its stability, upon light-accelerated autoxidation

Fatty acid esterification, common in naturally occurring astaxanthin, has been suggested to influence both colour stability and degradation of all-trans-astaxanthin. Therefore, astaxanthin stability was studied as influenced by monoesterification and diesterification with palmitate. Increased esterification decelerated degradation of all-trans-astaxanthin (RP-UHPLC-PDA), whereas, it had no influence on colour loss over time (spectrophotometry). This difference might be explained by the observation that palmitate esterification influenced the cis-trans equilibrium. Free astaxanthin produced larger amounts of 9-cis isomer whereas monopalmitate esterification resulted in increased 13-cis isomerization. The molar ratios of 9-cis:13-cis after 60min were 1:1.7 (free), 1:4.8 (monopalmitate) and 1:2.6 (dipalmitate). The formation of 9-cis astaxanthin, with its higher molar extinction coefficient than that of all-trans-astaxanthin, might compensate for colour loss induced by conjugated double bond cleavage. As such, it was concluded that spectrophotometry is not an accurate measure of the degradation of the all-trans-astaxanthin molecule.

Investigation of the aroma of commercial peach (Prunus persica L. Batsch) types by Proton Transfer Reaction–Mass Spectrometry (PTR-MS) and sensory analysis

The aim of this study was to investigate the aroma and sensory profiles of various types of peaches (Prunus persica L. Batsch.). Forty-three commercial cultivars comprising peaches, flat peaches, nectarines, and canning peaches (pavías) were grown over two consecutive harvest years. Fruits were assessed for chemical aroma and sensory profiles. Chemical aroma profile was obtained by proton transfer reaction-mass spectrometry (PTR-MS) and spectral masses were tentatively identified with PTR-Time of Flight-MS (PTR-Tof-MS). Sensory analysis was performed at commercial maturity considering seven aroma/flavor attributes. The four types of peaches showed both distinct chemical aroma and sensory profiles. Flat peaches and canning peaches showed most distinct patterns according to discriminant analysis. The sensory data were related to the volatile compounds by partial least square regression. γ-Hexalactone, γ-octalactone, hotrienol, acetic acid and ethyl acetate correlated positively, and benzeneacetaldehyde, trimethylbenzene and acetaldehyde negatively to the intensities of aroma and ripe fruit sensory scores.

Preliminary UHPLC-PDA-ESI-MS screening of light-accelerated autoxidation products of the tetrapyrrole biliverdin.

The application of phycobiliproteins, e.g. blue C-phycocyanin, as natural water-soluble food colourants is emerging. The chromophore of these proteins comprises a number of tetrapyrroles (or phycocyanobilins), which have an extensive conjugated system, vulnerable to autoxidation. To assess the autoxidation products, a simplified model system was used in which the free tetrapyrrole biliverdin, instead of phycobiliprotein, was subjected to light-accelerated autoxidation. Degradation products of biliverdin were subsequently annotated by reversed-phase ultra-high performance liquid chromatography with a photo diode array and positive mode in-line electrospray ionization mass spectrometry. To facilitate the analysis of degradation products, autoxidation of the three methine bridges in biliverdin was mimicked in silico. It was found that both the peripheral and central methine bridges of biliverdin were susceptible to light-accelerated autoxidation. Scission products tentatively annotated with MS(2) and MS(3) were propionic acid-containing pyrroles. From this, it can be speculated that tetrapyrroles attached to phycobiliproteins are susceptible to autoxidative degradation.

Analysis of Palmitoyl Apo-astaxanthinals, Apo-astaxanthinones, and their Epoxides by UHPLC-PDA-ESI-MS

Food products enriched with fatty acid-esterified xanthophylls may result in deviating dietary apo-carotenoids. Therefore, free astaxanthin and its mono- and dipalmitate esters were subjected to two degradation processes in a methanolic model system: light-accelerated autoxidation and hypochlorous acid/hypochlorite (HOCl/OCl(-)) bleaching. Reversed phase ultrahigh-performance liquid chromatography photodiode array with in-line electrospray ionization mass spectrometry (RP-UHPLC-PDA-ESI-MS) was used for assessment of degradation products. Apo-astaxanthinals and -astaxanthinones containing 3 (apo-9) to 10 (apo-8) conjugated double bonds were found upon autoxidation for all three types of astaxanthin (except free apo-8-astaxanthinal). Fragmentation of [M + H](+) and [M + Na](+) parent masses of apo-astaxanthins from dipalmitate astaxanthin indicated palmitate esterification. Astaxanthin monopalmitate degradation resulted in a mixture of free and palmitate apo-astaxanthins. HOCl/OCl(-) rapidly converted the astaxanthins into a mixture of epoxy-apo-9- and epoxy-apo-13-astaxanthinones. The palmitate ester bond was hardly affected by autoxidation, whereas for HOCl/OCl(-) the ester bond of the apo-astaxanthin palmitoyl esters was degraded.

New approaches towards discrimination of fresh/chilled and frozen/thawed chicken breasts by HADH activity determination: Customized slope fitting and chemometrics

Fresh/chilled chicken breasts retail at a higher price than their frozen/thawed counterparts. Verification of the fresh/thawed status of chicken meat is determined by measuring β-hydroxyacyl-Coenzyme A-hydrogenase (HADH) activity present in meat intra-cellular liquids spectrophotometrically. However, considerable numbers of reference samples are required for the current arithmetic method, adding to laboratory costs. Therefore, two alternative mathematical approaches which do not require such reference samples were developed and evaluated: curve fitting and multivariate classification. The approaches were developed using 55 fresh/thawed fillet samples. The performance of the methods was examined by an independent validation set which consisted of 16 samples. Finally, the approach was tested in practice in a market study. With the exception of two minor false classifications, both newly proposed methods performed equally well as the classical method. All three methods were able to identify two apparent fraudulent cases in the market study. Therefore, the experiments showed that the costs of HADH measurements can be reduced by adapting alternative mathematics.