Michael Murkovic

Changes in chemical composition of pumpkin seeds during the roasting process for production of pumpkin seed oil. (Part 1: non-volatile compounds)

Abstract Pumpkin seed oil is a common salad oil in Austria. It is not only of interest because of its typical taste but also because of its potential in curing prostate disease. Besides the fatty acids, the micronutrients, which comprise vitamin E, phytosterols and lignans, are of special interest. Since the seeds are roasted before pressing of the oil, changes occur in the composition of the fatty acids and micronutrients. The oxidation-sensitive linoleic acid decreases from 54.6 to 54.2% whereas the concentrations of the vitamin E isomers show a decrease during the first 40 min of about 30% followed by an increase during the last 20 min to about the same level as at the beginning of the roasting process. The concentrations of α-tocopherol and γ-tocopherol in the fresh dried seeds are 37.5 and 383 μg/g, respectively. The concentration of the tocotrienols is about one third of the corresponding tocopherols. The initial concentration of the total sterols (1710 μg/g) increases to 1930 μg/g. The increases of the sterols and vitamin E during the roasting process could be attributed to the changes of the seed meal, since at the end of the roasting the oil emerges from the seeds resulting in altered chemical behaviour of the extraction process. Secoisolariciresinol, which is only detectable at the beginning with a concentration of 3.8 μg/g, is destroyed after 20 min.

Dietary exposure to 5-hydroxymethylfurfural from Norwegian food and correlations with urine metabolites of short-term exposure.

5-Hydroxymethylfurfural (HMF) is formed in carbohydrate-rich food during acid-catalysed dehydration and in the Maillard reaction from reducing sugars. HMF is found in mg quantities per kg in various foods. HMF is mainly metabolised to 5-hydroxymethyl-2-furoic acid (HMFA), but unknown quantities of the mutagenic 5-sulphoxymethylfurfural (SMF) may also be formed, making HMF potentially hazardous to humans. We determined the HMF content in Norwegian food items and estimated the dietary intake of HMF in 53 volunteers by means of 24h dietary recall. The estimated intakes of HMF were correlated with urinary excretion of HMFA. Coffee, prunes, dark beer, canned peaches and raisins had the highest levels of HMF. The 95th percentile of the estimated daily dietary intake of HMF and the 24h urinary excretion of HMFA were 27.6 and 28.6mg, respectively. Coffee, dried fruit, honey and alcohol were identified as independent determinants of urinary HMFA excretion. Most participants had lower estimated HMF intake than the amount of HMFA excreted in urine. In spite of this there was a significant correlation (r=0.57, P<0.001) between the estimated HMF intake and urinary HMFA. Further studies are needed to reveal alternative sources for HMF exposure.

Mechanisms of cell death induction by L-amino acid oxidase, a major component of ophidian venom

L-amino acid oxidase (LAAO) from the Malayan pit viper induces both necrosis and apoptosis in Jurkat cells. Cell death by necrosis is attributed to H2O2 produced by oxidation of α-amino acids. In the presence of catalase that effectively scavenges H2O2, a switch to apoptosis is observed. The major factors contributing to apoptosis are proposed to be: (i) generation of toxic intermediates from fetal calf serum (ii) binding and internalization of LAAO. The latter process appears to be mediated by the glycan moiety of the enzyme as desialylation reduces cytotoxicity. D-amino acid oxidase (DAAO), which catalyzes the same reaction as LAAO but lacks glycosylation, triggers necrosis as a consequence of H2O2 production but not apoptosis in the presence of catalase. Thus induction of cell death by LAAO appears to involve both the generation of H2O2 and the molecular interaction of the glycan moiety of the enzyme with structures at the cell surface.

Variability of fatty acid content in pumpkin seeds (Cucurbita pepo L.)

Pumpkin (Cucurbita pepo L.) seed oil is a common salad oil which is produced in Slovenia, Hungary and the southern parts of Austria. It is dark green and has a high content of free fatty acids. The seed itself can be eaten. Due to its colour and the foam formation, the oil cannot be used for cooking. The content of vitamin E, especiallyγ-tocopherol, is very high. The oil content of the pumpkin seed is about 50%. The variability in the oil content is very high resulting from a broad genetic diversity. Thus a breeding programme for increasing the oil productivity is very promising. The four dominant fatty acids are palmitic, stearic, oleic and linoleic acids. These four fatty acids make up 98±0.13% of the total amount of fatty acids, others being found at levels well below 0.5%.

Antioxidative activity of sage (Salvia officinalis L.), savory (Satureja hortensis L.) and borage (Borago officinalis L.) extracts in rapeseed oil

The antioxidant activity (AA) of acetone oleoresins (AcO) and deodorised acetone extracts (DAE) of sage (Salvia officinalis L.), savory (Satureja hortensis L.) and borage (Borago officinalis L.) were tested in refined, bleached and deodorised rapeseed oil applying the Schaal Oven Test and weight gain methods at 80 °C and the Rancimat method at 120 °C. The additives (0.1 wt-%) of plant extracts stabilised rapeseed oil efficiently against its autoxidation; their effect was higher than that of the synthetic antioxidant butylated hydroxytoluene (0.02%). AcO and DAE obtained from the same herbal material extracted a different AA. The activity of sage and borage DAE was lower than that of AcO obtained from the same herb, whereas the AA of savory DAE was higher than that of savory AcO. The effect of the extracts on the oil oxidation rate measured by the Rancimat method was less significant. In that case higher concentrations (0.5 wt-%) of sage and savory AcO were needed to achieve a more distinct oil stabilisation.

Variability of vitamin E content in pumpkin seeds (Cucurbita pepo L.).

Pumpkin (Cucurbita pepo L.) seed oil is a common salad oil which is produced in the southern parts of Austria, Slovenia and Hungary. It is dark green and has a high content of free fatty acids. Due to its colour, the oil cannot be used for cooking. The content of vitamin E, especiallyγ-tocopherol, is very high. The oil content of the pumpkin seed is about 50%. The seed itself can be eaten. Therefore a pumpkin variety with high vitamin E content is desirable. The aim of this work was to find a variety ofCucurbita pepo which has a high oil yield and a high vitamin E content. A total of 100 breeding lines were tested for their tocopherol content. The tocopherols and tocotrienols are extracted with hexane and analysed by NP-HPLC/FLD with hexane/dioxan (96/4) as eluent, with fluorescence detection at 292/335 nm. The/gg-tocopherol content, which is about 5–10 times as much as that of α-tocopherol varies over a broad range (41–620 mg/kg dry pumpkin seeds).β- andδ-tocopherol are found at low levels.

Determination of acrylamide during roasting of coffee.

In this study different Arabica and Robusta coffee beans from different regions of the world were analyzed for acrylamide after roasting in a laboratory roaster. Due to the complex matrix and the comparably low selectivity of the LC-MS at m/ z 72, acrylamide was analyzed after derivatization with 2-mercaptobenzoic acid at m/ z 226. Additionally, the potential precursors of acrylamide (3-aminopropionamide, carbohydrates, and amino acids) were studied. The highest amounts of acrylamide formed in coffee were found during the first minutes of the roasting process [3800 ng/g in Robusta ( Coffea canephora robusta) and 500 ng/g in Arabica ( Coffea arabica)]. When the roasting time was increased, the concentration of acrylamide decreased. It was shown that especially the roasting time and temperature, species of coffee, and amount of precursors in raw material had an influence on acrylamide formation. Robusta coffee contained significantly larger amounts of acrylamide (mean = 708 ng/g) than Arabica coffee (mean = 374 ng/g). Asparagine is the limiting factor for acrylamide formation in coffee. 3-Aminopropionamide formation was observed in a dry model system with mixtures of asparagine with sugars (sucrose, glucose). Thermal decarboxylation and elimination of the alpha-amino group of asparagine at high temperatures (>220 degrees C) led to a measurable but low formation of acrylamide.

Formation of the food associated carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in model systems

Abstract Food-related mutagens play a major role in human carcinogenesis. Heterocyclic aromatic amines which are formed in meat or fish during cooking contribute to food-related carcinogenesis, at least in rodents. The mechanism of formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was clarified by using 13 C-labelled phenylalanine as a reaction partner in a model system containing additionally creatinine. Isolation of the labelled reaction product (PhIP) and 13 C-NMR experiments showed that the carbon atoms of phenylalanine form a part of the pyridine moiety. Carbon atoms C-5, C-6 and C-7 in PhIP originated quantitatively from phenylalanine, leading to the conclusion that PhIP is formed by a defined mechanism and that two phenylalanine molecules are needed to form PhIP in this model system. In the proposed mechanism phenylacetaldehyde plays a key role which undergoes an aldol condensation with creatinine. The six-membered pyridine ring is completed by formation of a Schiff’s base and cyclisation.

Stability of pumpkin seed oil

In Austria pumpkins are grown primarily for the production of pumpkin seeds that can be used for eating or the production of salad oil. Pumpkin seed oil is dark green and its fatty acid composition consists typically of linoleic acid and oleic acid as the dominant fatty acids. The saturated fatty acids palmitic and stearic acid occur at lower levels. The samples for this study were taken from a breeding program that intends to increase the seed and oil productivity. 15 samples with different contents of linoleic acid (40—57%) and vitamin E (100—600 μg/g) were selected. The stability of the oil was measured in a Rancimat that oxidizes the oil at 120 ?C and measures the induction time that is needed for the oxidation. The correlation analysis showed that only the ratio of linoleic acid to oleic acid had a significant influence on the oxidative stability of the oil. Vitamin E did not show any correlation. When α-tocopherol was added to the oil a strong pro-oxidative effect was observed.

Hydroxymethyl-substituted furans: mutagenicity in Salmonella typhimurium strains engineered for expression of various human and rodent sulphotransferases.

5-Hydroxymethylfurfural (HMF) and furfuryl alcohol (FFA) are present in numerous foodstuffs at high levels. FFA is also used for the production of polymers. Both compounds had demonstrated some evidence of carcinogenic activity in 2-year bioassays. We tested these compounds and four congeners for mutagenicity in Salmonella typhimurium TA100 and TA100-derived strains expressing human or rodent sulphotransferases (SULTs). 5-Hydroxymethylfuroic acid, a metabolite of HMF, was not mutagenic in any strain. 3-Hydroxymethylfuran was weakly mutagenic in all strains independently of SULT expression. HMF, 2,5-(bishydroxymethyl)furan (metabolite of HMF), FFA and 5-methyl-FFA were inactive in TA100 but strongly mutagenic when human SULT1C2 was expressed. This form has been detected in ovary, kidney and foetal tissues. Human SULT1A1, SULT1A2 and SULT1A3 as well as murine Sult1a1 and Sult1d1 also activated some hydroxymethyl-substituted furans to varying degrees. Whereas chemically synthesised 5-sulphooxymethylfurfural was mutagenic in TA100, furfuryl sulphate was bacteriotoxic, only leading to marginal increases in the number of revertants. Furfuryl acetate, an uncharged ester of FFA, used as fragrance and food flavouring, was clearly mutagenic. We determined half-life times of 120 min, 20 s and 10 h, respectively, for 5-sulphooxymethylfurfural, furfuryl sulphate and furfuryl acetate at 37°C in water. It is likely that the short lifespan of furfuryl sulphate, together with its charge, led to insufficient penetration of the bacteria when added externally, although it was mutagenic when generated by appropriate SULTs from FFA within the cell.