The Effect of Milking Frequency, Breed, and Stage of Lactation on the Milk Fat Globule Size and Fatty Acid Composition in Sheep's Milk.

This study examined the effects of milking frequency, breed, and stage of lactation on the milk fat globules (MFG) size and fatty acids (FA) composition of sheep milk. Milk from Karagouniko (n = 13) and Chios (n = 13) ewes was sampled postpartum on the 93rd, 101st, 108th, 121st, 156th, and 188th days of lactation. On the 108th day, the ewes were divided randomly into two milking groups: Once daily at 06:00 a.m. or twice daily at 06:00 a.m. and 16:00 p.m. Morphometric characteristics of MFG and FA composition were determined for each sample. Once versus twice daily milking had no effect on MFG dimensions, which tended to vary according to breed (smaller MFG were secreted from Chios with p = 0.065), while the stage of lactation had a significant effect (p < 0.001). FA composition differed significantly according to the stage of lactation and breed. The FA profile of the Karagouniko breed showed higher concentrations of short-chain FA. The milk samples from late lactation were characterized by higher concentrations of mono-unsaturated FA (MUFA) compared to early and mid-lactation. Moreover, correlations were found between the average diameter of MFG and FA concentrations, where the size of MFG was positively correlated with saturated FA (SFA) and negatively correlated with MUFA.

Mechanism of antimicrobial activity of honeybee (Apis mellifera) venom on Gram-negative bacteria: Escherichia coli and Pseudomonas spp.

Honeybee venom (Apitoxin, BV), a secretion substance expelled from the venom gland of bees, has being reported as antimicrobial against various bacterial species; however, the mechanism of action remains uncharacterized. In this study, the antibacterial activity of BV was investigated on hygiene indicator Escherichia coli and the environmental pathogen and spoilage bacterial species, Pseudomonas putida and Pseudomonas fluorescens. An array of methods was combined to elucidate the mode of action of BV. Viability by culture on media was combined with assessing cell injury with flow cytometry analysis. ATP depletion was monitored as an indicator to metabolic activity of cells, by varying BV concentration (75, 225and 500 µg/mL), temperature (25 [Formula: see text] and 37 [Formula: see text]), and time of exposure (0 to 24 h). Venom presented moderate inhibitory effect on E. coli by viability assay, caused high membrane permeability and significant ATP loss where the effect was increased by increased concentration. The viability of P. putida was reduced to a greater extent than other tested bacteria at comparable venom concentrations and was dictated by exposure time. On the contrary, P. fluorescens appeared less affected by venom based on viability; however, flow cytometry and ATP analysis highlighted concentration- and time-dependent effect of venom. According to Transmission Electron Microscopy results, the deformation of the cell wall was evident for all species. This implies a common mechanism of action of the BV which is as follows: the cell wall destruction, change of membrane permeability, leakage of cell contents, inactivation of metabolic activity and finally cell death.

Use of Transposon Directed Insertion-Site Sequencing to Probe the Antibacterial Mechanism of a Model Honey on E. coli K-12.

Antimicrobial resistance is an ever-growing health concern worldwide that has created renewed interest in the use of traditional anti-microbial treatments, including honey. However, understanding the underlying mechanism of the anti-microbial action of honey has been hampered due to the complexity of its composition. High throughput genetic tools could assist in understanding this mechanism. In this study, the anti-bacterial mechanism of a model honey, made of sugars, hydrogen peroxide, and gluconic acid, was investigated using genome-wide transposon mutagenesis combined with high-throughput sequencing (TraDIS), with the strain Escherichia coli K-12 MG1655 as the target organism. We identified a number of genes which when mutated caused a severe loss of fitness when cells were exposed to the model honey. These genes encode membrane proteins including those involved in uptake of essential molecules, and components of the electron transport chain. They are enriched for pathways involved in intracellular homeostasis and redox activity. Genes involved in assembly and activity of formate dehydrogenase O (FDH-O) were of particular note. The phenotypes of mutants in a subset of the genes identified were confirmed by phenotypic screening of deletion strains. We also found some genes which when mutated led to enhanced resistance to treatment with the model honey. This study identifies potential synergies between the main honey stressors and provides insights into the global antibacterial mechanism of this natural product.

Use of a model to understand the synergies underlying the antibacterial mechanism of H2O2-producing honeys.

Honey has been valued as a powerful antimicrobial since ancient times. However, the understanding of the underlying antibacterial mechanism is incomplete. The complexity and variability of honey composition represent a challenge to this scope. In this study, a simple model system was used to investigate the antibacterial effect of, and possible synergies between, the three main stressors present in honey: sugars, gluconic acid, and hydrogen peroxide (H2O2), which result from the enzymatic conversion of glucose on honey dilution. Our results demonstrated that the synergy of H2O2 and gluconic acid is essential for the antibacterial activity of honey. This synergy caused membrane depolarization, destruction of the cell wall, and eventually growth inhibition of E. coli K-12. The presence of H2O2 stimulated the generation of other long-lived ROS in a dose-dependent manner. Sugars caused osmosis-related morphological changes, however, decreased the toxicity of the H2O2/gluconic acid. The susceptibility of catalase and general stress response sigma factor mutants confirmed the synergy of the three stressors, which is enhanced at higher H2O2 concentrations. By monitoring cellular phenotypic changes caused by model honey, we explained how this can be bactericidal even though the antimicrobial compounds which it contains are at non-inhibitory concentrations.