To establish a co-expression system combining episomal and integrative vectors, enhance the expression efficiency of antimicrobial peptides in Pichia pastoris, and address the issue of insufficient production caused by copy number limitations and expression instability.

The 2 kanamycin-resistant episomal vectors, pPICKαA-panARS-JS21T and pGAPKαA-panARS-JS21T, were constructed using the Kluyveromyces lactis panARS autonomously replicating sequence. These were co-electroporated with the integrative vector pPICZαA-JS21T into Pichia pastoris X-33. Positive transformants were selected on Zeocin plus G418 double-antibiotic plates. Shake-flask cultures were run for more than 120 h with methanol supplementation every 24 h to maintain induction. Secreted total protein was quantified by the Bradford assay, target bands were visualized by trimethylglycine-sodium dodecyl sulfate polyacrylamide gel electrophoresis (Tricine-SDS-PAGE), and antimicrobial activity against Staphylococcus aureus was measured by agar diffusion.

After 120 h of fermentation, the integrative plus episomal pGAPKαA-panARS-JS21T co-expression group yielded 107.22 µg/mL of extracellular total protein, 52% higher than 70.56 µg/mL of the integrative-only control. The corresponding inhibition-zone assay showed a 129% increase in antimicrobial activity (P<0.05). The integrative plus pPICKαA-panARS-JS21T group also achieved a 15.8% improvement over the integrative-only group.

A combined episomal/integrative co-expression strategy synergistically elevates JS21T antimicrobial peptide levels in Pichia pastoris, outperforming single-vector systems and offering a new route for high-level production of small peptide molecules.

To analyze the effects of different termination methods on the yield of theaflavins.

Theaflavins were prepared through the enzymatic action of potato polyphenol oxidase (PPO). A high performance liquid chromatography (HPLC) analysis was conducted to detect theaflavin compounds. The effects of 6 kinds of different termination methods on theaflavin yield were then analyzed.

Following treatment with microwave medium power, microwave high power, a 100 °C water bath and 3 kinds of inhibitors, the concentration of theaflavins was measured. The results of the HPLC analysis indicated that the 100 °C water bath treatment resulted in the highest theaflavin content, with theaflavin-3,3’-gallate (TFDG) reaching 32.47 µg/mL and theaflavin substances at 119.12 µg/mL.

The concentration of theaflavins is highest after treatment in a 100 °C water bath.The research findings may provide valuable reference for rapid detection and industrial-scale production of theaflavins.

To develop a meat component detection method based on handheld quantitative real-time polymerase chain reaction (PCR) for rapid on-site identification of meat sources.

This study focused on the pork, chicken and duck-derived components, explored the influence of the single factors of meat sample pretreatment tools, lysate composition, lysate concentration and lysate addition amount on the extracted DNA, and constructed a rapid pretreatment method for meat samples; screened specific primers and probes, used the handheld quantitative teal-time PCR instrument to verify specificity, sensitivity and limit of detection, and drew amplification standard curves; and detected the real single and mixed meat samples.

The established rapid pretreatment method for meat samples could be completed within 5 min, the DNA extraction took 10 min, and the extracted DNA could meet the requirements of subsequent experiments. The detection methods exhibited high specificity for pork,chicken and duck samples. The limits of detection of pork, chicken and duck-derived DNA components were 0.010,0.001 and 0.001 ng/µL. The standard curve showed a sound linear relationship. The amplification standard curve of pig DNA was: Y=–3.956X+38.803, r²=0.9956. The amplification standard curve of chicken DNA was: Y=–3.499X+40.491,r²=0.9909. The amplification standard curve of duck DNA was: Y=–3.961X+39.192, r²=0.9962. The method was successfully applied to detect 28 meat samples with a 100% success rate.

These results indicate that the rapid meat sample pretreatment method established in this experiment can be used for the rapid and accurate detection of pork, chicken and duck meat on a handheld quantitative real-time PCR all-in-one machine. This approach provides robust technical support for monitoring and controlling meat adulteration at the retail or market level.

To establish a method for rapid detection of iron content in tap water and food samples by paper-based microfluidic chip.

The paper-based chips were cut out using an embossing gadget. And the paper chip was prepared by soaking. Samples to be tested could be dropped onto the prepared chips directly. When the chip showed a stable color based on O-dinitrogen colorimetric method, the chip could be placed on a self-made platform for taking photo. Then the gray value of the picture could be read and the iron concentration could be acquired. The study selected the type of filter paper, and optimized the concentrations of 1,10-phenanthroline and hydroxylamine hydrochloride during soaking, as well as the soaking time and coloring time.

Whatman 3#filter paper was selected to prepare the paper chip. And the cut paper chip was soaked in acetate buffer solution containing 8.00 g/L of 1,10-phenoroline and 50.00 g/L of hydroxylamine hydrochloride (pH=4.50) for 3 minutes.After the sample was dropped onto the paper chip for 15 minutes, photos could be taken for detection. When the concentration of Fe³⁺ in sample solution was 5.00×10^–3 mmol/L to 7.00 mmol/L, it had a good linear relationship between the gray value and the concentration. The limit of detection the paper chip method was 0.004 mmol/L. When the 13 kinds of interfering metal ions, including 1 mg/L Cd²⁺, Cu²⁺ and the like, were coexisting in the sample solution, they did not affect the detection of Fe³⁺. The adding standard recoveries were 86.0% to 114.7%. The relative standard deviations of the same sample 6 test results within the same day were 1.7% to 7.5%. And the relative standard deviations of 6 consecutive days detection results were 3.4% to 7.9%. The prepared paper chip could be stored for at least for 3 months under the condition of avoiding light at 4 ℃. When the actual samples were detected,the relative deviation between the paper chip method and the flame atomic absorption method was less than or equal to 2.404%.

The iron detection method based on paper-based chips established in this research is simple to operate, accurate and reliable, which can be a strong support for on-site rapid detection of iron content in food detection field.

To establish a rapid detection method suitable for the determination of chlorpyrifos residues in vegetables based on the colloidal gold immunochromatographic technology.

The pretreatment conditions such as extraction solvent, extraction time and detection temperature were systematically optimized to determine the optimal rapid detection parameters. Additionally, the performance indicators of this rapid detection method, including false negative rate, false positive rate and relative accuracy, were confirmed.

Using 8.0 mL of 0.1 mol/L phosphate buffer saline solution as the sample extraction solvent, setting the extraction time to 3 min, and controlling the detection temperature within the range of 15–35 ℃ were determined as the optimal parameters for rapid detection. The experimental results showed that the false negative rate and false positive rate of this method were both 0%, with a relative accuracy of 100%; the rapid detection results of 20 vegetable samples were consistent with those obtained by the liquid chromatography-tandem mass spectrometry method.

This method demonstrates excellent detection performance and is suitable for on-site rapid detection and large-scale screening of chlorpyrifos residues in vegetables.

To establish a method for quantitative determination of 2 kinds of novel tadalafil derivatives in blended wine, coffee and table candy by ultra performance liquid chromatography-tandem mass spectrometry.

Waters HSS T₃ (2.1 mm×100 mm, 1.8 μm) reversed-phase chromatographic column was used for gradient elution with 0.1% formic acid water and acetonitrile as the mobile phase, and was detected by mass spectrometry with multiple reaction monitoring.

In the mass concentration range of 1.0–20.0 ng/mL, there was a good nonlinear relationship between 2 kinds of new tadalafil derivatives, diethylaminopretadalafil and dipropylaminopretadalafil (r²>0.99); the limits of detection of the method were all 0.05 mg/kg and the limits of quantitation were all 0.10 mg/kg. The average recovery rate range at the 3 levels of 1, 2 and 10 times limits of quantitation were 82.5% to 95.6%, and the relative standard deviation were 1.9%–7.8% (n=6). The method had good accuracy.

This method is simple, accurate and sensitive, and can be used for the accurate and rapid determination of the content of diethylaminopretadalafil and dipropylaminopretadalafil in blended wine, coffee and table candy.