Improved analysis of 230 pesticide residues in three fermented soy products by using automated one-step accelerated solvent extraction coupled with GC-MS/MS.

Consumer concerns over healthy diets are increasing as a result of the toxicity and persistence of pesticide residues in foodstuffs. Developing sensitive and high-throughput monitoring techniques for these trace residues is seen as an essential step in ensuring food safety. An automatic and sensitive multi-residue analytical method was developed and validated for the simultaneous determination of 230 compounds, including pesticides and their hazardous metabolites, in fermented soy products. The method included preparing the sample using on-line extraction and clean-up system based on accelerated solvent extraction (ASE), then determining the analytes using GC-MS/MS techniques. The homogenized samples (soy sauce, douchi, and sufu) were automatically extracted at 80 °C and 10.3 MPa and at the same time, in situ cleaned by 300 mg of primary secondary amine (PSA) combined with 20 mg of hydroxylated multi-walled carbon nanotubes in an extraction cell. The method obtained excellent calibration linearity (r > 0.9220) and a satisfactory analysis of the targeted compounds, which were evaluated with matrix-matched calibration standards over the range of 5-500 µg L-1. The limit of detections (LODs) of analytes were in the range of 0.01-1.29 µg kg-1, 0.01-1.39 µg kg-1, and 0.01-1.34 µg kg-1 in soy sauce, douchi, and sufu, respectively. The limit of quantifications (LOQs), which defined as the lowest spiking level, were set at 5.0 µg kg-1. The recoveries were within 70-120 % for over 95 % of the analytes, and the relative standard deviations (RSDs) were below 13.6 %. Moreover, a positive detection rate of 47 % were obtained when the proposed method was used on 15 real fermented soy products. These results suggested that the developed high-throughput method is highly feasible for monitoring of these target analytes in trace level.

Genome-Wide Identification and Expression Pattern Profiling of the Aquaporin Gene Family in Papaya (Carica papaya L.).

Aquaporins (AQPs) are mainly responsible for the transportation of water and other small molecules such as CO2 and H2O2, and they perform diverse functions in plant growth, in development, and under stress conditions. They are also active participants in cell signal transduction in plants. However, little is known about AQP diversity, biological functions, and protein characteristics in papaya. To better understand the structure and function of CpAQPs in papaya, a total of 29 CpAQPs were identified and classified into five subfamilies. Analysis of gene structure and conserved motifs revealed that CpAQPs exhibited a degree of conservation, with some differentiation among subfamilies. The predicted interaction network showed that the PIP subfamily had the strongest protein interactions within the subfamily, while the SIP subfamily showed extensive interaction with members of the PIP, TIP, NIP, and XIP subfamilies. Furthermore, the analysis of CpAQPs' promoters revealed a large number of cis-elements participating in light, hormone, and stress responses. CpAQPs exhibited different expression patterns in various tissues and under different stress conditions. Collectively, these results provided a foundation for further functional investigations of CpAQPs in ripening, as well as leaf, flower, fruit, and seed development. They also shed light on the potential roles of CpAQP genes in response to environmental factors, offering valuable insights into their biological functions in papaya.

[Cloning, expression and purification of fructose-2, 6-bisphosphatase gene CpF2KP in papaya].

Papaya, which is mainly cultivated in the southeastern region of China, is one of the four famous fruits in Lingnan. It is favored by people because of its edible and medicinal value. Fructose-6-phosphate, 2-kinase/fructose-2, 6-bisphosphatase (F2KP) is a unique bifunctional enzyme with a kinase domain and an esterase domain that catalyzes the synthesis and degradation of fructose-2, 6-bisphosphate (Fru-2, 6-P2), an important regulator of glucose metabolism in organisms. In order to study the function of the gene CpF2KP encoding the enzyme in papaya, it is particularly important to obtain the target protein. In this study, the coding sequence (CDS) of CpF2KP, with a full-length of 2 274 bp, was got from the papaya genome. The amplified sequence of full-length CDS was cloned into the vector PGEX-4T-1 which was double digested with EcoR I and BamH I. The amplified sequence was constructed into a prokaryotic expression vector by genetic recombination. After exploring the induction conditions, the results of SDS-PAGE showed that the size of the recombinant GST-CpF2KP protein was about 110 kDa. The optimum IPTG concentration and temperature for CpF2KP induction were 0.5 mmol/L and 28 ℃, respectively. The purified sin[A1] gle target protein was obtained after purifying the induced CpF2KP protein. In addition, the expression level of this gene was detected in different tissues, and showed that the gene was expressed at the highest level in seeds and the lowest in pulp. This study provides an important basis for further revealing the function of CpF2KP protein and studying the involved biological processes of this gene in papaya.

Development of a fully automated analytical platform based on static headspace-gas chromatography-tandem mass spectrometry for the analysis of five N-nitrosamines in dried aquatic products of animal origin.

BACKGROUND: The development of rapid and sensitive monitoring methods for trace N-nitrosamines (NAs) in foodstuffs is essential for mitigating the potential health risks to consumers. In the present study, an analytical platform based on one step fully automated static headspace sampling and gas chromatography-tandem mass spectrometry (GC-MS/MS) was developed and validated for the analysis of N-nitrosamines in dried aquatic products of animal origin. The samples and sodium chloride solution mixture were incubated in a heated headspace vial for analyte evaporation, coupled to automatic sampling and online GC-MS/MS analysis. The proposed method requires minimal sample preparation and organic solvent consumption. Five N-nitrosamines including N-nitroso dimethylamine, N-nitroso methyl ethylamine, N-nitroso pyrolidine, N-nitroso piperidine and N-nitroso diphenylamine were selected as model compounds to optimize the significant factors by a using Box-Behnken design. RESULTS: The optimum conditions achieved limits of detections in the range 0.08-0.29 µg kg-1 , with correlation coefficient over 0.998. Relative recoveries in dried aquatic product sample were in the range 76.9-92.4%, with relative SDs of 1.9-7.2%. CONCLUSION: These results confirm the reliability of the developed method for further application in trace level monitoring of the target analytes in foodstuffs. © 2022 Society of Chemical Industry.

Auxin and cytokinin mediated regulation involved in vitro organogenesis of papaya.

In vitro organogenesis is a multistep process which is largely controlled by the balance between auxin and cytokinin. Previous studies revealed a complex network regulating in vitro organogenesis in Arabidopsis thaliana; however, our knowledge of the molecular mechanisms underlying de novo shoot formation in papaya (Carica papaya) remains limited. Here, we optimized multiple factors to achieve an efficient and reproducible protocol for the induction of papaya callus formation and shoot regeneration. Subsequently, we analyzed the dynamic transcriptome profiles of samples undergoing this process, identified 5381, 642, 4047, and 2386 differentially expressed genes (DEGs), including 447, 66, 350, and 263 encoding transcription factors (TFs), in four stage comparisons. The DEGs were mainly involved in phytohormone modulation and transduction processes, particularly for auxin and cytokinin. Of these, 21 and 7 candidate genes involved in the auxin and cytokinin pathways, respectively, had distinct expression patterns throughout in vitro organogenesis. Furthermore, we found two genes encoding key TFs, CpLBD19 and CpESR1, were sharply induced on callus induction medium and shoot induction medium, indicating these two TFs may serve as proxies for callus induction and shoot formation in papaya. We therefore report a regulatory network of auxin and cytokinin signaling in papaya according to the one previously modeled for Arabidopsis. Our comprehensive analyses provide insight into the early molecular regulation of callus initiation and shoot formation in papaya, and are useful for the further identification of the regulators governing in vitro organogenesis.