Purification and characterization of lysozyme from Chinese Lueyang black-bone Silky fowl egg white.

Lysozyme, an important antibacterial protein, is an enzyme that cleaves the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine of peptidoglycan in cell walls. The novel lysozyme was purified and characterized from Chinese Lueyang black-bone silky fowl (CBSF) egg white, and its N-terminal amino acid sequence, enzymatic properties, and antibacterial activity were investigated. The CBSF lysozyme was purified using adsorption chromatography, ammonium sulfate precipitation, ion exchange chromatography, and size-exclusion chromatography. The purification fold and yield were 3.28 and 14.69%, respectively. The purified lysozyme was revealed as a single protein band with SDS-PAGE and had a MALDI-TOF/TOF molecular weight of 14305.57 Da and a final specific activity of 3.49 × 105 U/mg protein using Micrococcus lysodeikticus as a substrate. The optimum temperature and pH of the lysozyme were 50 °C and 6.0, respectively. The 20 N-terminal amino acid residues of the purified lysozyme were determined to be KVFGRCELAAAMKRHGLDNY, showing some homology to the N-terminus of the odontophoridae egg white lysozyme. The purified lysozyme exerted a potent antimicrobial activity toward indicator microorganisms, including Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, and Escherichia coli ATCC 25922. However, its inhibition of gram-negative activity was weaker than that of the Gram-positive bacteria.

Deep eutectic solvent extraction of myricetin and antioxidant properties.

In this study, a response surface method (RSM) was used to optimise the ultrasonic-assisted deep eutectic solvent (DES) extraction of myricetin from myricetin leaves. The results demonstrated that the DES-5 (choline chloride-oxalic acid) system exhibited better extraction results than the other seven DESs prepared. The optimum extraction conditions for myricetin were a DES-5 system with 19% water content of DES, a liquid-to-solid ratio of 37 : 1 mL g-1, an extraction time of 45 min, and an extraction temperature of 72 °C. Under these conditions, the extraction amount of myricetin was 22.47 mg g-1. To optimise the extraction process, the crude myricetin extract was purified, and the optimal conditions were as follows: an AB-8 macroporous adsorption resin was used with an anhydrous ethanol desorption agent. The adsorption rate was 1 BV per h (bed volume per hour), the desorption rate was 1 BV per h, and the desorption capacity was 2 BV (bed volume). The antioxidant properties of the myricetin were also investigated. The results demonstrated that, with an increase in concentration, the scavenging rates of DPPH and ˙OH free radicals increased. Compared to Vc, myricetin had a better scavenging ability for DPPH free radicals, whereas purified myricetin had a better antioxidant effect. At the same concentration, the radical-scavenging rate of the ˙OH radical was slightly higher in myricetin purified by the macroporous adsorption resin than in Vc, and that of the unpurified myricetin was the smallest. Myricetin was purified using a macroporous adsorption resin to improve its antioxidant properties.

Application of Graphene and its Derivatives in Detecting Hazardous Substances in Food: A Comprehensive Review.

Graphene and its derivatives have been a burning issue in the last 10 years. Although many reviews described its application in electrochemical detection, few were focused on food detection. Herein, we reviewed the recent progress in applying graphene and composite materials in food detection during the past 10 years. We pay attention to food coloring materials, pesticides, antibiotics, heavy metal ion residues, and other common hazards. The advantages of graphene composites in electrochemical detection are described in detail. The differences between electrochemical detection involving graphene and traditional inherent food detection are analyzed and compared in depth. The results proved that electrochemical food detection based on graphene composites is more beneficial. The current defects and deficiencies in graphene composite modified electrode development are discussed, and the application prospects and direction of graphene in future food detection are forecasted.

Extraction and isolation of the active ingredients of dandelion and its antifungal activity against Candida albicans.

In this study, six compounds were isolated and purified from dandelion, and only sample I exhibited notable antifungal effect on Candida albicans (CA). high­performance liquid chromatography­diode­array detector­electrospray ionization­tandem mass spectrometry analysis showed that sample I comprised 4­coumaric acid, ferulic acid, quercetin pentoside, 3,5­di­O­caffeoylquinic acid, 4,5­di­O­caffeoylquinic acid, luteolin, and two unknown compounds, at a relative percent composition of 11.45, 3.96, 10.48, 34.24, 3.91, 11.80, 3.65 and 4.21%, respectively. Further antimicrobial experiments showed that the minimum inhibitory concentration of sample I was 32.0 mg/ml, and sample I mainly acts on bacterial growth in the exponential phase of CA growth. Optical density and infrared analyses conclusively suggested that sample I damages the structure of CA cells, particularly the cell wall and cell membrane, resulting in macromolecule leakage of intracellular nucleic acids and cell metabolism disruption. In conclusion, dandelion sample I was reported to increase CA cell membrane permeability by affecting the glycosidic bond in ß­(1­3)­D glucan and destroying the cell wall, ultimately leading CA to death.

Protective effect of wild Corni fructus methanolic extract against acute alcoholic liver injury in mice.

BACKGROUND: In Chinese folk medicine, Corni fructus (C. fructus) has traditionally been used to improve liver function, although the mechanism underlying its activity remains unclear. The aim of the present study was to evaluate the protective effects of wild C. fructus methanolic extract against acute alcoholic liver injury. METHODS: Alcohol was administered to mice for three consecutive days, either alone or in combination with C. fructus methanolic extract (50, 100, or 200 mg/kg body weight/d). Serum and liver tissue were collected from the animals and subjected to biochemical and histopathological analyses. RESULTS: C. fructus significantly alleviated alcohol-induced liver injury by reducing serum alanine aminotransferase, aspartate aminotransferase, and thiobarbituric acid reactive species, inhibiting hydroxyl radicals (•OH), and increasing total superoxide dismutase, glutathione peroxidase, and glutathione in the liver (P < 0.05). In addition, the C. fructus treatment inhibited the expression and activity of cytochrome P450 2E1 (P < 0.05). CONCLUSIONS: C. fructus could be a promising natural substance for ameliorating acute alcohol-induced oxidative stress and hepatic injury.

Voltage-Dependent Anion Channel-1, a Possible Ligand of Plasminogen Kringle 5.

Kringle 5, the fifth fragment of plasminogen, is known to be important for inhibiting the proliferation and migration of vascular endothelial cell (VEC), while not having any effects on normal endothelial cells. Therefore, it may be a potential tumor therapy candidate. However, the ligand of the Kringle 5 in VEC has not yet been identified. In this study, the possible ligand of Kringle 5 in vitro was screened and validated using Ph.D.-7 phage display peptide library with molecular docking, along with surface plasma resonance (SPR). After four rounds of panning, the specific clones of Kringle 5 were confirmed using enzyme-linked immunosorbent assay (ELISA). The gene sequence analysis showed that they expressed the common amino sequence IGNSNTL. Then, using a NCBI BLAST, 103 matching sequences were found. Following the molecular docking evaluation and considering the acting function and pathway of the plasminogen Kringle 5 in the human body, the most promising candidate was determined to be voltage-dependent anion channel-1 (VDAC-1), which was able to bind to Kringle 5 at -822.65 J·mol-1 of the binding energy at the residues of Lys12, Thr19, Ser57, Thr188, Arg139, Asn214, Ser240 and Lys274. A strong dose-dependent interaction occurred between the VDAC-1 and Kringle 5 (binding constant 2.43 × 103 L·mol-1) in SPR observation. Therefore, this study proposed that VDAC-1 was a potential ligand of plasminogen Kringle 5, and also demonstrated that the screening and validation of protein ligand using phage display peptide library with the molecular docking, along with SPR, was a practicable application.

Cloning, expression and identification of an isoform of human stromal cell derived factor-1α.

Human stromal cell derived factor-1α (hSDF-1α), a chemotactic factor of stem cells, regulates inflammation, promotes the mobilization of stem cells and induces angiogenesis following ischemia. Six SDF-1 isoforms, SDF-1α, SDF-1ß, SDF-1γ, SDF-1δ, SDF-1ε and SDF-1ϕ, which all contain a signal peptide at the N-terminus, have been reported. In the present study a special isoform of hSDF-1α is described that does not contain the N-terminal signal peptide sequence. The hSDF-1α gene was cloned with the recombinant plasmid pCMV-SPORT6-hSDF1 as the template, and the prokaryotic expression vector pET15b-hSDF-1α was constructed. This hSDF-1α was successfully expressed as an inclusion body in Escherichia coli BL21(DE3). The recombinant hSDF-1α was refolded in vitro and separated by cation exchange chromatography. Following these two steps the purity of the hSDF-1α was able to reach >85%. The recombinant hSDF-1α was then purified by size-exclusion chromatography. SDS-PAGE analysis demonstrated that the purity of the hSDF-1α was >95%, which meets almost all the requirements of a protein experiment. Chemotactic activity of the recombinant hSDF-1α was analyzed by Transwell migration assay and it was found that the recombinant hSDF-1α was able to stimulate THP-1 cell migration. These data suggest that the procedure of producing recombinant hSDF-1α proteins with chemotactic activity was feasible and the N-terminal signal peptide of hSDF-1α has little effect on the chemotactic activity of hSDF-1α.