Conjugation of Lysozyme and Epigallocatechin Gallate for Improving Antibacterial and Antioxidant Properties.

One of the main interests in the food industry is the preservation of food from spoilage by microorganisms or lipid oxidation. A novel alternative is the development of additives of natural origin with dual activity. In the present study, a chemically modified lysozyme (Lys) with epigallocatechin gallate (EGCG) was developed to obtain a conjugate (Lys-EGCG) with antibacterial/antioxidant activity to improve its properties and increase its application potential. The modification reaction was carried out using a free radical grafting method for the Lys modification reaction, using ascorbic acid and hydrogen peroxide as radical initiators in an aqueous medium. The synthesis of Lys-EGCG conjugate was confirmed by spectroscopic (FT-IR, 1H-RMN, and XPS) and calorimetry differential scanning (DSC) analyses. The EGCG binding to the Lys biomolecule was quantified by the Folin-Ciocalteu method; the antibacterial activity was evaluated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MCB) against Staphylococcus aureus and Pseudomonas fluorescens; the antioxidant activity was evaluated by ABTS, DPPH, and FRAP. The spectroscopic results showed that the Lys-EGCG conjugate was successfully obtained, and the DSC analysis revealed a 20 °C increase (P < 0.05) in the denaturation temperature of Lys due to EGCG modification. The EGCG concentration in Lys-EGCG was 97.97 ± 4.7 µmol of EGCG/g of sample. The antibacterial and antioxidant activity of the Lys-EGCG conjugate was higher (P < 0.05) than pure EGCG and Lys. The chemical modification of Lys with EGCG allows for the bioconjugate with a dual function (antibacterial/antioxidant), broadening the range of Lys and EGCG applications to different areas such as food, cosmetic, and pharmaceutical industries.

Antibiotic resistance of ESKAPE group-microorganisms in health institutions from Hermosillo and Ciudad Obregón, Sonora, México.

Introduction: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. are microorganisms referred as the ESKAPE group pathogens. These microorganisms have generated great concern in health institutions around the world since most of them have resistance to multiple antibiotics and cause most infections associated with healthcare, as well as community infections. The aim of this study was the analysis of antibiotic resistance in microorganisms of the ESKAPE group, recovered from clinical samples in 11 health institutions from Hermosillo and Ciudad Obregón in the State of Sonora, México, during the period from 2019 to 2020. Methods: A cross-sectional, descriptive, observational, and temporality epidemiological study was carried out. A comparative and statistical analysis of antibiotic resistance was carried out using the chi-square test, and small values were analyzed using Fisher's exact test p ≤ 0.05. Results and discussion: All the ESKAPE group microorganisms showed significant differences in antibiotic resistance percentages between both cities. High resistance percentages for some antibiotics, like cephalosporins and ciprofloxacin were detected for Klebsiella pneumoniae and Acinetobacter baumannii.

Sustainable-green synthesis of silver nanoparticles using safflower (Carthamus tinctorius L.) waste extract and its antibacterial activity.

Silver nanoparticles have high potential for application in food industry, as they have the ability to inhibit a wide range of bacteria of pathogenic and spoilage origin. They can be obtained from different methods classified in physical and chemical and which are aggressive with the environment since they produce toxic waste. Nowadays, environmentally friendly methods such as green synthesis can be used, through the use of agri-food waste. The use of these wastes is a more sustainable method, because it reduces the environmental pollution, at the same time that silver nanoparticles are obtained. The aim of the present study is the green synthesis of silver nanoparticles using safflower (Carthamus tinctorius L.) aqueous extract from waste and its antibacterial activity on Staphylococcus aureus (Gram positive) and Pseudomonas fluorescens (Gram negative). The analyses by TEM showed that the as-synthesized silver nanoparticles were uniform and spherical particles with an average diameter of 8.67 ± 4.7 nm and confirmed by SEM. The electron diffraction and TEM analyses showed the characteristic crystallinity of silver nanoparticles. FTIR spectroscopy confirmed that various functional groups were responsible for reducing and stabilizing during the biosynthesis process. Nanoparticles inhibited the growth of both types of bacteria from the lowest concentration evaluated (0.9 µg/mL). We conclude that silver nanoparticles synthesized in the present study have potential application as antibacterial agents in food and medicine industry.

Characterization of Epigallocatechin-Gallate-Grafted Chitosan Nanoparticles and Evaluation of Their Antibacterial and Antioxidant Potential.

Nanoparticles based on chitosan modified with epigallocatechin gallate (EGCG) were synthetized by nanoprecipitation (EGCG-g-chitosan-P). Chitosan was modified by free-radical-induced grafting, which was verified by Fourier transform infrared (FTIR). Furthermore, the morphology, particle size, polydispersity index, and zeta potential of the nanoparticles were investigated. The grafting degree of EGCG, reactive oxygen species (ROS) production, antibacterial and antioxidant activities of EGCG-g-chitosan-P were evaluated and compared with those of pure EGCG and chitosan nanoparticles (Chitosan-P). FTIR results confirmed the modification of the chitosan with EGCG. The EGCG-g-chitosan-P showed spherical shapes and smoother surfaces than those of Chitosan-P. EGCG content of the grafted chitosan nanoparticles was 330 µg/g. Minimal inhibitory concentration (MIC) of EGCG-g-chitosan-P (15.6 µg/mL) was lower than Chitosan-P (31.2 µg/mL) and EGCG (500 µg/mL) against Pseudomonas fluorescens (p < 0.05). Additionally, EGCG-g-chitosan-P and Chitosan-P presented higher Staphylococcus aureus growth inhibition (100%) than EGCG at the lowest concentration tested. The nanoparticles produced an increase of ROS (p < 0.05) in both bacterial species assayed. Furthermore, EGCG-g-chitosan-P exhibited higher antioxidant activity than that of Chitosan-P (p < 0.05) in 2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and ferric-reducing antioxidant power assays. Based on the above results, EGCG-g-chitosan-P shows the potential for food packaging and biomedical applications.

Combined hypoxia and high temperature affect differentially the response of antioxidant enzymes, glutathione and hydrogen peroxide in the white shrimp Litopenaeus vannamei.

Low oxygen concentration in water (hypoxia) and high temperature are becoming more frequent due to climate change, forcing animals to endure stress or decease. Hypoxia and high temperature stress can lead to reactive oxygen species (ROS) accumulation and oxidative damage to the organisms. The shrimp Litopenaeus vannamei is the most cultivated crustacean worldwide. The aim of this study was to evaluate the expression and enzymatic activity of glutathione peroxidase (GPx), catalase (CAT) and cytosolic manganese superoxide dismutase (cMnSOD) in gills and hepatopancreas from L. vannamei in response to two combined stressors: hypoxia and reoxygenation at control and high temperature (28 vs 35 °C, respectively). In addition, glutathione and hydrogen peroxide content were analyzed. The changes were mainly tissue-specific. In gills, cMnSOD expression and enzymatic activity increased in response to the interactions between oxygen variation and thermal stress, while GPx and CAT were maintained. More changes occurred in GPx, CAT and MnSOD in hepatopancreas than in gills, mainly due to the effect of the individual stress factors of thermal stress or oxygen variations. On the other hand, the redox state of glutathione indicated that during high temperature, changes in the GSH/GSSG ratio occurred due to the fluctuations of GSSG. Hydrogen peroxide concentration was not affected by thermal stress or oxygen variations in hepatopancreas, whereas in gills, it was not detected. Altogether, these results indicate a complex pattern of antioxidant response to hypoxia, reoxygenation, high temperature and their combinations.

Refolding and Activation from Bacterial Inclusion Bodies of Trypsin I from Sardine (Sardinops sagax caerulea).

BACKGROUND: Trypsin from fish species is considered as a cold-adapted enzyme that may find potential biotechnological applications. In this work, the recombinant expression, refolding and activation of Trypsin I (TryI) from Monterey sardine (Sardinops sagax caerulea) are reported. METHODS: TryI was overexpressed in Escherichia coli BL21 as a fusion protein of trypsinogen with thioredoxin. Refolding of trypsinogen I was achieved by dialysis of bacterial inclusion bodies with a recovery of 16.32 mg per liter of Luria broth medium. RESULTS: Before activation, the trypsinogen fusion protein did not show trypsin activity. Trypsinogen I was activated by adding 0.002 U of native TryI purified from the sardine pyloric caeca (nonrecombinant). The activated recombinant trypsin showed three times more activity than the nonrecombinant trypsin alone. CONCLUSION: The described protocol allowed obtaining sufficient amounts of recombinant TryI from Monterey sardine fish for further biochemical and biophysical characterization of its coldadaptation parameters.

Structure of nucleoside diphosphate kinase from pacific shrimp (Litopenaeus vannamei) in binary complexes with purine and pyrimidine nucleoside diphosphates.

Nucleoside diphosphate kinase (NDK; EC 2.7.4.6) is an enzyme that catalyzes the third phosphorylation of nucleoside diphosphates, leading to nucleoside triphosphates for DNA replication. Expression of the NDK from Litopenaeus vannamei (LvNDK) is known to be regulated under viral infection. Also, as determined by isothermal titration calorimetry, LvNDK binds both purine and pyrimidine deoxynucleoside diphosphates with high binding affinity for dGDP and dADP and with no heat of binding interaction for dCDP [Quintero-Reyes et al. (2012), J. Bioenerg. Biomembr. 44, 325-331]. In order to investigate the differences in selectivity, LvNDK was crystallized as binary complexes with both acceptor (dADP and dCDP) and donor (ADP) phosphate-group nucleoside diphosphate substrates and their structures were determined. The three structures with purine or pyrimidine nucleotide ligands are all hexameric. Also, the binding of deoxy or ribonucleotides is similar, as in the former a water molecule replaces the hydrogen bond made by Lys11 to the 2'-hydroxyl group of the ribose moiety. This allows Lys11 to maintain a catalytically favourable conformation independently of the kind of sugar found in the nucleotide. Because of this, shrimp NDK may phosphorylate nucleotide analogues to inhibit the viral infections that attack this organism.

Shrimp oncoprotein nm23 is a functional nucleoside diphosphate kinase.

Biosynthesis of nucleoside triphosphates is critical for bioenergetics and nucleic acid replication, and this is achieved by nucleoside diphosphate kinase (NDK). As an emerging biological model and the global importance of shrimp culture, we have addressed the study of the Pacific whiteleg shrimp (Litopenaeus vannamei) NDK. We demonstrated its activity and affinity towards deoxynucleoside diphosphates. Also, the quaternary structure obtained by gel filtration chromatography showed that shrimp NDK is a trimer. Affinity was in the micro-molar range for dADP, dGDP, dTDP and except for dCDP, which presented no detectable interaction by isothermal titration calorimetry, as described previously for Plasmodium falciparum NDK. This information is particularly important, as this enzyme could be used to test nucleotide analogs that can block white spot syndrome virus (WSSV) viral replication and to study its bioenergetics role during hypoxia and fasting.