Red clover-rich grassland increases equol concentration in eggs from free-range laying hens.

1. The aim of this study was to evaluate the production of equol (4',7-isoflavandiol; a bacterial polyphenol metabolite which is an isoflavandiol oestrogen metabolised from daidzein from plants) enriched eggs from free-range hens fed different pasture species. Four species were tested: red clover, white clover, ryegrass and chicory.2. The study was conducted from June to September 2017 on eight free range, outdoor areas, each containing fifteen laying hens and sown with a single pasture species3. Precursors of equol (daidzein, formononetin) were analysed every fortnight from the fresh pasture cover in each area, as well as equol and daidzein levels in eggs.4. Daidzein and formononetin concentrations in the fresh pasture samples differed significantly according to species (P < 0.001), whereby red clover had the highest concentrations of daidzein and formononetin (85 and 996 µg/g DM, respectively).5. Equol concentration in eggs differed according to pasture species (P < 0.001). Equol concentrations reached about 1,200 ng/g DM in eggs from hens with access to red clover. These eggs can represent a valuable source of equol in the human diet.

Effect of the dietary combination of flaxseed and Ricinodendron heudelotii or Punica granatum seed oil on the fatty acid profile of eggs.

The health promoting omega-3, -7, and -5 fatty acids, α-linolenic acid (ALA), docosahexaenoic acid (DHA), rumenic acid (RmA), and α-eleostearic acid (α-ESA)/punicic acid (PunA), are not currently combined in frequently consumed food items. We have evaluated the impact of supplementing laying hens' feeds with flaxseeds combined with oil derived from seeds of either Ricinodendron heudelotii, an α-ESA source, or Punica granatum, a PunA source, on the egg fatty acid profile. The supplemented diets increased the egg content in ALA, DHA, RmA, as well as α-ESA or PunA. The combination of dietary lipids did not affect the conversion rate of ALA into DHA. Hens fed on R. heudelotii or P. granatum seed oil both accumulated RmA in egg yolk, indicating an efficient conversion from the α-ESA or PunA precursors through a Δ-13 reductase activity. The accumulation of PunA in eggs was largely higher than that of α-ESA.

The effect of oak tannin (Quercus robur) and hops (Humulus lupulus) on dietary nitrogen efficiency, methane emission, and milk fatty acid composition of dairy cows fed a low-protein diet including linseed.

The objective of this study was to test the effects of inclusion of hop pellets (HP) and oak tannin extracts (OT) alone or in combination on N efficiency, methane (CH4) emission, and milk production and composition in 2 experiments with dairy cows fed low-N rations supplemented with linseed. In both experiments, 6 lactating Holstein cows were assigned to 3 dietary treatments in a 3 × 3 duplicated Latin square design (21-d periods). Cows were fed a total mixed ration at a restricted level to meet their nutrient requirements. In experiment 1, 169 g dry matter (DM) of OT or 56 g DM of HP was included separately in the control diet (C1). In experiment 2, the additives were included together (OT-HP) in the control diet (C2) similar to C1. Diet C2 was compared with a control without linseed (C0). In experiment 1, the supplementation of the control diet with OT decreased urinary N excretion by 12%. In experiment 2, the combination of OT and HP decreased urinary N by 7%. Oak tannin extracts and HP alone or in combination did not influence the daily enteric CH4 production of cows. Cows fed diet C0 produced 17% more enteric CH4 daily than those fed diet C2. Intake of diet C2, which contained 6.7% extruded linseed on a DM basis (experiment 2), decreased the sum of 6:0 to 14:0 fatty acids (-16%) and palmitic acid (-26%) and increased the stearic acid (+50%), oleic acid (+36%), vaccenic acid (trans-11 18:1; +285%), rumenic acid (cis-9,trans-11 18:2; +235%), and α-linolenic acid (+100%) in milk fat. The supplementation of diet C2 with the OT-HP mixture further improved the milk's fatty acid composition. Intake of the OT alone increased α-linolenic acid by 17.7% (experiment 1). The results of this study show that at the economically acceptable dose we tested, hops had no effect on urinary N excretion, CH4 emission, milk production, and milk composition. By contrast, supplementation of diets with oak tannin extract can be considered for reducing urinary N excretion. The combination of oak tannin and hops had no more effect than oak tannin alone except on the milk fatty acid profile, which was favorably influenced from a nutritional point of view.

Ochratoxin a in human breast milk, maternal and placental blood from Cochabamba-Bolivia / Presencia de ocratoxina a en leche, placenta y sangre materna en Cochabamba-Bolivia

The nephrotoxic and carcinogenic mycotoxin Ochratoxin A (OTA) is a contaminant in a wide variety of foods. Mothers who ingest food contaminated with OTA during the gestation and lactation period can transfer this mycotoxin to the fetus as to the infant, respectively. To quantify the transmission of Ochratoxin A from mother to newborn and to infant, samples were taken from maternal blood and umbilical cord of women at the moment of delivery and breast milk of ladies in lactation period in the city of Cochabamba-Bolivia. From a total of 31 samples of maternal plasma and its respective cord, 4 were positive in both sample types (incidence 13%, 12% allowable relative error to the population at 95% confidence). Maternal plasma samples had a range of 0.3-10.2 ng of Ochratoxin A/ml with a mean of 3.0 ng/ml (+/- SD=3,0). Similarly, in the umbilical cord plasma Ochratoxin A was also detected with values of 0.3 to 1.7 ng/ml and with a mean of 0.7+/- 0.65 ng/ml. In two samples the concentrations of ochratoxin A were higher in umbilical cord blood (0,5 and 1.7 ng/mL) than their corresponding pairs of maternal blood (0.3 and 1.3 ng /ml, respectively) with an average concentration ratio of 1.5 ng/ml. Two other samples presented positive in ochratoxin A in umbilical cord blood and negative in their corresponding pairs of maternal blood confirming the transfer of the mycotoxin through human placental barrier. Out of 29 samples the breast milk samples from the Bolivian mothers collected during the summer period, 13 (44.8%) were found positive in a ranged from 20.2 to 146.1 ng/l. Concerning the samples collected in the winter season, only one (2.4%) out of 42 had a measurable ochratoxin A concentration of 28.3 ng/1, and seven samples showed traces of the toxin, below the detection limit

La micotoxina nefrotóxica y cancerígena Ocratoxina A (OTA) es un contaminante en una amplia variedad de alimentos. Las madres que ingieren alimentos contaminados con OTA durante el período de gestación y lactancia pueden transferir esta micotoxina al feto y al bebé, respectivamente. Para cuantificar la transmisión de la ocratoxina A de la madre al feto y al niño, se tomaron muestras de sangre materna y cordón umbilical de mujeres en el momento del parto y de leche materna de madres en el período de lactancia en la ciudad de Cochabamba-Bolivia. De un total de 31 muestras de plasma materno y su respectivo cordón umbilical, 4 resultaron positivas en ambos tipos de muestra, (incidencia 13%, error admisible de 12% respecto a la población con 95% de confianza). Las muestras de plasma materno se encontraron en un rango de 0,3 a 10,2 ng/ml con una media de 3,0 ng/ml (+/- SD = 3,0). De igual manera, en el plasma de cordón umbilical se detectó también OTA en cuatro muestras, con valores de 0,3 a 1,7 ng/ml y con una media de 0,7 ng/ml (+/- SD=0.65). En dos muestras las concentraciones de ocratoxina A eran mayores en la sangre del cordón umbilical (0,5 y 1,7 ng/ml) que sus correspondientes pares de la sangre materna (0,3 y 1,3 ng/ml, respectivamente) con una relación de concentraciones promedio de 1,5. Otras dos muestras presentaron positivo en OTA en la sangre del cordón umbilical y negativo en sus correspondientes pares de sangre materna. Confirmándose así el traspaso de esta micotoxina a través de la barrera placentaria humana. De otra parte, de un total de 29 muestras de leche materna humana colectadas de madres bolivianas durante la estación de verano, 13 (44.8 %) se encontraron positivas en un rango de 20,2 a 146,1 ng/l. Respecto a las muestras de leche colectadas durante la estación de invierno solo 1 (2,4%) de 42 fueron positivas en ocratoxina a una concentración de 28,3 ng/l y siete muestras mostraron trazas de la toxina por debajo del límite de detección

Cloning and functional characterization of Δ6 fatty acid desaturase (FADS2) in Eurasian perch (Perca fluviatilis).

The Eurasian perch (Perca fluviatilis) is a freshwater carnivorous species of high interest to diversify inland aquaculture. However, little is known about its ability to bioconvert polyunsaturated fatty acids (PUFAs) from plant oils into long chain polyunsaturated fatty acids (LC-PUFAs). In this study, special attention has been given to the fatty acid desaturase 2 (FADS2) which is commonly described to be a rate-limiting enzyme of the LC-PUFA biosynthesis. This work reports on the cloning, tissue expression and functional characterization of the Eurasian perch fads2, but also on the cloning of two alternative splicing transcripts named fads2-AS1 and fads2-AS2. The fads2 cDNA cloned is composed of an open reading frame (ORF) of 1338 nucleotides (nt) and encodes a protein of 445 amino acids. This deduced amino acid sequence displays the typical structure of microsomal FADS2 including two transmembrane domains and an N-terminal cytochrome b5 domain with the "HPGG" motif. Quantitative real-time PCR assay of fads2, fads2-AS1 and fads2-AS2 expressions revealed that the fads2 transcript was mainly expressed in the liver and intestine and exhibited a typical gene expression pattern of freshwater species while fads2-AS1 and fads2-AS2 genes were highly expressed in the brain, followed by the liver and intestine. Functional characterization of Eurasian perch FADS2 in transgenic yeast showed a fully functional Δ6 desaturation activity toward C18 PUFA substrates, without residual Δ5 and Δ8 desaturase activities.

Effects of dietary linseed oil on innate immune system of Eurasian perch and disease resistance after exposure to Aeromonas salmonicida achromogen.

This study was designated to investigate the effects of dietary fish oil (FO diet) replacement by linseed oil (LO diet) on regulation of immune response and disease resistance in Eurasian perch (Perca fluviatilis). A control diet containing fish oil (FO = cod liver oil) and characterized by high levels of n-3 high LC-PUFA (6% EPA, 7.5% of total fatty acids (FAs)) was compared to linseed oil diet (LO diet) composed of low LC-PUFA contents (1% EPA, 2.3% DHA of total FAs) but high C18 fatty acids levels. The experiment was conducted in quadruplicate groups of 80 fish each. After 10 weeks of feeding, the innate immune status was evaluated in various organs (liver, spleen, and head-kidney) (feeding condition). Two days later, a bacterial challenge was performed on fish from 2 rearing conditions: fish infected with Aeromonas salmonicida (bacteria condition) and fish injected with sterile medium but maintained in the same flow system that fish challenged with bacteria (sentinel condition). Three days after injection of bacteria, a significant decrease of lymphocyte, thrombocyte and basophil populations was observed while neutrophils were not affected. In addition, plasma lysozyme activity and reactive oxygen species production in kidney significantly increased in fish challenged with A. salmonicida while the plasma alternative complement pathway activity was not affected. Increase of plasma lysozyme activity as well as reactive oxygen species production in spleen and kidney of sentinel fish suggest that these immune defenses can also be activated, but at lower bacteria concentration than infected fish. No differences in leucocyte populations, plasma lysozyme and alternative complement pathway activities were observed between dietary treatments. Similarly, expression of genes related to eicosanoid synthesis in liver were not affected by the dietary oil source but were strongly stimulated in fish challenged with A. salmonicida. These findings demonstrated that the use of linseed oil does not deplete the innate immune system of Eurasian perch juveniles.

Dietary Linseed Oil Reduces Growth While Differentially Impacting LC-PUFA Synthesis and Accretion into Tissues in Eurasian Perch (Perca fluviatilis).

The aim of this study was to evaluate the impact of replacing dietary fish oil (FO) with linseed oil (LO) on growth, fatty acid composition and regulation of lipid metabolism in Eurasian perch (Perca fluviatilis) juveniles. Fish (17.5 g initial body weight) were fed isoproteic and isoenergetic diets containing 116 g/kg of lipid for 10 weeks. Fish fed the LO diet displayed lower growth rates and lower levels of DHA in the liver and muscle than fish fed the FO diet, while mortality was not affected by dietary treatment. However, DHA content recorded in the liver and muscle of fish fed the LO diet remained relatively high, despite a weight gain of 134 % and a reduced dietary level of long-chain polyunsaturated fatty acids (LC-PUFA), suggesting endogenous LC-PUFA biosynthesis. This was supported by the higher amounts of pathway intermediates, including 18:4n-3, 20:3n-3, 20:4n-3, 18:3n-6 and 20:3n-6, recorded in the liver of fish fed the LO diet in comparison with those fed the FO diet. However, fads2 and elovl5 gene expression and FADS2 enzyme activity were comparable between the two groups. Similarly, the expression of genes involved in eicosanoid synthesis was not modulated by dietary LO. Thus, the present study demonstrated that in fish fed LO for 10 weeks, growth was reduced but DHA levels in tissues were largely maintained compared to fish fed FO, suggesting a physiologically relevant rate of endogenous LC-PUFA biosynthesis capacity.

Effectiveness of extruded rapeseed associated with an alfalfa protein concentrate in enhancing the bovine milk fatty acid composition.

Linseed and rapeseed, good sources of 18:3 n-3 and cis9-18:1, respectively, have been shown to improve the bovine milk fatty acid (FA) profile. However, rapeseed, unlike linseed, has little effect on the concentration of 18:3 n-3 in milk fat. Alfalfa protein concentrate (APC), besides being a valuable protein source for milk production, contains lipids rich in 18:3 n-3. Therefore, this experiment aimed at (1) evaluating the transfer efficiency of unsaturated FA (UFA), especially 18:3 n-3, of APC to bovine milk fat, and (2) evaluating whether extruded rapeseed (ER) associated with APC is as effective as extruded linseed (EL) in enhancing the bovine milk fat composition. Six lactating Holstein cows were used in a replicated 2 × 2 Latin square design with 2 iso-energy, iso-nitrogen and iso-FA corn silage-based diets (EL and ER-APC) and two 21-d periods. Extruded linseed, as main UFA source, was included in the first diet, whereas ER, as main UFA source, and APC, as supplemental 18:3 n-3, were included in the second diet. Diets were distributed as a restricted total mixed ration. Compared with the EL diet, the ER-APC diet, where ER was associated with APC, increased milk concentration of 18:3 n-3 (1.18 vs. 1.31% of FA) and cis9-18:1 (18.35 vs. 20.01% of FA). The apparent transfer efficiency of 18:3 n-3 from diet to milk was almost twice as much for the ER-APC diet than for the EL diet (7.4 vs. 3.8% of intake). Extruded linseed accounted for 84% of 18:3 n-3 provided in the EL diet, whereas ER and APC accounted for 33 and 38% of 18:3 n-3 provided in the ER-APC diet, respectively. Because both EL and ER underwent extrusion in similar conditions, these results suggest that 18:3 n-3 of EL in the EL diet and ER in the ER-APC diet were subjected to more extensive ruminal biohydrogenation than 18:3 n-3 of APC in the ER-APC diet. This experiment shows that corn silage-based diets supplemented with ER as the main UFA source, associated with APC as supplemental 18:3 n-3, are as effective as corn silage-based diets supplemented with EL as the main UFA source, in increasing bovine milk UFA and 18:3 n-3 contents. Furthermore, at similar levels of dietary incorporation, this experiment shows that the ruminal bypass of 18:3 n-3 is higher for APC compared with EL.

Potential of an in vitro toolbox combined with exposure data as a first step for the risk assessment of dietary chemical contaminants.

In vitro risk assessment of dietary contaminants has become a priority in human food safety. This paper proposes an in vitro approach associating different complementary tools in an original toolbox and aims to improve the assessment of the toxicological impact of dietary contaminants at realistic human exposure levels, with a special focus on the intestinal compartment. The system is based on the use of four complementary cellular tools, namely stress gene induction in transgenic strains of Escherichia coli, modulation of the activity of key biotransformation enzymes (cytochrome P-450 (CYP) 1A1 and 3A4) in a human intestinal cell line, and activation of aryl hydrocarbon receptor (AhR) and oestrogenic receptor (ER)-dependent genes in agonistic and antagonistic assays with luciferase reporter cells. It was applied to four chosen model molecules: ochratoxin A (OTA) and deoxynivalenol (DON), two common food-borne mycotoxins, and imazalil (IMA) and benomyl (BEN), two fungicides widely occurring in foodstuffs. All these assays were performed at or around a realistic intestinal concentration, determined through a deterministic approach based on the calculation of a theoretical maximum daily intake (TMDI). Using the four model molecules, it is clearly highlighted that induction of CYP1A1 activity and inhibition of CYP3A4 activity occurred in Caco-2 cells at a realistic intestinal concentration of IMA. Furthermore, some bacterial stress genes were induced in a range of realistic concentrations, following exposure to DON and IMA. In addition, BEN clearly provoked an ER agonistic activity in a human oestrogen sensitive reporter cell line. All these results are in accordance with the literature, suggesting that the in vitro toolbox constitutes an interesting approach in order to obtain a first 'fingerprint' of dietary contaminants at realistic human exposure for further risk assessment.

Screening, identification and quantification of glucosinolates in black radish (Raphanus sativus L. niger) based dietary supplements using liquid chromatography coupled with a photodiode array and liquid chromatography-mass spectrometry.

The glucosinolate profile of black radish (Raphanus sativus L. niger) based dietary supplements has been investigated by HPLC-PDA, LC-ESI-MS/MS and LC-APCI-MS/MS systems. Optimization of the MS/MS parameters and LC conditions was performed using sinigrin reference standard and rapeseed certified reference material (BC190) respectively. An LC-ESI-MS/MS system was used to detect (screen) and identify the naturally occurring intact glucosinolates (GLs). The intact GLs identified were then desulfated and quantified on an HPLC-PDA system as desulfo-glucosinolates (DS-GLs). Prior to quantification, the DS-GLs were identified using an APCI-MS/MS. The HPLC-PDA method performance criteria were evaluated using glucotropaeolin potassium salt. The validated method was applied for the analysis of six dietary supplements. In total, six glucosinolates were identified and quantified in the dietary supplements; glucoraphasatin (0.2-0.48 mg/g), glucosisaustricin (0.37-0.91 mg/g), glucoraphenin (0.84-1.27 mg/g), glucoputrajivin (0.14-0.28 mg/g), glucosisymbrin (0.70-0.99 mg/g) and gluconasturtiin (0.06-0.12 mg/g). Glucoraphenin was the most abundant glucosinolate in all samples.

Modulation of CYP1A1 activity by a Ginkgo biloba extract in the human intestinal Caco-2 cells.

Ginkgo biloba is a widely consumed dietary supplement. Some dietary active compounds modulate the activity of biotransformation enzymes inside the enterocytes and more interestingly of cytochrome P-450 1A1 (CYP1A1). This enzyme is of a particular interest because of its implication in the metabolism of some exogenous pro-carcinogens or endogenous molecules. In the present work, we have used Caco-2 cells to study the effect of a standard reference material of a Ginkgo biloba extract (GBE) (10-400 µg/ml), as well as of its major individual active compounds (kaempferol, quercetin, isorhamnetin, ginkgolides and bilobalide), alone or in mixtures, at realistic intestinal concentrations, on the induction of CYP1A1 activity, in the presence or absence of benzo[a]pyrene (B[a]P) (0.1 µg/ml), a well-known CYP1A1 inducer. 3-O-rutinosides of kaempferol, quercetin and isorhamnetin were also tested. We have demonstrated a strong induction (p < 0.005) of CYP1A1 activity and a slight, but significant (p < 0.005), decrease of this activity in the presence of B[a]P by the GBE at the realistic exposure level of 100 µg/ml. The inductive effect was explained, in part, by quercetin and kaempferol after 24h exposure while unknown compounds seem to be responsible for the strong CYP1A1 induction observed after 6h exposure. The inhibitory potency of flavonols on CYP1A1 activity in presence of B[a]P was much stronger for the aglycones than for the 3-O-rutinosides, explaining the slight effect observed with the GBE, mainly composed of glycosylated flavonoids. These results indicate that GBEs may disturb intestinal CYP1A1 activity and, in turn, affect the metabolism of other compounds. The present paper thus highlights the necessity to take these side effects into account when administrating Ginkgo biloba herbal supplements.

Linseed oil stabilisation with pure natural phenolic compounds.

Linseed has been used for a very long time in human and animal nutrition. Currently, there is an increasing interest in linseed oil because of its particularly high content in α-linolenic acid (ALA), an omega-3 fatty acid (FA). Unfortunately, ALA turns also the oil extremely sensitive to oxidation. This study aimed at assessing four pure representative phenolic compounds, myricetin (flavonol), (+)-catechin (flavanol), genistein (isoflavone), and caffeic acid (hydroxycinnamic acid) at a concentration of 555µmol/kg as antioxidants in refined linseed oil (RLO). Their protective effect was assessed by monitoring the hydroperoxide formation, the FA profile and the residual antioxidant concentration in RLO, along its storage at 60°C according to the Schaal oven test procedure. Caffeic acid, (+)-catechin and myricetin were found to be more efficient than butylated hydroxyanisole (BHA), a synthetic antioxidant. Interestingly, myricetin strongly reduced ALA oxidation. These results confirm that the chemical structure of the phenolic compounds plays a major role in their antioxidant properties.

Effectiveness of mycotoxin sequestration activity of micronized wheat fibres on distribution of ochratoxin A in plasma, liver and kidney of piglets fed a naturally contaminated diet.

A study was carried out to determine the ability of dietary micronized wheat fibres (MWF) to decrease the levels of ochratoxin A (OTA) in plasma, kidney and liver of piglets fed a naturally contaminated diet. A total of 96 piglets (weighting 11.4+/-1.5 kg) were fed one of four different diets for 28 days. Diets included (1) control diet, (2) control diet with MWF (1%), (3) OTA naturally contaminated diet (117.45+/-4.74 ng/g), (4) OTA naturally contaminated diet (118.13+/-2.85 ng/g) with MWF (1%). No difference in feed efficiency (P>0.05) could be observed between the different diets. The absolute weight of kidneys and liver were significantly higher in pigs fed the OTA-contaminated diet (diet 3) as compared to the control diet (diet 1) or to the control diet amended with MWF (diet 2) (P<0.05). However the use of MWF (diet 4) significantly protected against these weight changes. A significant protective effect of MWF was also observed in terms of OTA concentration in plasma (45.6% decrease), kidney (40.8% decrease) and liver (26.5% decrease). These results suggest that the addition of MWF is effective in decreasing the bioavailability of OTA from contaminated diets in piglets.

Study of the gastrointestinal biotransformation of zearalenone in a Caco-2 cell culture system with liquid chromatographic methods.

A high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) was developed and validated for the detection of zearalenone (ZON), alpha-zearalenol (alpha-ZOL) and beta-zearalenol (beta-ZOL) in in vitro biological samples. Furthermore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the detection of ZON, alpha-ZOL, beta-ZOL, alpha-zearalanol (alpha-ZAL) and beta-zearalanol (beta-ZAL) in in vitro biological samples. Zearalanone (ZAN) was used as internal standard in both methods. The limit of detection/limit of quantitation (LOD/LOQ) values for ZON, alpha-ZOL and beta-ZOL were 2/7, 2/7 and 4/13 microg l(-1), respectively, for the HPLC-FLD method. For the LC-MS/MS method LOD/LOQ values for ZON, alpha-ZOL, beta-ZOL, alpha-ZAL and beta-ZAL were 6/20, 5/17, 4/14, 9/30 and 6/19 microg l(-1), respectively. Within-day and between-day precision were less then 11 and 14%, respectively for the HPLC-FLD method, and both less then 20% for the LC-MS/MS method. The recovery of ZON and its metabolites ranged between 73 and 89% for the HPLC-FLD method and between 69 and 112% for the LC-MS/MS method. The methods were used for the detection of the compounds in in vitro biological samples, obtained with human intestinal Caco-2 cells culture experiments. The 8-days post-confluent Caco-2 cells were treated with ZON or a mixture of ZON and imazalil (IMA). After an incubation time of 24 h the samples were analysed with the HPLC-FLD method. Neither ZON nor its derivatives were detected in the samples. The disappearance of ZON could possibly point out the formation of phase II metabolites like glucuronide conjugates. Therefore, samples were pretreated with beta-glucuronidase before LC-MS/MS analysis. The LC-MS/MS results showed that ZON, alpha-ZOL and beta-ZOL could only be detected in the beta-glucuronidase pretreated samples. This confirmed the formation of glucuronide conjugates and the hydroxylation of ZON during the incubation with Caco-2 cells.

Liquid chromatographic methods for biotransformation studies of ochratoxin A.

Liquid chromatographic methods were used for the detection of ochratoxin A (OTA) and its metabolites ochratoxin alpha (OTalpha), 10-hydroxy OTA (10-OHOTA), 4R-hydroxy OTA (4R-OHOTA) and the ethyl ester of OTA (OTC) in in vitro samples, obtained with Caco-2 cell culture experiments and in in vivo urine samples from sheep. A high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method were developed and validated for the detection of OTA and its metabolites OTalpha, 10-OHOTA, 4R-OHOTA and OTC, which was used as internal standard. The LOD/LOQ values for OTalpha, 4R-OHOTA and OTA were 0.63/2.11, 0.99/3.31 and 0.84/2.81 microg/L, respectively, for the HPLC-FLD method and 0.98/3.28, 1.11/3.72 and 0.88/2.96 microg/L, respectively for the LC-MS/MS method. Within-day and between-day precision were both <12% for the HPLC-FLD method, and <10% for the LC-MS/MS method. The recovery of OTA and its metabolites ranged between 71 and 111% for the HPLC-FLD method and between 79 and 110% for the LC-MS/MS method. In the first experiment only OTA was added to the Caco-2 cells while in the second experiment 3-methylcholanthrene (3MC) was also present in the cell culture systems. Besides OTA, which was recovered in all the samples, an unknown compound was also observed in the second experiment. When 3MC was added, the results showed that the OTA concentration in the basolateral samples was decreased by 50%. The methods were also implemented for the analysis of urine samples of sheep, fed increasing amounts of OTA. With the HPLC-FLD method it could be concluded that the concentration of OTA and OTalpha increased according to ingested amounts of OTA, with OTalpha being the most abundant compound. The results obtained with the LC-MS/MS method confirmed these results.

Distribution of ochratoxin A in plasma and tissues of rats fed a naturally contaminated diet amended with micronized wheat fibres: effectiveness of mycotoxin sequestering activity.

The effectiveness of micronized wheat fibres (MWF) alone or in association with yeast cell walls (YCW) as active adsorbents to decrease, in vivo, the levels of ochratoxin A (OTA) was checked in a total of 48 rats, equitably distributed into four groups: (1) control; (2) OTA naturally contaminated diet (2.2 microg/g); (3) OTA naturally contaminated diet (2.2 microg/g) amended with MWF (2%); (4) OTA naturally contaminated diet (2 microg/g) amended with MWF (1.8%) in association with YCW (0.2%). A 4 week experimental period corresponding to a daily intake in the range of 132.2-146.1 microg OTA/kg bw decreased the rat body weight gains, as compared to the controls. The adsorbents did not significantly alleviate the growth depression caused by the contaminated diet. However, a significant protective effect of MWF was observed in terms of OTA concentration in plasma (40.5% decrease), kidney (28.1% decrease) and liver (38.8% decrease). Mixing this sorbent with the YCW did not significantly improve its protective activity against OTA. The faecal OTA concentrations were higher for the MWF and MWF+YCW treated animals, as compared to the positive control (group II). Taken together, these results suggest that MWF are a promising tool to counteract the toxic effects of OTA naturally contaminated diets.

Influence of an increase in diet structure on milk conjugated linoleic acid content of cows fed extruded linseed.

This experiment studied the effect of a modest difference in diet structure value (SV) on milk conjugated linoleic acid (CLA) contents of cows fed diets supplemented with extruded linseed, in situations where the diets provided enough SV and therefore did not induce milk fat depression. Six lactating Holstein cows were used in a crossover design with two treatments ('SV 1.50' and 'SV 1.73') and two periods of 21 days. The 'SV 1.50' diet contained 59% maize silage, 13% soya bean meal, 13% sugar beet pulp and 14% Nutex Compact (containing 56% extruded linseed) (dry matter (DM) basis) and was offered as a restricted total mixed ration. For the 'SV 1.73' diet, 8% wheat straw (DM basis) was added to the 'SV 1.50' diet as an additional structure source. The two diets had a forage-to-concentrate ratio of 59 : 41 and 62 : 38. The inclusion of straw in the diet resulted in an additional intake of NDF (+1110 g/day), which accounted for 90% of the additional intake of OM, whereas additional intakes of the other nutrients were minor. Milk yield and composition did not differ among treatments. The inclusion of straw in the diet did not affect the milk levels of t10-18:1, 18:2n-6, c9-16:1, c9-18:1, c11-18:1, 6:0, 8:0, 20:4 and 20:5. It decreased the milk levels of c9,t11-CLA (2.13% v. 3.03% of fatty acids (FA) reported, P < 0.001), t11-18:1 (4.99% v. 7.10% of FA reported, P < 0.001), 18:3n-3, t9-16:1 and t9-18:1, while it increased the milk levels of 6:0-14:0 (20.90% v. 19.69% of FA reported, P < 0.01), 16:0 (26.55% v. 25.25% of FA reported, P < 0.01), 18:0 (13.54% v. 12.59% of FA reported, P < 0.001), 17:0, 20:0 and 22:5. Regarding the ratio between FA, the inclusion of straw increased the 18:0/total C18 FA ratio (37.74% v. 32.07%, P < 0.001), whereas it decreased the total trans-C18 FA/total C18 FA ratio (15.46% v. 20.34%, P < 0.001), the t11-18:1/total C18 FA ratio (13.70% v. 17.95%, P < 0.01) and the c9,t11-CLA/total C18 FA ratio (5.82% v. 7.64%, P < 0.001). We conclude from this experiment that even a modest increase in SV to a diet supplemented with extruded linseed, yet already providing enough SV, alters the rumen lipid metabolism and, hence, CLA levels in milk fat.

Contaminants in organically and conventionally produced winter wheat (Triticum aestivum) in Belgium.

A database has been compiled with the levels of important contaminants (mycotoxins, heavy metals and pesticides) measured from 2002 to 2005 in winter wheat (Triticum aestivum) grown in Belgium according to the organic and conventional farming systems. Assuming no further change in contaminant levels during cereal processing and during the preparation of foodstuffs, conservative intakes are estimated for the consumers of cereal-based products such as flour, bread, breakfast cereals, dough and pastry. The results show that for the consumer of organic foodstuffs, estimated daily intakes are 0.56 microg deoxynivalenol (DON), 0.03 microg zearalenone (ZEA), 0.19 microg Cd, 0.28 microg Pb and 0.0006 microg Hg kg(-1) body weight, taking into account the average contaminant levels in unprocessed grains and the average cereal products consumptions in Belgium. For the consumers of conventional foodstuffs, the corresponding estimated daily intakes are 0.99 microg DON, 0.06 microg ZEA, 0.17 microg Cd, 0.12 microg Pb and 0.0007 microg Hg kg(-1) body weight. In addition, it appears that for the consumers of conventional products, intakes of some post-harvest insecticides have to be taken into account (0.11 microg chlorpyriphos-methyl, 0.2 microg dichlorvos and 0.24 microg pirimiphos-methyl kg(-1) bw). When expressed as a percentage of the tolerable/acceptable daily intake (TDI/ADI), it seems that the corresponding estimated (conservative) intakes are the highest for DON (56% for organic and 99% for conventional cereal products), ZEA (16% for organic and 32% for conventional cereal products), and Cd (19% for organic and 17% for conventional cereal products), all other estimated intakes of contaminants (including pesticides) being lower than 10% of the TDI/ADI.

Intake of ochratoxin A and deoxynivalenol through beer consumption in Belgium.

Estimations of ochratoxin A (OTA) and 4-deoxynivalenol (DON) exposure of the Belgian population through beer consumption were made using the results of the recent Belgian food survey and the compiled data set of OTA and DON levels in conventionally and organically produced beers in 2003-05. For the consumers of organic beers, the daily intake of OTA was 0.86 (in 2003), 1.76 (in 2004) and 0.72 (in 2005) ng kg(-1) body weight (bw), considering the mean beer consumption (0.638 litres) and the average level of OTA in 2003, 2004 and 2005, respectively. Using the 97.5th percentile of beer consumption (1.972 litres), the corresponding OTA daily intakes were 2.65, 5.44 and 2.24 ng kg(-1) bw, which are close or above the tolerable daily intake (TDI) of 5 ng kg(-1) bw. For the consumers of conventional beers, the OTA intakes were low: 0.23, 0.23 and 0.11 ng kg(-1) bw day(-1) for the average beer consumption, in 2003, 2004 and 2005 against 0.72, 0.73 and 0.34 ng kg(-1) bw day(-1) when the 97.5th percentile level was considered. As for the DON intake, the estimates were quite low for both conventional and organic beer consumers when the provisional maximum TDI (PMTDI) of 1 microg kg(-1) bw was considered. Average consumption of organic beer led to daily intakes of 0.05 and 0.04 microg DON kg(-1) bw in 2003 and 2004, respectively, whilst for conventional beer, daily intakes were 0.07 and 0.05 microg DON kg(-1) bw. At the 97.5th percentile level of beer consumption, daily intakes of 0.15 and 0.13 microg kg(-1) bw were obtained for organic beers against 0.23 and 0.17 microg kg(-1) bw for conventional ones. The results showed that beer could be an important contributor to OTA exposure in Belgium, even though a declining trend seems to be apparent during the last year of monitoring. Therefore, efforts should be devoted to maintain the OTA levels as low as reasonably achievable, especially for organic beer.