Affinity-Based Analysis Methods for the Detection of Aminoglycoside Antibiotic Residues in Animal-Derived Foods: A Review.

With the increasingly serious problem of aminoglycoside antibiotic residues, it is imperative to develop rapid, sensitive and efficient detection methods. This article reviews the detection methods of aminoglycoside antibiotics in animal-derived foods, including enzyme-linked immunosorbent assay, fluorescent immunoassay, chemical immunoassay, affinity sensing assay, lateral flow immunochromatography and molecular imprinted immunoassay. After evaluating the performance of these methods, the advantages and disadvantages were analyzed and compared. Furthermore, development prospects and research trends were proposed and summarized. This review can serve as a basis for further research and provide helpful references and new insights for the analysis of aminoglycoside residues. Accordingly, the in-depth investigation and analysis will certainly make great contributions to food safety, public hygiene and human health.

First Report of Leaf Spot Caused by Phyllosticta capitalensis on Alpinia oxyphylla Miq. in China.

Alpinia oxyphylla Miq. is mainly distributed in Hainan, Guangdong and Guangxi provinces of China. Between July and August 2021, a leaf spot disease was observed in Ledong, Hainan Province, China (18°70'20.50″ N, 109°25'25.47″E) on A.oxyphylla. The incidence of infected leaves ranged from 8% to 10%, and the incidence rate of infected plants was about 50%. Symptoms appeared as primary yellow-brown withered spots on the diseased leaves, which further developed into irregular red-brown spots. The center of the lesions was gray-black, and the tissue was irregularly necrotic, ruptured or perforated, and there were yellow chlorotic halos around the edges of the lesions (Figure 1A). Tissues 5 mm in diameter were taken from the junction of diseased and healthy tissue for pathogen isolation, Successively, a total of 8 isolates were obtained from the affected leaves. Three single spore isolates (YZ-HN-001, YZ-HN-043 and YZ-HN-051) were obtained and confirmed to be identical based on morphological characteristics. Therefore, the representative isolate YZ-HN-001 was selected for morphological and molecular identification. On Potato Dextrose Agar(PDA), the colony was gray-white at first and gradually turned dark green to dark brown with lead gray on the back, growth was slow, and mycelium was short and dense (Figure 1B and Figure 1C). Pycnidia were epiphyllous, globose, brown (about 120-140 µm in diameter), and conidia were elliptical, colorless, single celled and smooth (8-12×4-7 µm) (Figure 1D). Molecular identification was performed by partially sequencing the internal transcribed spacer gene (ITS), 18S rRNA gene and the actin gene (ACT) by using the primers ITS1/ITS4 (White et al. 1990), EF4/Fungi5 (Khodaparase et al. 2005) and ACT-512F/ACT-783R (Carbone and Kohn. 1999). The sequences of the amplified fragments were deposited in GenBank, the ITS sequence (ON005130, 616 bp) showed 100% identity with Phyllosticta capitalensis strain CGMCC3.14345 (JN791605.1), the 18S rRNA sequence (ON005129, 541 bp) showed 99% identity with P. capitalensis isolate MUCC0029 (AB454185.1) and the ACT sequence (ON049348, 251 bp) showed 100% identity with P. capitalensis strain DZSN202005-2 (MW533248.1). A phylogenetic analysis was conducted in MEGA X using the neighbor-joining method and showed that isolate YZ-HN-001 clustered together with P. capitalensis (Figure 2). Based on the above morphological and molecular characteristics, the isolate was determined to be P. capitalensis. Pathogenicity tests were conducted in three replicates by inoculating surface-sterilized leaves of A. oxyphylla. The leaves were wounded and inoculated with colonized PDA plugs (5×5 mm) from 15-day-old cultures. Control leaves wounded in the same way and were inoculated with sterile PDA plugs (5×5 mm). Leaves were moisturized by spraying with sterile water every three days. After 20 days at room temperature (23 to 28℃), similar symptoms were observed in the inoculated leaves as in the field (Figure 1E), but no symptoms were observed on the control leaves (Figure 1F). The same P. capitalensis was reisolated in the inoculated leaves, confirming Koch's postulates. Phyllosticta capitalensis has been reported to cause leaf spots or black spots on various host plants around the world (Wikee et al. 2013), including on oil palm (Nasehi et al. 2020), tea plant (Cheng et al. 2019 ), and castor (Tang et al. 2020). Nevertheless, to our knowledge, this is the first report of leaf spot caused by P. capitalensis on A. oxyphylla worldwide.

Selective solid-phase extraction of four phenylarsonic compounds from feeds, edible chicken and pork with tailoring imprinted polymer.

The novel molecularly imprinted microspheres for four phenylarsonic compounds have been firstly prepared with the reversible addition-fragmentation chain transfer polymerization in a suspension system. The resulting polymeric microspheres were characterized by infrared spectrum, scanning electron microscope and differential scanning calorimetry. With serial adsorption experiments, the polymeric microspheres showed highly specific molecular recognition, fast mass transfer rate and robust adsorption of the substrates. Then, the imprinted polymer was used as the solid-phase extraction adsorbent to extract the phenylarsonic compounds from the feeds, edible chicken and pork. The cartridge was washed with 2 mL ethyl acetate and eluted with 3 mL of methanol- acetic acid (90:10, v/v). The recoveries of the molecularly imprinted solid-phase extraction (MISPE) column ranged from 83.4% to 95.1%. This work provided a versatile approach for the specific extraction of the organoarsenic compounds from complicated matrices and exhibited a bright future for the application of MISPE column.

Strain improvement of Trichoderma viride for increased cellulase production by irradiation of electron and (12)C(6+)-ion beams.

OBJECTIVES: To improve cellulase production and activity, Trichoderma viride GSICC 62010 was subjected to mutation involving irradiation with an electron beam and subsequently with a (12)C(6+)-ion beam. RESULTS: Mutant CIT 626 was the most promising cellulase producer after preliminary and secondary screening. Soluble protein production and cellulase activities were increased mutifold. The optimum temperature, pH and culture time for the maximum cellulase production of the selected mutant were 35 °C, pH 5 and 6 days. The highest cellulase production was obtained using wheat bran. The prepared cellulases from T. viride CIT 626 had twice the hydrolytic performance with sawdust (83 %) than that from the parent strain (42.5 %). Furthermore, molecular studies demonstrated that there were some key mutation sites suggesting that some amino acid changes in the protein caused by base mutations had led to the enhanced cellulase production and activity. CONCLUSIONS: Mutagenesis with electron and (12)C(6+)-ion beams could be developed as an effective tool for improvement of cellulase producing strains.

Molecularly imprinted polymer for specific extraction of hypericin from Hypericum perforatum L. herbal extract.

The molecularly imprinted polymers (MIPs) were prepared by an oxidation-reduction polymerization system using a non-covalent molecularly imprinting strategy with hypericin as the template, acrylamide as the functional monomer and pentaerythritol triacrylate as the cross-linker in the porogen of acetone. The UV spectrum revealed that a cooperative hydrogen-bonding complex between hypericin and acrylamide might be formed at the ratio of 1:6 in the prepolymerized system. Two classes of the binding sites were produced in the resulting hypericin-imprinted polymer with the dissociation constants of 16.61µgL(-1) and 69.35µgL(-1), and the affinity binding sites of 456.53µgg(-1) and 603.06µgg(-1), respectively. The synthesized MIPs were characterized by scanning electron microscope, thermogravimetric and differential thermal analysis. High-performance liquid chromatography was used to investigate the adsorption and recognition properties of the MIPs. Selective binding of the template molecule was demonstrated in comparison to the analog pseudohypericin. After the Hypericum perforatum L. plant being air dried and finely ground, an extract was prepared by shaking the powder in a methanol-water solution (80:20, v/v), vacuum filtration though a Büchner funnel, liquid-liquid extraction with ethyl ether and ethyl acetate, and evaporating on a rotary evaporator until dry. With the sorbents of the optimized MIPs, a molecularly imprinted solid-phase extraction (MISPE) procedure was developed for enrichment and separation of hypericin from the Hypericum extract in the presence of interfering substances. The selective extraction of hypericin from herbal medicine was achieved with the recovery of 82.30%. The results showed that MISPE can be a useful tool for specific isolation and effective clean-up of target compounds from natural products.

Molecularly imprinted monolith coupled on-line with high performance liquid chromatography for simultaneous quantitative determination of cyromazine and melamine.

We report a novel method for simultaneous determination of cyromazine and melamine based on a molecularly imprinted monolith on-line coupled with high performance liquid chromatography (HPLC). The imprinted monolith was prepared by in situ polymerization using 2,4-diamino-6-undecyl-1,3,5-triazine (DAUTA) as a mimic template. Due to the better solubility of DAUTA in chloroform, hydrogen bonds were effectively developed between the template and the functional monomer and resulted in the formation of highly specific cavities in the obtained imprinted monolith. With methanol as the loading solvent, cyromazine and melamine were both selectively retained by the obtained imprinted monolith, while the nonspecific adsorption on the non-imprinted monolith was negligible. The imprinted monolithic column was on-line coupled with HPLC for purification and concentration of the two analytes from milk samples. To minimize the peak broadening during the on-line transfer of the analytes from the imprinted monolith to the following analytical column, a successive desorption program was developed for the elution step, which enabled on-line stacking of the target compounds before being analyzed by HPLC. Low detection limits of 0.12 µg mL(-1) for melamine and 0.05 µg mL(-1) for cyromazine were achieved with only 0.3 mL of milk sample and a low sensitivity HPLC-UVD instrument. The method may be further extended to detect other analytes of interest in a large variety of samples.