Detection of Tyrosinase Activity and Inhibitor Validation Based on N-GQDs Fluorescence Sensor.

Tyrosinase inhibitors have the ability to resist melanin formation and can be used for clinical and cosmetic, so it is becoming extremely crucial to search a rapid and effective method for detecting t the activity of tyrosinase. In this study, a sensing probe based on Nitrogen-doped graphene quantum dots (N-GQDs) were prepared with carbamide and citric acid. Tyrosinase can oxidize dopamine to dopamine quinone, which can quench the fluorescence of N-GQDs based on the principle of fluorescence resonance energy transfer (FRET) process, and then the detection of tyrosinase activity can be achieved. The result demonstrated that the fluorescence intensity of N-GQDs was a linear correlation with the activity of tyrosinase. Wide detection linear ranges between 0.05 and 5 U/mL and high selectivity. The detection range of tyrosinase was 0.05 to 5 U/mL and LOD of 0.005 U/mL. According to the above, the fluorescence method established in this work could be successfully used for the trace analysis of tyrosinase and it was verified that KA is an inhibitor of tyrosinase.

Synergistic antimicrobial effect of nisin-octanoic acid nanoemulsions against E. coli and S. aureus.

Food safety is a major public health concern all over the world. Therefore, the prevention of food contamination is becoming extremely crucial. In this study, an antimicrobial nanoemulsion composed of water-soluble nisin and fat-soluble octanoic acid was successfully prepared. The results showed that the average particle size and the polymer dispersity index of the nisin-octanoic acid (NOA) nanoemulsion were around 52.21 nm and 0.253, respectively. The NOA nanoemulsion required less amounts of nisin and octanoic acid to achieve the effective antimicrobial effect against Escherichia coli and Staphylococcus aureus. In addition, the growth curves of E. coli and S. aureus were determined. The OD600 of NOA nanoemulsion was significantly lower than free nisin after being incubated for 24 h (p < 0.001), indicating that the antimicrobial effect of NOA nanoemulsion was outstanding. Meanwhile, the synergistic antimicrobial property of NOA nanoemulsion against E. coli and S. aureus was significantly better than free nisin under nonacid conditions (p < 0.05). Overall, the results of this study may provide guidance for the further application of nisin in more forms.

Synergistic antimicrobial system based on nisin and α-hydroxy organic acids.

Synergistic antimicrobial is a promising way to overcome microbial contamination in food and drugs. In the study, the synergistic effect between nisin and α-hydroxy organic acids on E. coli and S. aureus was investigated. The experimental results showed that the combined antibacterial ability of nisin-citric acid system was the most prominent. The FCI index also indicated that the combination of nisin and citric acid had synergistic effects on E. coli. When nisin was combined with citric acid, the inhibition rates of E. coli and S. aureus were increased to 4.43 and 1.49 times, respectively. Nisin-citric acid complex system could effectively slow down the proliferation of S. aureus and E. coli at lower concentrations, and can quickly destroy the cell membrane after 4 h of action. Therefore, the combination of nisin and citric acid is expected to be a potential solution for food and drug preservation.

Immobilization of lipase on silica nanoparticles by adsorption followed by glutaraldehyde cross-linking.

In this study, Candida antarctica lipase B was immobilized on silica (SiO2) nanoparticles by physical adsorption, and then cross-linked with glutaraldehyde (GA) to prepare cross-linked immobilized lipase (CLIL). During the condition of 1.28 mg/mL lipase concentration, 25 â„ƒ temperature, 2 h adsorption time, 0.01% GA (V/V) 7.5 mL and 2 h cross-linking time, the highest recovery activity of CLIL reached 87.82 ± 0.07% (22.55 ± 0.025 U/mg). Scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM) confirmed that lipase was immobilized on the surface of SiO2 nanoparticles. The changes in secondary structures of CLIL indicated that cross-linking changed the secondary structure of lipase protein, which made the structure of CLIL more stable. Compared with the free lipase, the thermal stability and storage stability of CLIL was significantly improved, and the t1/2 at 60 °C was extended. Studies had shown that it was a feasible method to obtain CLIL by cross-linking after adsorbing lipase on SiO2 nanoparticles.

Enantioseparation of N-methyl duloxetine, duloxetine, and fluoxetine by countercurrent chromatography using anionic ß-cyclodextrin as chiral selector.

Two anionic ß-cyclodextrins as chiral selectors were successfully applied in the enantioseparation of N-methyl duloxetine, duloxetine, and fluoxetine by countercurrent chromatography. Sulfobutyl ether-ß-cyclodextrin and carboxymethyl-ß-cyclodextrin showed opposite enantioselectivity for both duloxetine and N-methyl duloxetine enantiomers. Two biphasic solvent systems, n-hexane: 0.1 mol/L phosphate buffer pH 7.6 with 50 mmol/L of sulfobutyl ether-ß-cyclodextrin (1:1, v/v) and n-hexane: 0.1 mol/L phosphate buffer pH 7.2 with 50 mmol/L of carboxymethyl-ß-cyclodextrin (1:1, v/v), were selected for N-methyl duloxetine. Enantioseparation of duloxetine was achieved by recycling countercurrent chromatography using a solvent system composed of n-butyl acetate: 0.1 mol/L phosphate buffer pH 7.2 with 20 mmol/L of sulfobutyl ether-ß-cyclodextrin or carboxymethyl-ß-cyclodextrin (1:1, v/v). A solvent system composed of n-hexane: n-butyl acetate: 0.1 mol/L phosphate buffer pH 7.6 containing 20 mmol/L of sulfobutyl ether-ß-cyclodextrin (6:4:10, v/v) was selected for enantioseparation of fluoxetine.

[In vitro screening of natural compounds with antitumor activity by trypsin].

In order to investigate the feasibility of in vitro screening the antitumor activity of natural compounds by trypsin, porcine trypsin was used to for screening test, which is marked by inhibition of enzyme activity. Four compounds, namely daidzin, genistin, matrine and oxymatrine, were selected as test subjects. The natural antitumor drug camptothecin was used as the control. The inhibitory effect was detected by two experimental methods: direct detection of trypsin activity inhibition and hydrolysis of bovine serum albumin by trypsin. The results showed the inhibitory effects of the four natural compounds on trypsin, and the inhibition rates of the four natural compounds were significantly different. The enzyme activity assay showed that the inhibitory effect of matrine was better than that of oxymatrine, indicating that trypsin had a good screening resolution. The inhibitory effect was significantly increased with the increased ratio of sample to trypsin, suggesting the structure-activity correlation and dose-effect correlation of the screening methods. Altogether, the experimental method of screening antitumor activity of natural compounds by trypsin has good application values. Since porcine trypsin is similar to human trypsin in terms of molecular structure and performance, it is more applicable for screening of antitumor efficacy of natural pharmacodynamic compounds.

Enantioseparation of ondansetron by countercurrent chromatography using sulfobutyl ether-ß-cyclodextrin as chiral selector.

Ondansetron, a highly selective 5-hydroxytryptamine 3 receptor antagonist, was successfully enantioseparated by recycling countercurrent chromatography using sulfobutyl ether-ß-cyclodextrin as chiral selector. Important factors for the enantioseparation were optimized, including different organic solvent, type of substituted ß-cyclodextrin, pH of aqueous phase, concentration of chiral selector, and separation temperature. A biphasic solvent system composed of n-hexane: n-butyl acetate: 0.1 mol/L phosphate buffer solution pH 9.2 with 50 mmol/L of sulfobutyl ether-ß-cyclodextrin (2.5:7.5:10, v/v/v) was selected. Under optimized separation conditions, 5 mg of ondansetron was enantioseparated using recycling countercurrent chromatography, yielding 1.2 and 1.5 mg of ondansetron enantiomers with 97.5 and 95.8% purity and the recovery reached 48-60%.

Catalytic esterification performance of protease in micro-aqueous system.

OBJECTIVE: To evaluate the catalytic esterification performance of proteases in micro-aqueous systems and to study the suitable conditions for maintaining protease activity. RESULTS: It was found that the protease showed better enzyme catalytic activity in the micro-aqueous phase containing 4% boric acid-borax buffer than that of the pure organic phase. The protease activity was easily activated by 0.20 M boric acid-borax buffer, and the enzyme activity was still high for a long time in alkaline environment (pH 8.40-9.60) and under the temperature of 40-55 °C. Experiments using protease and Candida lipase to synthesize sucrose-6-ethyl ester showed that protease had better esterification activity than Candida lipase in the micro-aqueous phase.

Utilization of Nano-SiO2 as a Supporting Material for Immobilization of Porcine Pancreatic Lipase.

Nano-SiO2 is an inexpensive material for enzyme immobilization and has been frequently utilized for this purpose. In addition to its low cost, Nano-SiO2 has several advantages when used as a supporting material, including its lack of toxicity and chemical reactivity, allowing easy fixation of enzymes. In this article, Porcine Pancreatic lipase was non-covalently immobilized on Nano-SiO2. The properties of immobilized enzyme were then defined. The optimum pH and temperature for the immobilized PPL for hydrolysis of olive oil were determined as 7.6 and 40-50 °C, respectively. The immobilized enzyme possessed high stability in batch operation; with 73.5% of the initial activity still remaining after 8 times of repeated operation of the immobilized lipase. The immobilized enzyme was more stable than free enzyme and t1/2 was 25 d, while free lipase activity was lost 50% in 2 days. The apparent Km for the immobilized enzyme was significantly smaller than that of the free one.

Efficient kinetic resolution of (RS)-1-phenylethanol by a mycelium-bound lipase from a wild-type Aspergillus oryzae strain.

A mycelium-bound lipase from Aspergillus oryzae (AOL) exhibited excellent enantioselectivity for kinetic resolution of (RS)-1-phenylethanol ((RS)-1-PE) in organic solvent. The various reaction parameters affecting the conversion and enantioselectivity were studied, including type of acyl donor, solvent, molar ratio, temperature, enzyme amount, and substrate concentration. The optimum reaction conditions were found to be transesterification with vinyl acetate at 30 °C in methyl tert-butyl ether with a vinyl acetate: (RS)-1-PE molar ratio of 1:1 and an enzyme concentration of 60 g/L. At the optimum reaction conditions, the conversion could reach above 46% with >99% enantiomeric excess of the product, (R)-1-phenylethyl acetate, when the substrate concentration was below 1.4 M. The enzyme displayed an excellent enantioselectivity with an E-value of >200 and a strong tolerance for high substrate concentration of up to 1.8 M. Those results indicated that AOL was a promising biocatalyst in the kinetic resolution of (RS)-1-PE.

Optimization of the extraction and purification of the compatible solute ectoine from Halomonas elongate in the laboratory experiment of a commercial production project.

Optimization of compatible solutes (ectoine) extraction and purification from Halomonas elongata cell fermentation had been investigated in the laboratory tests of a large scale commercial production project. After culturing H. elongata cells in developed medium at 28 °C for 23-30 h, we obtained an average yield and biomass of ectoine for 15.9 g/L and 92.9 (OD600), respectively. Cell lysis was performed with acid treatment at moderate high temperature (60-70 °C). The downstream processing operations were designed to be as follows: filtration, desalination, cation exchange, extraction of crude product and three times of refining. Among which the cation exchange and extraction of crude product acquired a high average recovery rate of 95 and 96%; whereas a great loss rate of 19 and 15% was observed during the filtration and desalination, respectively. Combined with the recovering of ectoine from the mother liquor of the three times refining, the average of overall yield (referring to the amount of ectoine synthesized in cells) and purity of final product obtained were 43% and over 98%, respectively. However, key factors that affected the production efficiency were not yields but the time used in the extraction of crude product, involving the crystallization step from water, which spended 24-72 h according to the production scale. Although regarding to the productivity and simplicity on laboratory scale, the method described here can not compete with other investigations, in this study we acquired higher purity of ectoine and provided downstream processes that are capable of operating on industrial scale.

[Research on improving memory impairment of blue lavender volatile oil].

In order to study the potential application value of lavender volatile oil (LVO), the chemical composition of the volatile oil of lavender was analyzed by GC-MS, and the mouse model of Alzheimer's disease (AD) was established. Additionally, the antioxidant enzymes activity of T-SOD, GSH-PX, CAT and MDA content were studied. Experimental results showed that 55 kinds of chemical constituents including terpene, terpene alcohol and ester compounds from LVO were identified, and the content of linalool and linalyl acetate was the highest, accounting for 49.71% of the total volatile oil. The ability of mouse platform memory was improved significantly. The levels of GSH-PX, CAT and T-SOD of mouse brain tissue in the treatment group were significantly higher than those in the model group (P<0.05). The level of MDA reached the maximum value in the model group, while there was no notable difference between the levels of MDA in the drug group and the normal group. The result indicated the significant oxidative activity of LVO, the possibility of induced oxidative stress reduction in neurons, and the reversal effect of memory acquired disorder.

Biosynthesis of sucrose-6-acetate catalyzed by surfactant-coated Candida rugosa lipase immobilized on sol-gel supports.

Sucrose-6-acetate is an important intermediate in the preparation of sucralose (a finest sweetener). In our study, Candida rugosa lipase coated with surfactant was firstly immobilized on sol-gel supports. Then, the immobilized enzyme was used in the regioselective synthesis of sucrose-6-acetate by transesterification of sucrose and vinyl acetate. The screening results revealed that Tween 80 was an ideal surfactant to coat lipase immobilized in sol-gel and exhibited the highest yield of sucrose-6-acetate. Other factors that influenced the yield during the preparation process were also studied. Under optimal conditions, the yield of sucrose-6-acetate could reach up to 78.68 %, while free lipase was easily inactivated in polar solvent. Thermal and operational stabilities were also improved significantly. Surfactant-coated lipase immobilized in sol-gel remained stable when the temperature was higher than 60 °C. Moreover, they could maintain high catalytic activity after six recycles. This strategy is economical, convenient and promising for the food industry.

Optimization of technological parameters for preparation of lycopene microcapsules.

Lycopene belongs to the carotenoid family with high degree of unsaturation and all-trans form. Lycopene is easy to isomerize and auto oxide by heat, light, oxygen and different food matrices. With an increasing understanding of the health benefit of lycopene, to enhance stability and bioavailability of lycopene, ultrasonic emulsification was used to prepare lycopene microcapsules in this article. The results optimized by response surface methodology (RSM) for microcapsules consisted of four major steps: (1) 0.54 g glycerin monostearate was fully dissolved in 5 mL ethyl acetate and then added 0.02 g lycopene to form an organic phase, 100.7 mL distilled water which dissolved 0.61 g synperonic pe(R)/F68 as the aqueous phase; (2) the organic phase was pulled into the aqueous phase under stirring at 60 °C water bath for 5 min; (3) the mixture was then ultrasonic homogenized at 380 W for 20 min to form a homogenous emulsion; (4) the resulting emulsion was rotary evaporated at 50 °C water bath for 10 min under a pressure of 20 MPa. Encapsulation efficiency (EE) of lycopene microcapsules under the optimized conditions approached to 64.4%.

Magnetic nanoparticles immobilized thrombin ligand fishing to screen thrombin inhibitors in natural products.

In this study, thrombin was immobilized with magnetic particles modified by glutaraldehyde. The changes in secondary structures of immobilized enzyme revealed an increment in conformational rigidity and stability, which can be reflected in temperature and pH stability as well as the tolerance of organic reagents. The optimal reutilization times of magnetic particle immobilized thrombin were 7 times, and the half-life of enzyme activity preserved at room temperature was 5 days, which was 2.5 times higher than that of free enzyme. Ligusticum chuanxiong and Anemarrhenae Rhizoma with high enzyme inhibitory activity were selected for primary screening, and six potential inhibitors of thrombin were identified by HPLC/MS. The results showed that three compounds in Anemarrhenae Rhizoma had better predictive thrombin inhibitory activity. Through the in vitro thrombin activity inhibition experiment, it was also verified that mangiferin and neo-mangiferin had an ideal thrombin activity inhibition effect, which was consistent with the results of molecular docking.

The correlation of molecule weight of chitosan oligomers with the corresponding viscosity and antibacterial activity.

In order to explore the correlation between the viscosity of chitosan oligomers-acetic solution and its viscosity average molecular weight (Mv), and determine the Mv range with a strong bactericidal effect. A series of chitosan oligomers were obtained by degraded chitosan (728.5 kDa) with dilute acid and chitosan oligomer (101.5 kDa) was characterized by FT-IR, XRD, H NMR and C NMR. The bactericidal effect of chitosan oligomers with different Mv on E. coli, S. aureus and C. albicans was measured by plate counting method. And the bactericidal rate was taken as the evaluation indicator, the optimum conditions were determined by single-factor experiments. The result showed that the molecular structure of chitosan oligomers and original chitosan (728.5 kDa) were similar. The viscosity of the chitosan oligomers in acetic acid solution was positively correlated with the Mv, and the chitosan oligomers with the Mv of 52.5-145.0 kDa had a strong bactericidal performance. In addition, the bactericidal rate of chitosan oligomers on experimental strains was more than 90% when the concentration of 0.5 g/L (bacteria) and 1.0 g/L (fungi), pH6.0, incubation time of 30 min. Thus, chitosan oligomers had a potential application value when the Mv was in the range of 52.5-145.0 kDa.

Separation and Purification of Bamboo Leaf Flavones by Polyvinylpolypyrrolidone Adsorption.

In view of the adsorption performance of polyvinylpolypyrrolidone (PVPP) to flavones, the adsorption and purification of bamboo leaf flavones (BLFs) by PVPP were studied. The flavones solution was adsorbed by PVPP column chromatography, and then establish a relatively effective method for elution and purification of flavones from bamboo leaf. The optimal separation conditions of column chromatography were determined as the following: the feed concentration of 10 mg/mL, the ratio of diameter to height of 1:1.9, eluents of deionized water (21 mL) and 70% ethanol (800 mL) with a flow rate of 0.33 mL/min. The purity of flavones obtained from ethanol eluents (80-480 mL) was 96.2%. This showed that the PVPP had an ideal adsorption and purification effect on BLFs.

Degree and distribution of substitution of hydroxypropyl-ß-cyclodextrin in enantioselective liquid-liquid extraction and countercurrent chromatographic enantioseparation.

Nine types of hydroxypropyl-ß-cyclodextrin (HP-ß-CD) with different degrees and distributions of substitution were synthesised, and nine racemates were selected to investigate the effect of different degrees and distributions of substitution of HP-ß-CD on the enantioseparation factor. 1H NMR and GC/MS were used to characterise the synthesised HP-ß-CD. The degree and distribution of substitution had a significant influence on enantioselective liquid-liquid extraction and enantioseparation by countercurrent chromatography. For most of the tested racemates, increasing both the degree of substitution and distribution of substitution at the C-2 position for HP-ß-CD would lead to an increasing enantioseparation factor; the optimal enantioseparation factor of 2-phenylbutyric acid, tropic acid, 2,3-diphenylpropionic acid, 2-(4-hydroxylphenyl) propanoic acid, and naproxen was increased to 1.77, 1.53, 1.67, 1.61, and 1.75, respectively. The enantioseparation of racemic naproxen, 2-(4-hydroxylphenyl) propanoic acid, and 2,3-diphenylpropionic acid by countercurrent chromatography was optimised using HP-ß-CD with a degree of substitution of 16.5, and peak resolution was significantly improved to 1.03, 1.35, and 1.01, respectively.

Hypoglycemic activity and mechanisms of myricetin.

Myricetin has been reported to have a wide variety of beneficial physiological functions. The present study was designed to investigate the mechanism of high purity myricetin, as a hypoglycemic functional component on high fat diet (HFD) fed streptozotocin (STZ) induced diabetic rats. Four-week antihyperglycemic effects of myricetin were assayed. The results showed that continuous administration of myricetin (50 and 200 mg/kg body weight) in HFD/STZ induced diabetic rats dose-dependently reduced the body serum glucose and insulin. Furthermore, administrations of myricetin significantly increased the expression of insulin receptor (InsR) and glucose transporter 4 (GLUT4) gene and increased the expression of glucose-6-phosphatase (G-6-Pase) and phosphoenolpyruvate carboxykinase (PEPCK) gene. Moreover, myricetin protected pancreatic tissue from HFD fed STZ induced apoptosis through regulation of Bcl-2 associated X (Bax) gene and B-cell lymphoma-2 (Bcl-2) gene. The experimental results show that myricetin has significant health benefits and can be explored as a potentially promising dietary supplement for auxiliary hypoglycemic.

The properties and kinetics of enzymatic reaction in the process of the enzymatic extraction of fish oil.

The technology of enzymatic extraction of fish oil has many advantages, such as moderate operating conditions, lower energy consumption and high efficiency. Moreover, it could reduce the breakage for the functional component of fish oil. In enzymatic extraction of oil, the investigation of the property of enzymatic reaction is propitious to improve the enzymolysis efficiency. In this study, the 1398 neutrase was used for hydrolyzing fish protein, then analyzed the impacts to the enzymolysis efficiency which were induced by the different initial substrate concentration and different initial enzyme concentration, the result showed that the higher substrate concentration generate inhibition to the enzyme. And then, the properties of enzymatic hydrolysis were studied by the Michaelis-Menten equation of substrate inhibition and the enzymatic hydrolysis kinetics equation which is derived by theory. By means of the verification to the enzymatic hydrolysis kinetics model, it could see that the model in line with the actual situation better at a lower degree of hydrolysis. Lastly, the critical enzyme concentration and critical substrate concentration of enzymatic reaction could be obtained by deducing the enzymatic hydrolysis kinetics model.