Validation of the performance of a GMO multiplex screening assay based on microarray detection

A new screening method for the detection and identification of GMO, based on the use of multiplex PCR followed by microarray, has been developed and is presented. The technology is based on the identification of quite ubiquitous GMO genetic target elements first amplified by PCR, followed by direct hybridisation of the amplicons on a predefined microarray (DualChip^® GMO, Eppendorf, Germany). The validation was performed within the framework of a European project (Co-Extra, contract no 007158) and in collaboration with 12 laboratories specialised in GMO detection. The present study reports the strategy and the results of an ISO complying validation of the method carried out through an inter-laboratory study. Sets of blind samples were provided consisting of DNA reference materials covering all the elements detectable by specific probes present on the array. The GMO concentrations varied from 1% down to 0.045%. In addition, a mixture of two GMO events (0.1% RRS diluted in 100% TOPAS19/2) was incorporated in the study to test the robustness of the assay in extreme conditions. Data were processed according to ISO 5725 standard. The method was evaluated with predefined performance criteria with respect to the EC CRL method acceptance criteria. The overall method performance met the acceptance criteria; in particular, the results showed that the method is suitable for the detection of the different target elements at 0.1% concentration of GMO with a 95% accuracy rate. This collaborative trial showed that the method can be considered as fit for the purpose of screening with respect to its intra- and inter-laboratory accuracy. The results demonstrated the validity of combining multiplex PCR with array detection as provided by the DualChip^® GMO (Eppendorf, Germany) for the screening of GMO. The results showed that the technology is robust, practical and suitable as a screening tool.     

Novel multiplex PCR-RFLP assay discriminates bovine,porcine and fish gelatin substitution in Asian pharmaceuticals capsule shells

Gelatin is widely used in pharmaceuticals as a protective coating, such as soft and hard capsule shells. However, the animal source of gelatin is a sensitive issue because certain gelatins such as porcine and bovine gelatins are not welcome in Halal, Kosher and Hindus’ consumer goods. Recently, we have documented DNA barcoding and multiplex PCR platforms for discriminating porcine, bovine and fish gelatins in various fish and confectionary products; but those assays were not self-authenticating and also not tested in highly refined pharmaceutical products. To address this knowledge gap, here we report a self-authenticating multiplex PCR-restriction fragment length polymorphism (RFLP) assay to identify animal sources of various gelatin in pharmaceutical capsules. Three different restriction enzymes, BsaAI, Hpy188I and BcoDI were used to yield distinctive RFLP patterns for gelatin-based bovine (26, 94 bp), fish (97, 198 bp) and porcine (17, 70 bp) DNA in control experiments. The specificity was cross-tested against 16 non-target species and the optimised assay was used to screen gelatin sources in 30 halal-branded pharmaceuticals capsule shells. Bovine and porcine DNA was found in 27 and 3 of the 30 different capsules products. The assay was suitable for detecting 0.1 to 0.01 ng total DNA extracted from pure and mixed gelatins. The study might be useful to authenticate and monitor halal, kosher, vegetarian and Hindu compliant pharmaceuticals, foods and cosmetics.     

Side‐Directed Transfer and Presystemic Metabolism of Selenoneine in a Human Intestinal Barrier Model

Scope : Selenoneine, a recently discovered selenium (Se) species mainly present in marine fish, is the Se analogue of ergothioneine, a sulfur‐containing purported antioxidant. Although similar properties have been proposed for selenoneine, data on its relevance to human health are yet scarce. Here, the transfer and presystemic metabolism of selenoneine in an in vitro model of the human intestinal barrier are investigated. Methods and results : Selenoneine and the reference species Se‐methylselenocysteine (MeSeCys) and selenite are applied to the Caco‐2 intestinal barrier model. Selenoneine is transferred in higher amounts, but with similar kinetics as selenite, while MeSeCys shows the highest permeability. In contrast to the reference species, transfer of selenoneine is directed toward the blood side. Cellular Se contents demonstrate that selenoneine is efficiently taken up by Caco‐2 cells. Moreover, HPLC/MS‐based Se speciation studies reveal a partial metabolism to Se‐methylselenoneine, a metabolite previously detected in human blood and urine. Conclusions : Selenoneine is likely to pass the intestinal barrier via transcellular, carrier‐mediated transport, is highly bioavailable to Caco‐2 cells and undergoes metabolic transformations. Therefore, further studies are needed to elucidate its possible health effects and to characterize the metabolism of selenoneine in humans.     

Ochratoxin A in raw materials and cooked meat products made from OTA-treated pigs

The aim of this study was to determine ochratoxin A (OTA) concentrations in the raw materials and cooked meat products made from pigs sub-chronically exposed to OTA. The treated animal group (n = 5) was administered with 300 μg OTA/kg of feed for 30 days, whereas the control group (n = 5) was left untreated. OTA concentrations were quantified using immunoassay (ELISA) and high performance liquid chromatography with fluorescence detection (HPLC-FD). OTA concentration was the highest in the kidney, followed by the lungs, liver, blood, spleen, heart, and adipose tissue. As for the final meat products, the highest average OTA concentration was detected in black pudding sausages (14.02 ± 2.75 μg/kg), then in liver sausages (13.77 ± 3.92 μg/kg), while the lowest was found in pâté (9.33 ± 2.66 μg/kg). The results pointed out that a sub-chronic pig exposure leads to the accumulation of OTA in raw materials and consequently in meat products, whose level of contamination is directly dependent on OTA contents in raw materials used for their production.     

Investigation of the in vivo antioxidative activity of Cynara scolymus (artichoke) leaf extract in the streptozotocin‐induced diabetic rat

The in vivo antioxidant activity of a quantified leaf extract of Cynara scolymus (artichoke) was studied. The aqueous artichoke leaf extract (ALE), containing 1.5% caffeoylquinic acid with chlorogenic acid being most abundant (0.30%), and luteolin‐7‐O‐glucoside as major flavonoid (0.15%), was investigated by evaluating the effect on different oxidative stress biomarkers, after 3 wk oral supplementation in the streptozotocin‐induced diabetic rat model. Apart from two test groups (0.2 g ALE/kg BW/day and 1 g ALE/kg BW/day, where BW is body weight), a healthy control group, untreated oxidative stress group, and vitamin E treated group (positive control) were included. A 0.2 g/kg BW/day of ALE decreased oxidative stress: malondialdehyde and 8‐hydroxydeoxyguanosine levels significantly diminished, whereas erythrocyte glutathione levels significantly increased. A 1.0 g/kg BW/day ALE did not show higher antioxidant activity.     

In vitro digestibility of phenolic compounds from edible fruits: could it be explained by chemometrics?

The health benefits of phenolic compounds depend on the ingested amount, molecular diversity and gastrointestinal digestibility. The phenolic profile of eight fruits (blackberry, blueberry, strawberry, raspberry, mulberry, pomegranate, green and red globe grapes) was chemometrically associated with their in vitro digestibility (oral, gastric, intestinal). Extractable phenols, flavonoids and anthocyanins strongly correlated with each other (r ≥ 0.84), proanthocyanidins with anthocyanins (r = 0.62) and hydrolysable phenols with both extractable phenols (r = 0.45) and proanthocyanidins (r = ?0.54). Two principal components explained 93% of the variance [61% (free‐phenols), 32% (bounded‐phenols)], and four clusters were confirmed by hierarchical analysis, based in their phenolic richness (CLT 1‐4: low to high) and molecular diversity. In vitro digestibility of extractable phenols and flavonoids was blackberry (CLT‐4)> raspberry (CLT‐2)> red grape (CLT‐1) related to their phenolic richness (r ≥ 0.96; P < 0.001), but anthocyanins’ digestibility was pH‐dependent. Chemometrics is useful to predict the in vitro digestibility of phenolic compounds in the assayed fruits.     

Neonatal treatment of rats with diethylstilboestrol (DES) induces stromal-epithelial abnormalities of the vas deferens and cauda epididymis in adulthood following delayed basal cell development

This study investigated whether transient, neonatal (days 2-12) treatment of rats with the potent oestrogen, diethylstilboestrol (DES), altered the structure of the cauda epididymis/vas deferens in adulthood, and if the changes observed related to altered development of basal cells in early puberty. Neonatal treatment with 10 microg DES resulted in the following during adulthood: (a) coiling of the normally straight initial vas deferens, (b) gross epithelial abnormalities, (c) 4-fold widening of the periductal non-muscle layer, (d) infiltration of immune cells across the epithelium into the lumen, and (e) reduction/absence of sperm from the vas deferens lumen. Amongst affected animals>75% exhibited reduced epithelial immunoexpression of androgen receptor and aberrant oestrogen receptor-alpha immunoexpression and 63% exhibited multi-layering of basal cells coincident with increased epithelial cell proliferation. None of the aforementioned changes occurred in rats treated neonatally with 0.1 microg DES.As basal cells play a key role in the development of epithelia such as that in the epididymis and vas deferens, we went on to investigate if neonatal DES treatment affected basal cell development. In controls, basal cells were first evident at day 10 (vas deferens) or day 18 (cauda). Rats treated with 10 microg, but not those treated with 0.1 microg, DES, showed approximately 90% reduction (P<0.001) in basal cell numbers at day 15 and day 18. This decrease coincided with gross suppression of testosterone levels; co-treatment of rats with 10 microg DES+testosterone maintained basal cell numbers at control levels at day 18. However, suppression of testosterone production (GnRH antagonist treatment) or action (flutamide treatment) did not alter basal cell numbers. It is concluded that neonatal exposure to high oestrogen levels coincident with reduced testosterone action results in abnormal changes in the adult cauda/vas deferens that are preceded by delayed differentiation of basal cells. These findings imply a role for androgens and oestrogens in basal cell development and suggest that this may be pivotal in determining normal epithelial (and stromal) development of the cauda/vas deferens.     

亮菌固态和液体发酵多糖及其醒酒作用研究

本实验对亮菌在固态、液体两种不同培养基上发酵生长的代谢产物 -- 亮菌多糖进行了研究,以热浸提和冷冻法提取亮菌多糖,苯酚-硫酸法测定亮菌多糖含量,同时进行了亮菌醒酒的动物实验.实验结果表明亮菌固态发酵62d时多糖含量最高,液体发酵5d时多糖含量高于发酵8d;亮菌多糖高、低剂量组均可以显著延长小白鼠对白酒的耐受时间(p＜0.05),显著缩短实验小白鼠的醉酒时间(p＜0.05),其结果亦优于阳性对照组.实验结果表明亮菌的解酒功效果显著.     

Identity and overall acceptance of two types of sour rye bread

Response surface methodology was employed to study the influence of four recipe variables (wheat: rye flour ratio, bread acidity, ash content of rye flour and sodium chloride content) on the identity and overall acceptance of two rye bread types (soft and crisp rye bread). The subjects ( n = 79) rated attribute intensities, the extent to which the salient sensory properties and the overall sample corresponded to their expectations of rye bread, and the overall acceptance (pleasantness and purchase intentions). The acidity and ash content contributed the most to the extent to which a sample met subjects' expectations. The NaCl content was not critical. Consumer acceptance was affected by ash content, and by the interactions, NaCl content × acidity and wheat:rye ratio × ash content. The non-significance of NaCl content should encourage the baking industry to put low-salt rye products on the market.