Ultrasound probe enhanced enzymatic hydrolysis for rapid separation of ß2-adrenergic agonists from animal urine and livestock wastewater: Applicability to biomonitoring investigation.

BACKGROUND: An increasing number of ß2-adrenergic agonists are illicitly used for growth promoting and lean meat increasing in animal husbandry in recent years, but the development of analytical methods has lagged behind these emerging drugs. RESULTS: Here, we designed and developed an ultrasound probe enhanced enzymatic hydrolysis reactor for quick separation and simultaneously quantification of 22 ß2-adrenergic agonists in animal urine and livestock wastewater. Owing to the enhancement of the conventional enzymatic digestion through the ultrasound acoustic probe power, only 2 min was required for the comprehensively separation of ß2-adrenergic agonists from the sample matrices, making it a much more desirable alternative tool for high-throughput investigation. The swine, bovine and sheep urines (n = 287), and livestock wastewater (n = 15) samples, collected from both the north and south China, were examined to demonstrate the feasibility and capability of the proposed approach. Six kinds of ß2-adrenergic agonists (clenbuterol, salbutamol, ractopamine, terbutaline, clorprenaline and cimaterol) were found in animal urines, with concentrations ranged between 0.056 µg/L (terbutaline) and 5.79 µg/L (clenbuterol). Up to nine ß2-adrenergic agonists were detected in wastewater samples, of which four were found in swine farms and nine in cattle/sheep farms, with concentration levels from 0.069 µg/L (tulobuterol) to 2470 µg/L (clenbuterol). SIGNIFICANCE: Interestingly, since ß2-adrenergic agonists are usually considered to be abused mainly in the pig farms, our data indicate that both the detection frequencies and concentrations of these agonists in the ruminant farms were higher than the pig farms. Furthermore, the findings of this work indicated that there is a widespread occurrence of ß2-adrenergic agonists in livestock farms, especially for clenbuterol and salbutamol, which may pose both food safety and potential ecological risks. We recommend that stricter controls should be adopted to prevent the illegally usage of these ß2-adrenergic agonists in agricultural animals, especially ruminants, and they should also be removed before discharging to the environment.

Quantum Dot Nanobead-Based Fluorescence-Linked Immunosorbent Assay for Detection of Glycinin in Soybeans and Soy Products.

Soybean glycinin, as a major soybean allergen, is difficult to accurately quantify due to its large molecular weight and complex structure. CdSe/ZnS quantum dot nanobead (QB) is a core/shell fluorescent nanomaterial with strong fluorescent signals and high sensitivity at 630 nm. An immunosorbent assay based on CdSe/ZnS quantum dot nanobeads (QBs-FLISA) was developed for the glycinin quantification in soybean and soybean products. Here, the purified glycinin was coated on the microporous plate to serve as the coating antigen, and CdSe/ZnS nanobead conjugated with anti-glycinin polyclonal antibodies was used as fluorescent detection probe. The target glycinin in the sample and the coated antigen on the plate competitively adsorbed the antibody labeled the CdSe/ZnS QBs probes. The limits of detection and quantitation for glycinin were 0.035 and 0.078 µg mL-1, respectively. The recoveries of the spiked samples ranged from 89.8% to 105.6%, with relative standard deviation less than 8.6%. However, compared with ELISA, the sensitivities of QBs-FLISA for the detection of glycinin were increased by 7 times, and the detection time was shortened by two-thirds. This QBs-FLISA method has been effectively applied to the detection of soybean seeds with different varieties and soy products with different processing techniques, which will provide a rapid screening method for soybean and soybean products with low allergens.

Different copper sources and levels affect growth performance, copper content, carcass characteristics, intestinal microorganism and metabolism of finishing pigs.

Copper (Cu) is an essential trace element in the production of swine. This study was conducted to investigate the effect of 3 different sources of Cu on growth performance, Cu metabolism, and intestinal microorganisms of finishing pigs, so as to estimate the bioavailability of the 3 sources for pigs. A total of 42 male finishing pigs (88.74 ± 5.74 kg) were randomly allocated to 7 treatments. The factors were 3 sources (CuSO4, Cu-glycine, Cu-proteinate) and 2 levels (5 and 20 mg/kg) of Cu, plus one negative control treatment (0 mg/kg added Cu level) for the entire 28-d experiment. The average daily gain (ADG) and feed to gain ratio (F:G) both increased when Cu was added. The Cu level in liver, bile, kidney, serum, lung, urine and feces rose (P < 0.001) with increasing dietary Cu level regardless of the source. Meanwhile, pigs receiving organic Cu (glycinate or proteinate) retained more Cu and excreted less Cu than those receiving inorganic Cu (CuSO4), which showed that organic forms were more bioavailable. At the transcriptional level, changes in the level and source of dietary Cu resulted in modulation of transporters. In the jejunal mucosa, import transporter high affinity copper uptake protein 1 (CTR1) and export transporter ATPase copper transporting alpha (ATP7A) in supplemental Cu treatments were down-regulated compared to the control. Also, peptide transporter 1 (PepT1) and lanine-serine-cysteine transporter, type-2 (ASCT2) were significantly (P < 0.01) up-regulated in 20 mg/kg Cu-proteinate and Cu-glycinate treatments, respectively. Microbial diversity was lowest in the 20 mg/kg CuSO4 treatment, and the ratio of Firmicutes to Bacteroidetes was higher in added Cu treatments, especially Cu-glycinate treatment. These results indicate that uptake of different Cu forms is facilitated by different transporters and transport mechanisms, and compared with inorganic Cu, organic Cu provides benefits to intestinal microflora and reduces Cu excretion.

Dual quantum dot nanobeads-based fluorescence-linked immunosorbent assay for simultaneous detection of aflatoxin B1 and zearalenone in feedstuffs.

A novel dual quantum dot nanobeads-based fluorescence-linked immunosorbent assay (QBs-FLISA) was successfully developed for simultaneously detecting aflatoxin B1 (AFB1) and zearalenone (ZEN) in feedstuffs. Dual CdSe/ZnS quantum dot nanobeads with different diameters that emit red and green fluorescence were conjugated with anti-AFB1 and anti-ZEN monoclonal antibodies to prepare fluorescent probes, which greatly enhance analytical performance. Under the optimal conditions, the limits of detection for AFB1 and ZEN were 9.3 and 102.1 pg mL-1, respectively. The recoveries ranged from 82.50% to 116.21% with relative standard deviation less than 11.3%. Compared with traditional enzyme-linked immunosorbent assay, detection sensitivities of AFB1 and ZEN using QBs-FLISA were increased 20 and 5 folds, respectively. In addition, results of feedstuff samples analyzed by QBs-FLISA and liquid chromatography tandem mass spectrometry showed a good agreement (R2 = 0.99).

Recent advances in immunoassays and biosensors for mycotoxins detection in feedstuffs and foods.

Mycotoxins are secondary metabolites produced by fungus. Many mycotoxin species are highly toxic and are frequently found in cereals and feedstuffs. So, powerful detection methods are vital and effective ways to prevent feed contamination. Traditional detection methods can no longer meet the needs of massive, real-time, simple, and fast mycotoxin monitoring. Rapid detection methods based on advanced material and sensor technology are the future trend. In this review, we highlight recent progress of mycotoxin rapid detection strategies in feedstuffs and foods, especially for simultaneous multiplex mycotoxin determination. Immunoassays, biosensors, and the prominent roles of nanomaterials are introduced. The principles of different types of recognition and signal transduction are explained, and the merits and pitfalls of these methods are compared. Furthermore, limitations and challenges of existing rapid sensing strategies and perspectives of future research are discussed.

Different Sources of Copper Effect on Intestinal Epithelial Cell: Toxicity, Oxidative Stress, and Metabolism.

Copper (Cu) is widely used in the swine industry to improve the growth performance of pigs. However, high doses of copper will induce cell damage and toxicity. The aim of this study was to evaluate toxicity, bioavailability, and effects on metabolic processes of varying copper sources using porcine intestinal epithelial cells (IPEC-J2) as a model. The IPEC-J2 were treated with two doses (30 and 120 µM) of CuSO4, Cu Glycine (Cu-Gly), and Cu proteinate (Cu-Pro) for 10 h, respectively. Cell damage and cellular copper metabolism were measured by the changes in cell viability, copper uptake, oxidative stress biomarkers, and gene/protein expression levels. The results showed that cell viability and ratio of reduced and oxidized glutathione (GSH/GSSG) decreased significantly in all treatment groups; intracellular copper content increased significantly in all treatment groups; total superoxide dismutase (SOD) activity increased significantly in the 120 µM exposed groups; SOD1 protein expression levels were significantly upregulated in 30 µM Cu-Pro, 120 µM Cu-Gly, and 120 µM Cu-Pro treatment groups; intracellular reactive oxygen species (ROS) generation and malondialdehyde (MDA) content increased significantly in 30 µM treatment groups and 120 µM CuSO4 treatment group. CTR1 and ATP7A gene expression were significantly downregulated in the 120 µM exposed groups. While upregulation of ATOX1 expression was observed in the presence of 120 µM Cu-Gly and Cu-Pro. ASCT2 gene expression was significantly upregulated after 120 µM Cu-Glycine and CuSO4 exposure, and PepT1 gene expression was significantly upregulated after Cu-Pro exposure. In addition, CTR1 protein expression level decreased after 120 µM CuSO4 and Cu-Gly exposure. PepT1 protein expression level was only upregulated after 120 µM Cu-Pro exposure. These findings indicated that extra copper supplementation can induce intestinal epithelial cell injury, and different forms of copper may have differing effects on cell metabolism.

Fluorometric lateral flow immunoassay for simultaneous determination of three mycotoxins (aflatoxin B1, zearalenone and deoxynivalenol) using quantum dot microbeads.

A fluorometric lateral flow immunoassay (LFA) is described for the simultaneous determination of the mycotoxins aflatoxin B1 (AFB1), zearalenone (ZEN) and deoxynivalenol (DON). The method is based on the use of CdSe/SiO2 quantum dot microbeads (QBs) with a mean diameter of 106 nm. These have strong red luminescence (with excitation/emission peaks at 365/622 nm) which results in enhanced sensitivity. The QBs binding with monoclonal antibodies (mAbs) as the signal probes can react specifically with AFB1, ZEN and DON, respectively. There is an inverse correlation between the fluorescence signal intensity of test line and the analyte content, which can realize the quantitative analysis of analytes within 15 min. The limits of detection in solution are 10, 80 and 500 pg mL-1 for AFB1, ZEN and DON, respectively. Besides, the average recoveries from spiked feed range from 85.5 to 119.0%, and the relative standard deviations are less than 16.4% for both intra- and inter-day assays. The method was used to analyze naturally contaminated feedstuff, and this resulted in a good agreement with data obtained by LC-MS/MS. Graphical abstractSchematic representation of a fluorometric method for the simultaneous determination of three mycotoxins. Quantum dot microbeads (QBs) binding with monoclonal antibodies (mAbs) are signal probes. There is an inverse correlation between the fluorescence intensity of test line and the analyte concentration.

Determination of Branched-Chain Keto Acids in Serum and Muscles Using High Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry.

Branched-chain keto acids (BCKAs) are derivatives from the first step in the metabolism of branched-chain amino acids (BCAAs) and can provide important information on animal health and disease. Here, a simple, reliable and effective method was developed for the determination of three BCKAs (α-ketoisocaproate, α-keto-ß-methylvalerate and α-ketoisovalerate) in serum and muscle samples using high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF/MS). The samples were extracted using methanol and separated on a 1.8 µm Eclipse Plus C18 column within 10 min. The mobile phase was 10 mmol L-1 ammonium acetate aqueous solution and acetonitrile. The results showed that recoveries for the three BCKAs ranged from 78.4% to 114.3% with relative standard deviation (RSD) less than 9.7%. The limit of quantitation (LOQ) were 0.06~0.23 µmol L-1 and 0.09~0.27 nmol g-1 for serum and muscle samples, respectively. The proposed method can be applied to the determination of three BCKAs in animal serum and muscle samples.