Heterologous expression and enzymatic characteristics of sulfatase from Lactobacillus plantarum dy-1.

Barley, rich in bioactive components including dietary fiber, polyphenolic compounds and functional proteins, exhibits health benefits such as regulating glucose and lipid metabolism. Previous studies have found that the content and composition of free phenolic acids in barley may be significantly changed by fermentation with the laboratory patented strain Lactobacillus plantarum dy-1 (L. p dy-1), but the mechanism of enzymatic release of phenolic acid remains to be elucidated. Based on this, this study aimed to identify the key enzyme in L. p dy-1 responsible for releasing the bound phenolic acid and to further analyze its enzymatic properties. The Carbohydrate-Active enZYmes database revealed that L. p dy-1 encodes 7 types of auxiliary enzymes, among which we have identified a membrane sulfatase. The enzyme gene LPMS05445 was heterologous to that expressed in E. coli, and a recombinant strain was induced to produce the target protein and purified. The molecular weight of the purified enzyme was about 59.9 kDa, with 578.21 U mg-1 enzyme activity. The optimal temperature and pH for LPMS05445 expression were 40 °C and 7.0, respectively. Furthermore, enzymatic hydrolysis by LPMS05445 can obviously change the surface microstructure of dietary fiber from barley bran and enhance the release of bound phenolic acid, thereby increasing the free phenolic acid content and improving its physiological function. In conclusion, sulfatase produced by Lactobacillus plantarum dy-1 plays a key role in releasing bound phenolic acids during the fermentation of barley.

Effect of structural stability of lipase in acetonitrile on its catalytic activity in EGCG esterification reaction: FTIR and MD simulation.

In this study, (-)-Epigallocatechin-3-O-gallate (EGCG) esterification reaction was catalyzed by Novozym 435, Lipozyme RM, Lipozyme TLIM, and lipase Amano 30SD in acetonitrile. Fourier transform infrared spectroscopy (FTIR) and molecular dynamic (MD) simulations were used to analyze the structural stability of different lipases in acetonitrile and their effect on EGCG esterification reaction. The results showed that conversion rate of EGCG catalyzed by Lipozyme RM was the highest, followed by Lipozyme TLIM. FTIR indicated that the secondary structure of Lipozyme RM was the most stable. MD simulations suggested that whole structural stability of Lipozyme RM in acetonitrile was superior to Novozym 435 and lipase Amano 30SD and similar to Lipozyme TLIM due to their similar conformation, while the active site of Lipozyme RM is more flexible than that of Lipozyme TLIM, which indicated that lipase with stable whole structure and flexible active site may be more conducive to the esterification of EGCG in acetonitrile. This study provided a direction for rapidly screening lipase to synthetize EGCG or other polyphenols esterified derivatives.

Responses of bitter melon saponins to oxidative stress and aging via the IIS pathway linked with sir-2.1 and hlh-30.

Saponins from bitter melon (BMS) exert potential bioactivities and pharmacological activities, including anti-oxidation and lifespan extension. However, the exact mechanisms of BMS in response to oxidative stress remain unknown. Results demonstrated that bitter melon saponins could strengthen locomotive activities (body bend and head thrashing) accompanied by delaying the muscle fiber damage with age in Caenorhabditis elegans. In addition, BMS inhibited the ROS accumulation, improved the activities of antioxidant enzymes like SOD (by 57.90% and 94.34% for 100 µg/ml and 200 µg/ml BMS, respectively) and CAT (by 51.45% and 56.91% for 100 µg/ml and 200 µg/ml BMS, respectively), and extend the lifespan of N2 and CL2006 worms under paraquat-induced oxidative stress. Mechanism study suggested that BMS modulated the mRNA expressions of oxidation-related regulators, like the upregulation of cat-1, hsf-1, sir-2.1, and hlh-30. Furthermore, gene-deficient mutants verified that IIS (insulin/insulin-like growth factor-1 signaling) pathway linked with sir-2.1 and hlh-30 factors were involved in the BMS's lifespan-extension effects under oxidative stress. In general, this study supplemented the explanation of BMS in promoting oxidation-resistance and lifespan-extension activities, which could be served as a potential candidate for anti-aging. PRACTICAL APPLICATIONS: Our previous studies have suggested that saponins from bitter melon exhibited fat-lowering activity in C. elegans. However, little was known about the mechanism underlying the anti-oxidation effects of BMS in C. elegans. Current results indicated that the IIS pathway linked with sir-2.1 and hlh-30 transcriptional factors jointly to increase the lifespan in BMS' responses to oxidative stress. Our findings are beneficial to understand the main nutritional ingredients in bitter melon, which are ideal and expected in functional foods for aging.

Rapid analysis of triazine herbicides in fruit juices using evaporation-assisted dispersive liquid-liquid microextraction with solidification of floating organic droplets and HPLC-DAD.

A rapid and convenient analytical procedure (evaporation-assisted dispersive liquid-liquid microextraction with solidification of floating organic droplets) is advanced for determining the concentrations of triazine herbicide residues (e.g. simazine and atrazine) in fruit juices via HPLC-DAD. The technique involves adding 1-dodecanol (low density) and dichloromethane (high density) to the test solution to act as the extraction and volatile solvents, respectively. Calcium oxide is added to generate heat to accelerate the evaporation of dichloromethane, whereupon the 1-dodecanol quickly disperses into small droplets to complete the microextraction process. Thus, there is no need to use a dispersive solvent and heating equipment is also not required. The floating 1-dodecanol is subsequently frozen using an ice bath to facilitate its separation from the sample. Under optimal conditions (250 µL of 1-dodecanol (extraction solvent), 150 µL of CH2Cl2 (volatile solvent), 1250 mg of CaO, and an extraction time of 60 s) the detection procedure is linear over the range 0.05-5 µg mL-1 (with R > 0.99). The limits of detection (LOD) and quantification (LOQ) were determined to be 0.0022-0.0034 µg mL-1 and 0.0073-0.0113 µg mL-1, respectively. The recovery of simazine and atrazine in three fruit juices ranged between 78.5% and 96.4% with a relative standard deviation <8.2%. Therefore, the proposed approach can be effectively adopted to analyze the triazine herbicide content in fruit juices. The method has been proved to be simple, reliable, and remarkably efficient.

Popping candy-assisted dispersive liquid-liquid microextraction for enantioselective determination of prothioconazole and its chiral metabolite in water, beer, Baijiu, and vinegar samples by HPLC.

To simultaneously determine the enantiomers of prothioconazole and its chiral metabolite prothioconazole-desthio in water, beer, Baijiu, and vinegar samples by HPLC, a simple, fast, environmentally-friendly popping candy-assisted dispersive liquid-liquid microextraction technique was developed. A green medium-chain fatty acid (decanoic acid) and popping candy could be used as the extractant and solid dispersant respectively to avoid the use of toxic organic solvents. Decanoic acid was collected after extraction by solidification at room temperature. The linear range of this technique was from 27.1 to 1000 µg L-1. The limits of detection and quantification were within the ranges of 8.1-11.2 µg L-1 and 27.1-37.3 µg L-1, respectively. The extraction recovery was 80.8% to 102.5% with the relative standard deviation ranged from 1.1 to 7.1%. This technique has been successfully applied to enantioselectively determine the residues of prothioconazole and prothioconazole-desthio in water, beer, Baijiu, and vinegar samples.

Analysis of pyrethroids in cereals by HPLC with a deep eutectic solvent-based dispersive liquid-liquid microextraction with solidification of floating organic droplets.

This work presents a novel and green analytical procedure involving a deep eutectic solvent-based dispersive liquid-liquid microextraction with solidification of floating organic droplets (DES-DLLME-SFOD) followed by HPLC to measure three pyrethroids (bifenthrin, ß-cypermethrin, and deltamethrin) in cereal samples. Firstly, a low-density hydrophobic DES was synthesized from thymol and octanoic acid in the molar ratio of 1/4 and this was applied as a green extraction solvent in the DLLME procedure to avoid the use of a toxic extractant. After centrifugation and placing it on an ice bath, it is transformed into a solid phase on the top of the sample solution to reduce the loss of extractant, conducive to convenient collection thereafter. This procedure required the optimal conditions (including the type, proportion, and amount of DES as the extractant, the volume of the dispersant acetonitrile, the amount of salt, and the pH value) to be evaluated. Under optimized variates, the proposed method provided good linearity with a correlation coefficient greater than 0.997 and limits of quantification within the range of 6.6-8.9 µg kg-1. The recoveries of pyrethroids in corn, wheat, barley, and oats were 75.6-87.2%, and the relative standard deviation was less than 3.6%. The method, therefore, offers a green, efficient, and convenient approach for the determination of pesticides in cereals.

Dispersive liquid-liquid microextraction based on the solidification of floating organic droplets for HPLC determination of three strobilurin fungicides in cereals.

In this paper, a dispersive liquid-liquid microextraction method based on the solidification of floating organic droplets, combined with high-performance liquid chromatography (DLLME-SFOD-HPLC), was developed for the detection of strobilurin fungicides (azoxystrobin, pyraclostrobin, and trifloxystrobin) in cereals. Natural fatty acids were used as an extractant and have low toxicity, density, and freezing point. The extractant nonanoic acid was evenly dispersed as droplets in sample solution and was then solidified in the upper layer of sample solution after centrifugation and ice bath, which improved the extraction and collection efficiency. The dispersive liquid-liquid microextraction procedure was optimised by univariate analysis and the Box-Behnken response surface methodology. Optimum conditions were as follows: the volume of nonanoic acid was 82 µL, the volume of acetonitrile was 620 µL, and the amount of salt was 256 mg. Under optimised conditions, the method had good linearity with a correlation coefficient higher than 0.997, and the limit of detection was 2.57-4.87 µg kg-1. The recoveries of azoxystrobin, pyraclostrobin, and trifloxystrobin in rice, corn, and wheat were 82.0%-93.2%, and the relative standard deviations were 1.6%-7.4%. Therefore, the method was successfully applied to detect target fungicides in cereals.