In Silico Screening of Bioactive Peptides in Stout Beer and Analysis of ACE Inhibitory Activity.

Stout beer was selected as the research object to screen angiotensin-converting enzyme (ACE) inhibitory peptides. The peptide sequences of stout beer were identified using ultra-performance liquid chromatography-quadrupole-Orbitrap mass spectrometry with de novo, and 41 peptides were identified with high confidence. Peptide Ranker was used to score the biological activity and six peptides with a score ≥ 0.5 were screened to predict their potential ACE inhibitory (ACEI) activity. The toxicity, hydrophilicity, absorption, and excretion of these peptides were predicted. In addition, molecular docking between the peptides and ACE revealed a significant property of the peptide DLGGFFGFQR. Furthermore, molecular docking conformation and molecular dynamics simulation revealed that DLGGFFGFQR could be tightly bound to ACE through hydrogen bonding and hydrophobic interaction. Lastly, the ACEI activity of DLGGFFGFQR was confirmed using in vitro evaluation and the IC50 value was determined to be 24.45 µM.

Effects of electron-beam irradiation on volatile flavor compounds of salmon fillets by the molecular sensory science technique.

Vacuum-packed salmon was treated by electron beam irradiation preservation technology, to study the effects of electron-beam irradiation on odor active compounds of salmon by two types of methods for extraction: headspace-solid phase micro extraction (HS-SPME) and solvent assisted flavor evaporation (SAFE). Volatile flavor compounds examined by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-olfactometry (GC-O), combined with aroma extract dilution method (AEDA) and odor activity value (OAV) for identification of important odorants. In addition, the correlation between sensory attributes and volatile compounds of salmon irradiated at different doses was analyzed by partial least squares regression (PLSR). The results showed that after SPME and SAFE extraction, a total of 49 and 70 volatile flavor compounds were detected in salmon before and after electron beam irradiation. AEDA and OAV were further identified, among which 10 odorants were considered as important volatile flavor compounds and played an important role in the formation of aroma contours such as meaty, fatty, and grassy in salmon. In addition, methanethiol, 3-methyl butyraldehyde, 3-methyl propyl aldehyde, dimethyl disulfide, dimethyl trisulfide, and 2-pentyl furan were identified as the important volatile flavor compounds in salmon irradiated with 4 kGy, and were also the unique compounds that constituted irradiation off-odor. In general, salmon irradiated with 1 kGy showed the best aroma profile. PRACTICAL APPLICATION: SPME and SAFE were used as two types of extraction methods for volatile compounds of salmon, which complemented each other. Additionally, combined with AEDA and OAV, characteristic flavor compounds were identified. Furthermore, the odor fingerprint of salmon with E-beam irradiation was established for the first time.

New Monoclonal Antibodies against a Novel Subtype of Shiga Toxin 1 Produced by Enterobacter cloacae and Their Use in Analysis of Human Serum.

Shiga toxin (Stx) is a major virulence factor of several bacterial pathogens that cause potentially fatal illness, including Escherichia coli and Shigella spp. The continual emergence of new subtypes of Stxs presents challenges for the clinical diagnosis of infections caused by Stx-producing organisms. Here, we report the development of four new monoclonal antibodies (MAbs) against Stx1e, a novel subtype of Stx1 that was produced by an Enterobacter cloacae strain and had limited reactivity with existing anti-Stx1 antibodies. Western blot analysis indicates that these MAbs were Stx1 specific, bound to the A subunit, and had distinct preferences for subtypes of Stx1. Of the four MAbs, Stx1e-2 was capable of partially neutralizing cytotoxicities derived from Stx1e in Vero cells. Enzyme-linked immunosorbent assays assembled with these high-affinity MAbs detected Stx1e at concentrations as low as 4.8 pg/ml in phosphate-buffered saline and 53.6 pg/ml in spiked human serum samples and were also capable of distinguishing Stx1e-producing strains in enriched cultures. These assays may therefore have clinical value in diagnosing Stx1e-producing bacterial infection. Additionally, characteristics of Stx1e, such as the origin of stx1e genes, conditions for toxin expression, receptor binding, and cytotoxicity, were investigated with the new antibodies developed in this study. This information should be useful for further understanding the clinical significance and prevalence of Stx1e-harboring E. cloacae and other organisms. IMPORTANCE Stxs are among the most clinically important virulence factors of Shigella and enterohemorrhagic Escherichia coli. There are many varieties of Stx, and although Stx1a and Stx2a are the most common and widely distributed types of Stx, new variants of Stx are continually emerging. These new variants of Stx can be challenging to detect, since most Stx detection kits are optimized for the detection of Stx1a and Stx2a. Stx1e, recently discovered in an atypical host (Enterobacter cloacae), is undetectable by many Stx assays. To formulate new assays for the detection of Stx1e, we generated four new MAbs that recognize this Stx subtype. Using these antibodies, we generated an assay capable of detecting Stx1e at low picogram-per-milliliter concentrations. This assay is also compatible with a human serum matrix, suggesting that it may have utility for the clinical detection and diagnosis of Stx1e-associated infections.

A New Immunoassay for Detecting All Subtypes of Shiga Toxins Produced by Shiga Toxin-Producing E. coli in Ground Beef.

BACKGROUND: Shiga toxin (Stx) is a common virulence factor of all Shiga toxin producing E. coli (STEC) that cause a wide spectrum of disease, including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Although several commercial kits are available for detection of Stx produced by STEC, none of them are capable of recognizing all subtypes of Stxs, which include three subtypes of Stx1 and seven subtypes of Stx2. METHODS AND FINDINGS: New monoclonal and polyclonal antibodies against Stx1 and Stx2 were developed. A universal sandwich ELISA capable of detecting all known subtypes of Stx1 and Stx2 was established using a pool of newly developed antibodies. To precisely monitor the sensitivity of the assay for each subtype of Stxs, recombinant toxoids were created and used as standards in ELISAs. Because of the high affinity of the antibodies incorporated, the ELISA assay is highly sensitive with a limit of detection for the different subtypes of Stx1a and Stx2a between 10 and 50 pg/mL in phosphate buffered saline (PBS). The assay was also able to identify STEC based on the production of Stxs using the supernatants of culture fluids or even single colonies on agar plates without lengthy enrichment in liquid medium. When applied to ground beef samples, this newly developed ELISA was capable of distinguishing beef samples spiked with a single bacterial cell. CONCLUSIONS: A highly sensitive and universal assay for all subtypes of Stx1 and Stx2 was developed. It has significantly improved upon the current technologies by avoiding false negative results due to the narrow detection range of the assay. The assay developed in this study can be useful for prompt detection of new and emerging serotypes and screening ground beef samples for contamination of STEC at an early stage in the food supply chain, thus avoiding the need for possible recall.