In vitro inhibition of Citrobacter freundii, a red-leg syndrome associated pathogen in raniculture, by indigenous Lactococcus lactis CRL 1584

Red-leg syndrome (RLS) is one of the main infectious diseases that cause economic losses in Lithobates catesbeianus hatcheries, Citrobacter freundii being an etiological agent. Treatment or prevention with therapeutics or chemicals results in modifications of the indigenous microbiota, development of antibiotic resistance, presence of their residues in food and enhancement of production costs. Thus, probiotics could be used as an alternative therapy. Lactic acid bacteria are part of the indigenous microbiota of healthy frogs and can prevent pathogen colonization by different mechanisms, including the production of antagonistic substances. In this work, the evaluation and characterization of the inhibition of C. freundii CFb by Lactococcus lactis subsp. lactis CRL 1584, a potentially probiotic candidate, were carried out. This strain produced lactic acid, H(2)O(2) and bacteriocin in static and shaken conditions and inhibited pathogen growth in associative cultures, with an earlier inhibition under agitated conditions. The elimination of each of the antimicrobial metabolites partially abolished the inhibition of the pathogen, suggesting that the inhibitory effect could be attributed to a combined action of the three antagonistic molecules. Electron microphotographs revealed the damage caused by L. lactis CRL 1584 supernatants to C. freundii cells. The addition of pure lactic acid, H(2)O(2) and bacteriocin to the culture media showed that each metabolite caused different morphological modifications in C. freundii, in agreement with the effect on viable cell counts. The results support the possibility that L. lactis CRL 1584 might be considered as a probiotic to be used in the prevention of RLS in raniculture.     

QuEChERS multiresidue method validation and mass spectrometric assessment for the novel anthranilic diamide insecticides chlorantraniliprole and cyantraniliprole

The gas-phase dissociation reactions of chlorantraniliprole (Rynaxypyr) and cyantraniliprole (Cyazypyr) have been studied in triple-quadrupole, ion trap, and orbitrap mass spectrometers equipped with electrospray and desorption electrospray ion sources, revealing the formation of odd-electron fragment ions, the structures of which were elucidated. The odd-electron fragments were unusually abundant, and their formation is proposed to occur via a tricyclic intermediate. The applicability of the QuEChERS multiresidue method for the quantitation of chlorantraniliprole and cyantraniliprole was also assessed in this study. Four matrices representative of oily, watery, acidic, and dry crop groups were tested, with a targeted limit of quantitation (LOQ) of 0.01 mg/kg. Average recoveries ranged between 87 and 107%, with relative standard deviations (RSD) of ≤ 8%. Linear calibration functions with correlation coefficients r > 0.99 were obtained. The study provides an expansion of the QuEChERS method to include anthranilic diamides and a mass spectrometric assessment for these two novel agrochemical active ingredients.     

Post-mortem recovery,in vitro maturation and fertilization of fallow deer (Dama dama,Linnaeus 1758) oocytes collected during reproductive and no reproductive season

Habitat degradation leads to small and fragmented populations, lower genetic variability and fertility overtime. Assisted reproductive techniques represent important tools to cope with the dramatic loss of biodiversity. Fallow deer (Dama dama), beyond its high commercial value and wide distribution, may represent the most suitable model to study endangered cervids. In this study, oocytes were recovered post-mortem from fallow deer during the breeding and no breeding seasons and were in vitro matured (IVM). The ability of cryopreserved thawed sperm samples recovered by electroejaculation from four adult males was tested by in vitro fertilization of IVM oocytes. The number of oocytes collected per ovary did significantly vary across seasons from 6.2 ± 0.92 during breeding season to 10.4 ± 1.26 during no breeding season (p = .006). Oocytes collected during the breeding season showed higher in vitro fertilization rate compared to the no breeding season (p = .045). However, no embryos reached the blastocyst stage. Semen samples obtained by electroejaculation were successfully cryopreserved, although the cryopreservation process negatively affected most kinetic parameters, mainly at 2 hr post-thawing. Moreover, the percentage of rapid spermatozoa significantly decreased between fresh samples and at 2 hr post-thawing, whereas the percentage of slow spermatozoa increased across the same period (p < .05). Our study provides the logistic steps for the application of assisted reproductive techniques in fallow deer and might be of great interest for genetic resource bank planning.     

Use of CLSA and SPME-headspace techniques followed by GC-MS analysis to extract and identify the floral odorants

Flowers of Ziziphus spina christi are known to be attractive for parasitoids and predators. In Y-tube olfactometer experiments, the dried flowers attracted significantly (p < 0.001) the female parasitoids Aphelinus abdominalis. The flower volatile compounds were analyzed to understand which compounds could be specifically responsible for this attractiveness. The volatile compounds of Ziziphus flowers were extracted by closed-loop-stripping-analysis (CLSA) and also by solid phase microextraction (SPME) followed by gas chromatography-mass spectrometry (GC-MS) analysis. The main chemical classes of the volatile compounds are aldehydes, monoterpene-alcohols, ketones and hydrocarbons. Flower extract and some specific compounds will be further tested for their responsiveness to predators and parasitoids in behavioural and electrophysiological experiments.     

Evaluation of the Lime-Cooking and Tortilla Making Properties of Quality Protein Maize Hybrids Grown in Mexico

Eleven experimental and three commercial white quality protein maize (QPM) hybrids and two regular endosperm controls were planted at Celaya, Guanajuato, Mexico with the aim of comparing grain physical characteristics, protein quality, lime-cooking and tortilla making properties. All genotypes were planted under irrigation using a density of 80,000 plants/ha and fertilized with 250 kg N-60 P-60 K per hectare. When compared with the controls these QPM genotypes had lower test (77.4 vs. 76.5 kg/hL) and 1,000 kernel weights (327 vs. 307 g), softer endosperm texture (2.5 vs. 1.8 where 1 = soft, 2 intermediate and 3 hard endosperm), lower protein (10.0 vs. 8.0%), higher nixtamal water uptake after 30 min lime-cooking (50.0 vs. 53.1% moisture) and lower pericarp removal scores. The lower thousand-kernel weight and softer endosperm texture observed in the QPM genotypes lowered the optimum lime-cooking time as estimated with regression equations. Most QPM genotypes had higher amounts of lysine, tryptophan and albumins/globulins when compared with the controls. QPMs HEC 424973, HEC 774986 and HEC 734286 had the best grain traits for nixtamalization and therefore the best potential for industrial utilization. The commercial use of these QPM hybrids should benefit Mexicans who depend on tortillas as the main staple.     

Antiproliferative activity of Citrus juices and HPLC evaluation of their flavonoid composition

The antiproliferative activity of fresh fruit juices extracted from Citrus sinensis (cv. Washington Navel and cv. Sanguinello), C. deliciosa cv. Avana, C. clementina cv. Nules, C. aurantium subsp. myrtifolia , was evaluated against K562 (human chronic myelogenous leukemia), HL-60 (human leukemia) and MCF-7 (human breast adenocarcinoma) cell lines. All the juices tested showed antiproliferative activity. Moreover, the pattern of the main flavanone compounds in the juices has been determined by HPLC analysis.     

Polyphenolics and Antioxidant Capacity of White and Blue Corns Processed into Tortillas and Chips

White and blue corns of Mexican and American origins were lime-cooked to obtain nixtamals with optimal moisture (48–50%) for tortillas and chips. Blue kernels had less bulk density, softer endosperm and, consequently, required less cooking time than the white kernels. The optimum cooking regime for the white kernels was 100°C for 20 min, while the optimum for both pigmented genotypes was 90°C for 0 min (until the lime-cooking solution reached 90°C). Doughs, tortillas, and chips were characterized by total soluble phenolics (TSP), anthocyanins (ACN), and antioxidant capacity (AOX). A dough acidification procedure using fumaric acid (pH 5.2) was assessed as a means to improve TSP, ACN, and AOX retention. The Mexican blue corn had higher AOX (16%) than the American blue genotype, although the latter had a threefold higher TSP content (12.1 g/kg, dwb). Mexican and American blue corns had higher AOX capacity (29.6 and 25.6 μM trolox equivalents [TE]/g dwb), respectively, than the white corn (17.4 μM TE/g). White corns did not have detectable amounts of ACN, while blue Mexican and American kernels contained 342 and 261 mg/kg. Lime cooking had the greatest negative impact on the stability of TSP, ACN, and AOX. However, the acidification reduced ACN, TSP, and AOX losses by 8–23, 3–14, and 4–15%, respectively. Similar ACN losses were observed for both types of blue kernels when processed into nixtamal/dough (47%); however, ACN losses in tortillas and chips manufactured from the American blue genotype were higher (63 and 81%, respectively) than those of Mexican blue corn products (54 and 75%). ACN losses were highly correlated to TSP (r = 0.91) and AOX capacity losses (r = 0.94).     

RFLP markers are an effective tool for the identification of creeping bentgrass (Agrostis stolonifera L.) cultivars

The potential use of RFLP molecular markers for the identification of four creeping bentgrass (Agrostis stolonifera L.) cultivars and one variety of A. tenuis Sibth. used as control, was investigated. Seven probes out of the total 44 screened were able to differentiate all five cultivars at once. On the basis of their genetic similarity the varieties bred at the Pennsylvania State University were grouped closely together, whereas the variety Prominent and the A. tenuis control were more distantly related.