Applied Biological Chemistry

The inhibitory activity of an angiotensin I-converting enzyme (ACE), a key regulatory enzyme of blood pressure from the fermented anchovy sauce, was evaluated, and ACE inhibitory peptides were purified. The ACE activity significantly increased with an increase in salt concentration. In addition, the ACE inhibitory activity of the fermented anchovy sauce containing high salt content (25 %) significantly increased with an increase in fermentation time. The maximum activity (96 %) was reached after 15 months of fermentation. Four ACE inhibitors [Pro-Lys (PK), Gly-Cys-Lys (GCK), Asn- His-Pro (NHP), and Asp-Gly-Gly-Pro (DGGP)] in the fermented anchovy sauce were purified through various chromatographic techniques and identified by electrospray ionization tandem mass spectrometer analysis. Four newly identified peptides were synthesized and analyzed for ACE inhibitory activity in order to confirm that the purified peptides were actually ACE inhibitors. The IC50 values for ACE inhibitory activities of synthesized peptides DGGP, GCK, NHP, and PK were 164, 178, 1172, and 4092 μM, respectively.

The present study was conducted to find the effect of vitamin E and selenium (Se) on physiological, hormonal and antioxidant status of Damani and Balkhi sheep under high ambient temperature. Forty Damani and Balkhi healthy multiparous, non-pregnant sheep having similar initial body weight were selected. The sheep in each breed were further equally divided into control and treated groups. The sheep were fed a dietary plan recommended by the National Research Council (Nutrient requirements of small ruminants: sheep, goats, cervids, and new world camelids, National Academies Press, Washington, DC, 2007). In addition, the sheep in the treated groups were supplemented with Se (0.3 mg) and vitamin E (50 mg) per kg of diet for 4 weeks. The results indicated that respiration and pulse rate decreased significantly (P < 0.05) on day 28 compared to the first day of the study in the treatment groups. The concentration of T3 and T4 was significantly (P < 0.01) high in Damani sheep compared to Balkhi. In addition, except follicle-stimulating hormone (FSH) and progesterone, T3 and T4 were significantly (P < 0.01) high in the treated sheep compared to the control. Malondialdehyde (MDA), cortisol and heat-shock protein (HSP-70) increased significantly (P < 0.05) in Balkhi sheep compared to Damani. In the treated sheep, MDA, cortisol and HSP-70 were significantly (P < 0.01) low, while superoxide dismutase (SOD) and glutathione peroxidase (GPx) were significantly (P < 0.01) high in the treatment sheep. It was concluded that vitamin E and Se at the present doses improved the physiological, hormonal and antioxidant status in Damani and Balkhi sheep. In addition, Damani sheep were more tolerant to heat stress than Balkhi sheep.

The Bacillus subtilis endo-β-1,4-glucanase gene (beg), which encodes the enzyme BEG that comprises 499 amino acid residues, was mutated by error-prone polymerase chain reaction and DNA shuffling to make variants with improved functionalities. The mutated DNAs were transformed into Escherichia coli DH5α, and among the 1370 transformants, a positive clone 8-20 was obtained finally based on a halo assay and thermostability and alkaline tolerance analyses. The mutated enzyme BEG8-20 of clone 8-20 was changed at seven amino acid residues compared to the wild-type enzyme BEGwt: K45E, I102Y, M112V, D226Y, D295E, L423S, and D460G. The optimum temperature and pH of BEG8-20 were nearly the same as those of BEGwt. However, the thermostability of BEG8-20 was increased 1.53-fold (21.4 min vs. 14.0 min) based on the half-life of the residual activity at 70 °C. This increase in enzyme thermostability is advantageous for the hydrolysis of lignocellulosic materials to produce fermentable sugars. In the activity staining experiment, only a truncated smaller enzyme was observed for BEG8-20, whereas two forms, a matured (52 kDa) and a truncated smaller (34.5 kDa) enzyme, were observed for BEGwt owing to proteolytic internal cleavage at the linker region between the 297th and 356th amino acid residues. These results indicate that amino acid substitutions in the mutant enzyme have rendered the protein prone to cleavage at the C-terminal region.

The accumulation of advanced glycation end products (AGEs) plays critical roles in exacerbating obesity, arteriosclerosis, cardiovascular disease, diabetes, and their associated complications. Glycolaldehyde (GA) is the metabolic precursor of several AGEs, and its effects vary based on food and cooking methods. Here, 3T3-L1 adipocytes were used to examine the effects of GA on obesity and insulin resistance. We found that GA treatment did not increase lipid accumulation but increased the distribution of adipocyte differentiation. We also investigated the production of receptor for AGEs (RAGE) and reactive oxygen species (ROS) upon GA treatment, as well as the expression levels of peroxisome proliferator-activated receptors γ (PPARγ), CCAAT enhancer binding protein α (c/EBPα), and CCAAT enhancer binding protein β (c/EBPβ), which are transcription factors for adipogenesis, were significantly increased upon GA treatment in a concentration-dependent manner. GA arrested the cell cycle at the G0/G1 stage during the early phase of adipogenesis and suppressed the expression of p21 and p27. GA increased the expression of CDK2, phosphorylation of mitogen-activated protein kinases, and secretion of pro-inflammatory cytokines. Overall, these results suggest that GA can stimulate lipid metabolism, hence, we suggest that the stimulation of adipogenesis and insulin resistance by GA may be associated with the interaction between RAGE and adipogenic factors in adipocytes.

The gut microbiota plays an important role in the health and disease resistance of the host. Host health depends on the microbiota of the gastrointestinal tract, and imbalance in its composition may leads to certain diseases. This study analyzed the influence that dromedary camel milk has on the gut microbiota of mice. This study characterized the bacterial populations of untreated [Control (C) and camel milk-treated Raw (R), Pasteurized (P), and Fermented (F)] C57BL/6 J mice feces using high-throughput 16S rDNA sequencing on the Illumina MiSeq platform. In total, 286,606 tags were generated, with an average of 71,651 tags being generated per group, and these tags were clustered to the operational taxonomic units (OTUs) at 97% sequence similarity, resulting 1090 OTUs. Significant weight gain was observed among all of the groups, and the total cholesterol level declined in F group followed by in group P compared to group C. The F and P groups demonstrated a correlation between the beneficial microbiota structures that corresponded with lower cholesterol levels than those observed in the other groups. The major dominant bacteria correspond to the phyla Bacteroidetes and Firmicutes. The dromedary camel milk propagated the beneficial bacteria (Allobaculum and Akkermansia) and reduced harmful bacteria such as Proteobacteria, Erysipelotrichaceae, and Desulfovibrionaceae. This study provides a comparative analysis of the gut microbiota of mice based on camel milk, which may be helpful in understanding host health and diverse gut microbial conditions.

This study investigated the effects of storage temperatures, vacuum packaging, and high hydrostatic pressure (HHP) on the formation of biogenic amines (BAs) in Gwamegi. The Gwamegi samples were subjected to 400 MPa pressure for 10 min at 20 °C or vacuum packaging, and then stored at −16, 5, or 25 °C for 28 days. The amounts of six BAs (histamine, tyramine, cadaverine, putrescine, spermine, and spermidine) and the total plate counts were checked weekly. Compared with the control at 5 °C, the total plate counts were 2–3 log CFU lower for the HHP-treated samples and 1–3 log lower for the vacuum-packaged samples. Tyramine, spermine, and spermidine were found in Gwamegi stored at −16 °C and tyramine, cadaverine, spermine, and spermidine at 5 °C, whereas all six BAs were found at 25 °C. Histamine was relatively low in amount in all the treated samples. The BAs reached a high level during storage at 25 °C, with the total concentration reaching 1099 ppm in 28 days. After HHP treatment or vacuum packaging, the concentration of cadaverine and spermidine decreased, whereas that of tyramine and spermine increased. Tyramine was the dominant BA in all the samples and during storage at 5 °C in the control (0–56.67 ppm), HHP-treated (0–105 ppm), and vacuum-packaged (0–90 ppm) samples. Both HHP treatment and vacuum packaging effectively reduced the formation of some types of BAs and improved the microbial quality in Gwamegi. However, simultaneously, they also promoted the formation of others types of BAs such as tyramine and spermine.

Edible bird's nest (EBN) is a traditional food which was nourishing and functional. Particularly, there is the epidermal growth factor (EGF) in EBN, which is thought to play an important role in promoting skin repair. However, the type and content of EGF in EBN were not determined yet. In this study, the type of EGF in EBN was identified as bird EGF by enzyme-linked immunosorbent assay and this method was validated to be accurate and precise. Moreover, it was found that the content of EGF in raw-unclean EBN, raw-clean EBN and stewed EBN was 3000 pg/g–4000 pg/g and there were no significant differences, which suggested that the batches, origins, forms, stewing temperatures and stewing times of EBN had no effect on the content of EGF in EBN. However, it was due to that enzyme destroyed the primary structure of EGF, the EGF content of neutral protease and trypsin hydrolysates of EBN was lower than that of flavor enzymes, alkaline protease and pepsin hydrolysates of EGF. This study was the first to determine the type and content of EGF in EBN, and provided a theoretical basis for the selection and processing of EBN and using EBN as a source of EGF.

Monoacylglycerol lipase (MAGL) catalyzes the hydrolysis of monoacylglycerols (MAG) to free fatty acids and glycerol, which is the last step of triacylglycerol breakdown. Among sixteen members, Arabidopsis thaliana MAGL6 (AtMAGL6) and AtMAGL8 showed strong lipase activities, but several AtMAGLs including AtMAGL16 displayed very weak activities (Kim et al. in Plant. J 85:758–771, 2016). To understand the internal factors that influence Arabidopsis MAGL activities, this study investigated the significance of ‘GxSxS motif,’ which is conserved in MAGLs. First, we observed that the presence of a serine protease inhibitor, phenylmethylsulfonyl fluoride, decreased the enzyme activity of AtMAGL6 and AtMAGL8 by IC50 values of 2.30 and 2.35, respectively. Computational modeling showed that amino acid changes of the GxSxG motif in AtMAGL6 and AtMAGL8 altered the nucleophilic elbow structure, which is the active site of MAGLs. Mutating the GxSxG motif in the recombinant maltose binding protein (MBP):AtMAGL6 and MBP:AtMAGL8 proteins to SxSxG, GxAxG, and GxSxS motifs completely demolished the activities of the mutant MAGLs. In contrast, no significant differences were observed between the activities of AtMAGL16 wild type form harboring the SxSxG motif, and mutant AtMAGL16 containing the GxSxG motif. These results revealed that the glycine and serine residues of the GxSxG motif are essential for AtMAGL6 and AtMAGL8 enzyme activities, and that AtMAGL16 may not be involved in the hydrolysis of lipid substrates.