RESULTS: Findings revealed 40 aroma-active compounds with flavor dilution (FD) factor ranges of 2-1024. Of these, 22 compounds (FD ≥ 16) were quantified by stable isotope dilution assays (SIDA). Subsequent analysis of the 22 compounds by odor activity values (OAVs) revealed 14 compounds with OAVs ≥ 1 and the highest concentrations were obtained for 2,3-butanedione, 2-phenylethanol, 3-methylbutanal and acetoin respectively. Two recombination models of the bagels (i.e. 24 h and 48 h bagels) showed similarity to the corresponding bagels. Omission tests confirmed that 2,3-butanedione (buttery), acetoin (buttery), 2-acetyl-1-pyrroline (roasty), 5-methyl-2-furanmethanol (bread-like), (Z)-4-heptenal (biscuit-like) and 4-hydroxy-2,5-dimethyl-3(2H)-furanone, were the key aroma compounds. Additionally, acetic acid, butanoic acid, 2-phenylethanol (honey-like), 3-methylbutanoic acid, 2/3-methylbutanal, vanillin, 3-methylbutanol, methional were also important odorants of the bagel.
CONCLUSION: Whilst the long, cold fermented bagels exhibited roasty, malty, buttery, baked potato-like, smoky and biscuit-like notes, the control bagels produced similar but less intense odor notes.
RESULTS: The constructed integration system comprises of a lactococcal promoter (PnisA or P170), phage attachment site (attP) from bacteriophage TP901-1, a signal peptide (USP45 or SPK1) for translocation of the target protein, and a PrtP344 anchor domain in the case of the integrative vectors for surface display. There were eight successfully constructed integrative vectors with each having a different combination of promoter and signal peptide; pS1, pS2, pS3 and pS4 for secretion, and pSD1, pSD2, pSD3 and pSD4 for surface display of desired protein. The integration of the vectors into the host genome was assisted by a helper vector harbouring the integrase gene. A nuclease gene was used as a reporter and was successfully integrated into the L. lactis genome and Nuc was secreted or displayed as expected. The signal peptide SPK1 was observed to be superior to USP45-LEISSTCDA fusion in the secretion of Nuc. As for the surface display integrative vector, all systems developed were comparable with the exception of the combination of P170 promoter with USP45 signal peptide which gave very low signals in whole cell ELISA.
CONCLUSION: The engineered synthetic integrative vectors have the potential to be used for secretion or surface display of heterologous protein production in lactococcal expression system for research or industrial purposes, especially in live vaccine delivery.
METHODS: Thirty-six full-length pkmsp7D gene sequences (along with the reference H-strain: PKNH_1266000) obtained from clinical isolates of Malaysia, which were orthologous to pvmsp7H (PVX_082680) were downloaded from public databases. Population genetic, evolutionary and phylogenetic analyses were performed to determine the level of genetic diversity, polymorphism, recombination and natural selection.
RESULTS: Analysis of 36 full-length pkmsp7D sequences identified 147 SNPs (91 non-synonymous and 56 synonymous substitutions). Nucleotide diversity across the full-length gene was higher than its ortholog in Plasmodium vivax (msp7H). Region-wise analysis of the gene indicated that the nucleotide diversity at the central region was very high (π = 0.14) compared to the 5' and 3' regions. Most hyper-variable SNPs were detected at the central domain. Multiple test for natural selection indicated the central region was under strong positive natural selection however, the 5' and 3' regions were under negative/purifying selection. Evidence of intragenic recombination were detected at the central region of the gene. Phylogenetic analysis using full-length msp7D genes indicated there was no geographical clustering of parasite population.
CONCLUSIONS: High genetic diversity with hyper-variable SNPs and strong evidence of positive natural selection at the central region of MSP7D indicated exposure of the region to host immune pressure. Negative selection at the 5' and the 3' regions of MSP7D might be because of functional constraints at the unexposed regions during the merozoite invasion process of P. knowlesi. No evidence of geographical clustering among the clinical isolates from Malaysia indicated uniform selection pressure in all populations. These findings highlight the further evaluation of the regions and functional characterization of the protein as a potential blood stage vaccine candidate for P. knowlesi.