Redesigned pMAL expression vector for easy and fast purification of active native antimicrobial peptides.

AIMS: The aim of this study was to construct the improved pMAL expression vector to increase the efficacy of purification of small native peptides and their clear-cut separation from MBP tag. The modifications we introduced can be applied to many expression vectors. METHODS AND RESULTS: To improve the pMAL expression vector, we introduced the His6 tag and the enterokinase cleavage site (Ek) downstream from the MBP tag and Xa cleavage site on the original vector. For cloning of a desired peptide DNA, the enterokinase site contains a unique BsaBI restriction site adjacent to the original multi-cloning site. This redesigned pMAL vector was optimized for the purification of cytoplasmic (pMALc5HisEk) and periplasmic (pMALp5HisEk) peptides. The purification of native and active peptide (P) was obtained following two-step affinity chromatography. In the first step, the entire MBP-His6 -Ek-P fusion protein is purified using the Ni-NTA agarose column. This fusion protein was cleaved with active His6 tagged enterokinase. In the second step, the further purification was performed by column containing the mixture of amylose and Ni-NTA agarose resins. This removes both the MBP-His6 and His6 -enterokinase leaving pure native protein in solution. These new vectors and the two-step purification protocol were successfully applied in purification of active native small antimicrobial peptides (AMPs), lactococcin A and human ß-defensin. CONCLUSIONS: We constructed the improved pMAL expression vectors and established the pipeline and optimal conditions for their use in efficient purification of large amounts of active native small peptides. SIGNIFICANCE AND IMPACT OF THE STUDY: Choice of expression vector impacts on the efficiency of expression and purification of desired proteins. The idea of redesigning pMAL vector was driven by the need for rapid purification of larger amounts of active native AMPs. This newly improved pMAL vector, the cloning strategy, expression conditions and two-step purification protocol represent a unique simple approach which can be applied in every laboratory.

Determination of antilisterial effect of some microbial isolates from traditional zlatar cheese during the fermentation of soft white cheese.

The aim of this study was to select autochthonous lactic acid bacteria (LAB) isolates with antilisterial activity from Zlatar cheese and to evaluate the ability of selected LAB to control Listeria monocytogenes growth during soft white cheese production. The genotype characterization of isolated LAB (n = 93) was done using PCR method by 16S rRNA sequencing. In this way, the following isolates were detected: Lactococcus lactis ssp. lactis (40 isolates), Enterococcus faecalis (30), Lactobacillus plantarum (12), Leuconostoc mesenteroides ssp. mesenteroides (3) Lactobacillus garvieae (3), Lactobacillus curvatus (2), Lactobacillus casei (1), Enterococcus faecium (1) and Staphylococcus hominis (1). Each isolated LAB was tested for bacteriocin-producing ability. It was determined that two LAB isolates had bactericidal properties: Lactococcus lactis ssp. lactis SRB/ZS/094 and Enterococcus faecalis SRB/ZS/090. Semi-purified of enterococcal bacteriocin (enterocin) was isolated using precipitation procedures with ammonium sulphate. Its properties were determined (strength and range of activities). Isolated enterocin and bacteriocin-producing Lactococcus strain showed significant antimicribial activity against Listeria monocytogenes, but still the inhibition activity of Staphylococcus aurues and Escherichia coli was not detected. Based on the obtained laboratory results, in the second phase of the research, the antilisterial effect of bacteriocin isolated from Enterococcus faecalis SRB/ZS/090 and cells Lactococcus lactis ssp. lactis SRB/ZS/094 were determined, that are added as additives in the production of soft white cheese through five variants. Cheese supplemented with enterocin (E2) had the lowest aerobic mesophilic bacteria count, indicating that enterocin (E2) play an important role for bio-preservation.

The Intake of Phosphorus and Nitrites through Meat Products: A Health Risk Assessment of Children Aged 1 to 9 Years Old in Serbia.

This study provides the data on dietary exposure of Serbian children to nitrites and phosphorus from meat products by combining individual consumption data with available analytical data of meat products. A total of 2603 and 1900 commercially available meat products were categorized into seven groups and analysed for nitrite and phosphorous content. The highest mean levels of nitrite content, expressed as NaNO2, were found in finely minced cooked sausages (40.25 ± 20.37 mg/kg), followed by canned meat (34.95 ± 22.12 mg/kg) and coarsely minced cooked sausages (32.85 ± 23.25 mg/kg). The EDI (estimated daily intake) of nitrites from meat products, calculated from a National Food Consumption Survey in 576 children aged 1-9 years, indicated that the Serbian children population exceeded the nitrite ADI (acceptable daily intake) proposed by EFSA (European Food Safety Authority) in 6.4% of children, with a higher proportion in 1-3-year-old participants. The mean phosphorus concentration varied from 2.71 ± 1.05 g/kg to 6.12 ± 1.33 g/kg in liver sausage and pate and smoked meat products, respectively. The EDI of phosphorus from meat products was far below the ADI proposed by EFSA, indicating that the use of phosphorus additives in Serbian meat products is generally in line with legislation.