The aim of this study was to compare the effectiveness of gas chromatography (GC) and near‐infrared spectroscopy (NIR) spectroscopy in detecting butter adulteration. Butter samples with a known ...percentage of added margarine or pork fat were prepared. Chemometric techniques (principal component analysis and partial least squares regression) were applied on the NIR spectra and GC data, which enabled the qualitative separation of samples depending on the added proportion of adulterant, and the quantitative prediction of the proportion of added adulterant or certain fatty acid content. C12:0; C16:0; C18:0; C16:1; C18:1n9; C18:2n6 showed significant trend changes depending on the adulteration degree. NIR has proven to be a suitable technique for the rapid determination of butter adulteration.
Novelty impact statement
Fast and nondestructive technique as near‐infra red (NIR) spectroscopy can be used in qualitative and quantitative detection of butter originality. Obtained models by chemometrics methods allow prediction of proportion of added adulterant in butter and identify specific fatty acids. NIR may be applied in screening control of the correctness of butter labeling to improve the system of quality control in food fraud.
The consumption of raw milk from vending machine in Croatia has increased in recent years. However, its health safety is insufficiently investigated and controlled. The objective of this study was to ...investigate the suitability of raw milk consumption, from 28 vending machines from 3 counties in Croatia during one-year research period, based on the physicochemical and hygiene quality. Also, the simulation of psychrothrophic bacteria growth has been conducted in order to determine its optimal storage time because of their unfavorable effect on the milk quality. The physicochemical composition of milk samples (n=320) was determined by the infrared spectrometry (chemical composition) and cryoscopic method (freezing point), while hygiene quality was estimated with a total bacterial count (TBC) and a somatic cell count (SCC). Microbiological analyses included determining the presence of Enterococcus spp., Pseudomonas spp. and the pathogenic species. Satisfactory quality of raw milk was established in 95% (chemical quality) and 70% unadulterated samples (without added water). The prescribed hygiene quality was met by 36.6% (TBC) and 71.9% (SCC) samples. Of total 320 samples, the presence of bacteria in genus Enterococcus spp. and Pseudomonas spp. was confirmed in 93.4% and 96.9% samples, respectively, and bacteria Escherichia coli, Staphylococcus aureus, Lysteria monocytogenes was confirmed in 32.2%, 27.5% and 3.8% samples. The finding of Salmonella spp. and Yersinia enterocolitica was negative. At the end of the study improvement of the microbiological quality of raw milk from vending machines was established. The results indicate the need for systematic control and obligate thermal treatment of raw milk before consumption.
Dipeptidyl peptidase III (DPP III) is a zinc exopeptidase with an implied role in the mammalian pain-modulatory system owing to its high affinity for enkephalins and localisation in the superficial ...laminae of the spinal cord dorsal horn. Our study revealed that this human enzyme hydrolyses opioid peptides belonging to three new groups, endomorphins, hemorphins and exorphins. The enzymatic hydrolysis products of endomorphin-1 were separated and quantified by capillary electrophoresis and the kinetic parameters were determined for human DPP III and rat DPP IV. Both peptidases cleave endomorphin-1 at comparable rates, with liberation of the N-terminal Tyr-Pro. This is the first evidence of DPP III acting as an endomorphin-cleaving enzyme.
The number of aerobic mesophilic bacteria in milk is one of the indicators of the hygienic quality of milk. The aim of this work was to determine such aerobic mesophilic bacteria and their number in ...raw unpreserved milk and milk preserved with sodium azide. In 40 collected samples, the total number of aerobic mesophilic bacteria was determined using the classical method of counting colonies on a nutrient medium according to the international standard HRN EN ISO 4833-1:2013. The results showed a trend of decreasing the number of grown colonies in milk preserved with sodium azide. MALDI-TOF mass spectrometry also successfully identified 392 bacterial colonies in raw unpreserved milk samples and 330 colonies in preserved milk samples. Of these, 30 genera and 54 bacterial species were identified in the raw unpreserved milk samples, while 27 genera and 41 bacterial species were identified in the preserved samples. By using a collective approach, the present study provided a more detailed insight into milk’s hygienic quality and the presence of certain species before and after the preservation with sodium azide.
Dipeptidyl peptidase III (DPP III), a member of the metallopeptidase family M49, was considered as an exclusively eukaryotic enzyme involved in intracellular peptide catabolism and pain modulation. ...In 2003, new data on genome sequences revealed the first prokaryotic orthologs, which showed low sequence similarity to eukaryotic ones and a cysteine (Cys) residue in the zinc-binding motif HEXXGH. Here we report the cloning and heterologous expression of DPP III from the human gut symbiont Bacteroides thetaiotaomicron. The catalytic efficiency of bacterial DPP III for preferred synthetic substrate hydrolysis was very similar to that of the human host enzyme. Substitution of Cys450 from the active-site motif by serine did not substantially change the enzymatic activity. However, this residue was wholly responsible for the inactivation effect of sulfhydryl reagents. Molecular modeling indicated seven basic amino acid residues in the local environment of Cys450 as a possible cause for its high reactivity. Sequence analysis of 81 bacterial M49 peptidases showed conservation of the HECLGH motif in 68 primary structures with the majority of proteins lacking an active-site Cys originated from aerobic bacteria. Data obtained suggest that Cys450 of B. thetaiotaomicron DPP III is a regulatory residue for the enzyme activity.
It is well known that cheese yield and quality are affected by animal genetics, milk quality (chemical, physical, and microbiological), production technology, and the type of rennet and dairy ...cultures used in production. Major differences in the same type of cheese (i.e., hard cheese) are caused by the rennet and dairy cultures, which affect the ripening process. This review aims to explore current technological advancements in animal genetics, methods for the isolation and production of rennet and dairy cultures, along with possible applications of microencapsulation in rennet and dairy culture production, as well as the challenge posed to current dairy technologies by the preservation of biodiversity. Based on the reviewed scientific literature, it can be concluded that innovative approaches and the described techniques can significantly improve cheese production.
Human dipeptidyl peptidase III (DPP III) is a member of the metallopeptidase family M49, involved in protein metabolism and oxidative stress response. DPP III crystal structure shows the two ...lobe-like domains separated by a wide cleft. The human enzyme has a total of six cysteines, three in the lower (Cys19, Cys147, and Cys176) and three in the upper (Cys509, Cys519, and Cys654), catalytic, domain containing the active-site zinc ion. To elucidate the molecular basis of this enzyme’s susceptibility to sulfhydryl reagents, biochemical analysis of a set of Cys to Ala mutants was used, supported by mass spectrometry. Cys176, a residue 44 Å apart from the catalytic center of the ligand-free enzyme, was found responsible for the inactivation with the submicromolar concentration of an organomercurial compound, and three additional cysteines contri\xadbuted to sensitivity to aromatic disulfides. Upon treatment with oxidized glutathione glutathione disulfide (GSSG), cysteine residues at positions 147, 176, and 654 were found glutathionylated. The mutational analysis confirmed the involvement of Cys176 and Cys654 in human DPP III inactivation by GSSG. Observation that Cys176, a residue quite distant from the active center, contributes to enzyme inactivation, indicates that the substrate-binding site of human DPP III comprises both lower and upper protein domain.
Human dipeptidyl peptidase III (DPP III) is a member of the metallopeptidase family M49 with an implied role in the pain-modulatory system and endogenous defense against oxidative stress. Here, we ...report the heterologous expression of human DPP III and the site-directed mutagenesis results which demonstrate a functional role for Tyr318 at the active site of this enzyme. The substitution of Tyr318 to Phe decreased k cat by two orders of magnitude without altering the binding affinity of substrate, or of a competitive hydroxamate inhibitor designed to interact with S1 and S2 subsites. The results indicate that the conserved tyrosine could be involved in transition state stabilization during the catalytic action of M49 peptidases.
Binding of a competitive inhibitor Tyr-Phe-NHOH to the active site of human DPP III.
The role of the unique fully conserved tryptophan in metallopeptidase family M49 (dipeptidyl peptidase III family) ...was investigated by site-directed mutagenesis on human dipeptidyl peptidase III (DPP III) where Trp300 was subjected to two substitutions (W300F and W300L). The mutant enzymes showed thermal stability equal to the wild-type DPP III. Conservative substitution of the Trp300 with phenylalanine decreased enzyme activity 2–4 fold, but did not significantly change the
K
m
values for two dipeptidyl 2-naphthylamide substrates. However, the
K
m
for the W300L mutant was elevated 5-fold and the
k
cat
value was reduced 16-fold with Arg-Arg-2-naphthylamide. Both substitutions had a negative effect on the binding of two competitive inhibitors designed to interact with S1 and S2 subsites.
These results indicate the importance of the aromatic nature of W300 in DPP III ligand binding and catalysis, and contribution of this residue in maintaining the functional integrity of this enzyme’s S2 subsite.
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