Prolylleucine

Two different Lactococcus lactis host strains, L. lactis subsp. lactis MG1363 and L. lactis subsp. cremoris SK1128, both containing plasmid pNZ521, which encodes the extracellular serine proteinase (PrtP) from strain SK110, were used to study the medium and growth-rate-dependent activity of three different enzymes involved in the proteolytic system of lactococci. The activity levels of PrtP and both the intracellular aminopeptidase PepN and the X-prolyl-dipeptidyl aminopeptidase PepXP were studied during batch and continuous cultivation. In both strains, the PrtP activity level was regulated by the peptide content of the medium. The highest activity level was found during growth in milk, and the lowest level was found during growth in the peptide-rich laboratory medium M17. Regulation of the intracellular peptidase activity appeared to be a strain-dependent phenomenon. In cells of strain MG1363, the activity levels of PepN and PepXP were regulated in a similar way to that observed for PrtP. In cells of strain SK1128, the levels of both peptidases were not significantly influenced by the peptide content of the medium. The presence of specific concentrations of the dipeptide prolylleucine could mimic the low activity levels of the regulated proteolytic enzymes, even to the activity level found on M17 medium. The effect of the presence of the dipeptide prolylleucine in the medium on the activity level of the regulated proteolytic enzymes was confirmed at fixed growth rates in chemostat cultures.

Adulteration of high-quality meat products using lower-priced meats, such as pork, is a crucial issue that could harm consumers. The consumption of pork is strictly forbidden in certain religions, such as Islam and Judaism. Therefore, the objective of this research was to develop untargeted metabolomics using liquid chromatography-high resolution mass spectrometry (LC-HRMS) combined with chemometrics for analysis of pork in beef meatballs for halal authentication. We investigated the use of non-targeted LC-HRMS as a method to detect such food adulteration. As a proof of concept using six technical replicates of pooled samples from beef and pork meat, we could show that metabolomics using LC-HRMS could be used for high-throughput screening of metabolites in meatballs made from beef and pork. Chemometrics of principal component analysis (PCA) was successfully used to differentiate beef meatballs and pork meatball samples. Partial least square-discriminant analysis (PLS-DA) clearly discriminated between halal and non-halal beef meatball samples with 100% accuracy. Orthogonal projection to latent structures-discriminant analysis (OPLS-DA) perfectly discriminated and classified meatballs made from beef, pork, and a mixture of beef-pork with a good level of fitness (R2X = 0.88, R2Y = 0.71) and good predictivity (Q2 = 0.55). Partial least square (PLS) and orthogonal PLS (OPLS) were successfully applied to predict the concentration of pork present in beef meatballs with high accuracy (R2 = 0.99) and high precision. Thirty-five potential metabolite markers were identified through VIP (variable important for projections) analysis. Metabolites of 1-(1Z-hexadecenyl)-sn-glycero-3-phosphocholine, acetyl-l-carnitine, dl-carnitine, anserine, hypoxanthine, linoleic acid, and prolylleucine had important roles for predicting pork in beef meatballs through S-line plot analysis. It can be concluded that a combination of untargeted metabolomics using LC-HRMS and chemometrics is promising to be developed as a standard analytical method for halal authentication of highly processed meat products.

Acrylamide has been reported as an important dietary risk factor from carbohydrate-rich processing food. However, systemic biological effects on the serum metabolomics induced by acrylamide have poorly been understood. In the present study, we evaluated the metabolic profiles in a rat serum after exposure to acrylamide using ultrahigh-performance liquid chromatography combined with quadrupole-orbitrap high-resolution mass spectrometry. The serum biochemical parameters of the treated and control groups were also determined using an automatic biochemical analyzer. Compared with the control group, 10 metabolites were significantly upregulated, including citric acid, d-(-)-fructose, gluconic acid, l-ascorbic acid 2-sulfate, 2-hydroxycinnamic acid, valine, l-phenylalanine, prolylleucine, succinic acid, and cholic acid, while 5 metabolites were significantly downregulated, including 3-hydroxybutyric acid, 4-oxoproline, 2,6-xylidine, 4-phenyl-3-buten-2-one, and N-ethyl-N-methylcathinone in the serum of 4-week-old rats exposed to acrylamide in the high-dose group (all P < 0.05). Importantly, acrylamide exposure affected metabolites mainly involved in the citrate cycle, valine, leucine, and isoleucine biosyntheses, phenylalanine, tyrosine and tryptophan biosyntheses, and pyruvate metabolism. These results suggested that exposure to acrylamide in rats exhibited marked systemic metabolic changes and affected the cardiovascular system. This study will provide a theoretical basis for exploring the toxic mechanism and will contribute to the diagnosis and prevention of acrylamide-induced cardiovascular toxicity.