Yuan-Ming Sun

Raffino-oligosaccharide (ROS), the smallest oligosaccharide of the raffinose family, is a novel food ingredient. However, the anti-constipation effects of ROS remain obscure. This study investigates the anti-constipation effects of ROS based on the loperamide-induced mice model and reveals the underlying mechanism using constipation parameters, neurotransmitter level, 16S rRNA sequencing, and the targeted screening strategy. The prevention effects were firstly investigated by the gastro-intestinal transit rate experiment (50 mice) and defecation status experiment (50 mice), which were divided into five groups (n = 10/group): blank, model, and low-, medium- and high-dose ROS. Furthermore, the slow-transit constipation experiment (blank, model, and high-dose ROS, n = 10/group) was conducted to illustrate the underlying mechanism. The results showed that ROS aided in preventing the occurrence of constipation by improving the gastro-intestinal transit rate and the defecation frequency in mice, and ROS significantly reduced the serum levels of vasoactive intestinal peptide (VIP). In addition, ROS regulated the diversity and structure of intestinal flora. Among them, one specific family and six specific genera were significantly regulated in constipated mice. The targeted screening revealed that 29 targets related to the anti-constipation effects of ROS, indicating ROS may play a role by regulating multiple targets. Furthermore, the network pharmacology analysis showed that Akt1, Stat3, Mapk8, Hsp90aa1, Cat, Alb, Icam1, Sod2, and Gsk3b can be regarded as the core anti-constipation targets. In conclusion, ROS could effectively relieve constipation, possibly by inhibiting the level of neurotransmitters and regulating the gut flora in mice. This study also provides a novel network pharmacology-based targeted screening strategy to reveal the anti-constipation effects of ROS.

Dexamethasone (DEX) is widely used because of its anti-inflammatory, anti-endotoxin, anti-shock, and stress-enhancing response activities. It can increase the risk of diabetes and hypertension if it is abused or used improperly. However, there is a lack of sensitive and rapid screening methods for DEX in food. In this study, a time-resolved fluorescent microspheres immunochromatographic assay (TRFM-ICA) integrated with a portable fluorescence reader was developed for the quantitative detection of DEX in milk and pork. The cut-off values of the TRFM-ICA were 0.25 ng/mL and 0.7 µg/kg, respectively. The limits of quantitation (LOQs) were 0.003 ng/mL and 0.062 µg/kg, respectively. The recovery rates were 80.0–106.7%, and 78.6–83.6%, respectively, with the coefficients of variation ranging 6.3–12.5%, and 7.5–10.3%, respectively. A parallel experiment for 20 milk and 10 pork samples with LC-MS/MS was carried out to confirm the performance of the on-site application of the developed TRFM-ICA. The results of the two methods are basically the same; the correlation (R²) was >0.98. The establishment of TRFM-ICA will provide a new sensitive and efficient technical support for the rapid screening of DEX in food.

Enrofloxacin (ENR) is a widely used fluoroquinolone (FQ) antibiotic for antibacterial treatment of edible animal. In this study, a rapid and highly specific fluorescence polarization immunoassay (FPIA) was developed for monitoring ENR residues in animal foods. First, ENR was covalently coupled to bovine serum albumin (BSA) to produce specific polyclonal antibodies (pAbs). Three fluorescein-labeled ENR tracers (A, B, and C) with different spacers were synthesized and compared to obtain higher sensitivity. Tracer C with the longest arm showed the best sensitivity among the three tracers. The developed FPIA method showed an IC₅₀ (50% inhibitory concentration) of 21.49 ng·mL⁻¹ with a dynamic working range (IC₂₀–IC₈₀) of 4.30–107.46 ng·mL⁻¹ and a limit of detection (LOD, IC₁₀) of 1.68 ng·mL⁻¹. The cross-reactivity (CR) of several structurally related compounds was less than 2%. The recoveries of spiked pork liver and chicken samples varied from 91.3% to 112.9%, and the average coefficients of variation were less than 3.83% and 5.13%, respectively. The immunoassay took only 8 min excluding sample pretreatment. This indicated that the established method had high sensitivity, specificity, and the advantages of simplicity. Therefore, the proposed FPIA provided a useful screening method for the rapid detection of ENR residues in pork liver and chicken.

Immunoassay for pesticides is an emerging analytical method since it is rapid, efficient, sensitive, and inexpensive. In this study, a recombinant antigen-binding fragment (Fab) against a broad set of O,O-diethyl organophosphorus pesticides (DOPs) was produced and characterized. The κ chain and Fd fragment were amplified via PCR and inserted into the vector pComb3XSS and the soluble Fab on phagemid pComb3XSS was induced by isopropyl β-d-thiogalactoside in E. coli TOP 10F’. SDS-PAGE, Western blotting, and indirect competitive ELISA results indicated that Fab maintained the good characteristics of the parental mAb. To better understand antibody recognition, the three-dimensional (3D) model of Fab was built via homologous modeling and the interaction between Fab and DOPs was studied via molecular docking and dynamics simulations. The model clearly explained the interaction manner of Fab and DOPs, and showed that the Arg-L96 and Arg-H52 were mainly responsible for antibody binding. This work provided a foundation for further mutagenesis of Fab to improve its characteristics.

Histamine (HA) is an essential test item for fishery samples. However, the fast and effective determination of HA is difficult due to its simple structure and small molecular size. In this study, a sensitive and specific monoclonal antibody against p-nitrobenzoylated histamine (NPHA), which can be easily obtained from the reaction of HA and p-nitrobenzoic acid N-hydroxysuccinimide ester (PNBA-OSu) under mild conditions, was generated for the first time. Based on this mAb, an immunochromato-graphic assay strip (ICA strip) using a colloidal gold nano particles-antibody (GNPs) probe for rapid detection of HA in saury samples was established. After screening the coating antigens and optimization of analytical parameters, a heterologous coating based ICA strip exhibited the most excellent detection ability with a visual detection limit (VDL) of 6.0 mg kg⁻¹ in qualitative experiments and a detection limit (by a strip reader) of 1.0 mg kg⁻¹ in semi-quantitative experiments for saury samples, with no cross-reactivity with HA analogs. Good correlation between the ICA strip with liquid chromatography-tandem mass spectrometry was achieved for spiked and naturally contaminated saury samples. Overall, this method is suitable for screening of HA residue for large scale fish samples in a quick, simple and low-cost manner.

Novel haptens were designed and synthesized to prepare antibodies against free histamine, but none resulted in producing suitable antibodies for developing an enzyme-linked immunosorbent assay (ELISA). However, an antiserum was obtained having high specificity and affinity to p-nitrobenzoylated histamine (NPHA), which can be easily formed from reaction between histamine and p-nitrobenzoic acid N-hydroxysuccinimide ester (PNBA-OSu) under mild conditions. Based on rabbit polyclonal antibodies, a competitive indirect ELISA (ciELISA) for histamine determination in foods was developed. After ciELISA and derivatization optimization, the assay showed good sensitivity, with limits of detection of 1.8 mg/kg, 93.6 μg/L, and 93.6 μg/kg in fish, red wine, and yoghurt, respectively, with negligible cross-reactivity with related biogenic amines and amino acids. Average recovery of histamine in fortified food samples ranged from 80.9% to 110.1% with coefficients of variation below 16.3%. Good correlation between the ciELISA and liquid chromatography–tandem mass spectrometry was obtained for spiked food samples.