Assessment of the Microbiological Quality and Biochemical Parameters of Traditional Hard Xinotyri Cheese Made from Raw or Pasteurized Goat Milk

Traditional hard Xinotyri cheese was manufactured using raw or pasteurized goat milk, without starter cultures, and the changes in microbiological and biochemical characteristics were studied during ripening and storage. Mesophilic lactic acid bacteria (LAB) predominated (>8.5 log CFU/g) in freshly fermented Xinotyri cheeses (pH 4.5–4.6), regardless of milk pasteurization. Enterobacteria, pseudomonads and staphylococci were suppressed below 6 and 4–5 log CFU/g in fresh cheeses from raw and pasteurized milk, respectively. Salmonella and Listeria spp. were absent in 25 g cheese samples. Coagulase-positive staphylococci exceeded the 5-log safety threshold in fresh raw milk cheeses, which also had 10-fold higher levels of enterococci than pasteurized milk cheeses. Non-LAB groups declined <100 CFU/g, whereas yeasts increased to 5–6 log CFU/g in both cheeses during ripening. Milk pasteurization affected the protein, fat, ash, moisture, nitrogen fractions, total free fatty acids and total free amino acids content of cheeses. Primary proteolysis, detectable by urea-PAGE, was more intense in raw milk cheeses than in pasteurized milk cheeses. However, the hydrophilic and hydrophobic peptides and their ratio in the water-soluble fraction were similar in both cheeses. Cheeses discriminated clearly according to the milk kind (raw, pasteurized) and the stage of ripening, based on the examined biochemical characteristics.

Salt-Mediated Organic Solvent Precipitation for Enhanced Recovery of Peptides Generated by Pepsin Digestion

Conventional solvent-based precipitation makes it challenging to obtain a high recovery of low mass peptides. However, we previously demonstrated that the inclusion of salt ions, specifically ZnSO4, together with high concentrations of acetone, maximizes the recovery of peptides generated from trypsin digestion. We herein generalized this protocol to the rapid (5 min) precipitation of pepsin-digested peptides recovered from acidic matrices. The precipitation protocol extended to other organic solvents (acetonitrile), with high recovery from dilute peptide samples permitting preconcentration and purification. Mass spectrometry profiling of pepsin-generated peptides demonstrated that the protocol captured peptides as small as 800 u, although with a preferential bias towards recovering larger and more hydrophobic peptides. The precipitation protocol was applied to rapidly quench, concentrate, and purify pepsin-digested samples ahead of MS. Complex mixtures of yeast and plasma proteome extracts were successfully precipitated following digestion, with over 95% of MS-identified peptides observed in the pellet fraction. The full precipitation workflow—including the digestion step—can be completed in under 10 min, with direct MS analysis of the recovered peptide pellets showing exceptional protein sequence coverage.

Molecular Dynamics Simulations of hydrophobic peptides that form β-hairpin structures in solution

Peptides designed with residues that have high propensity to occur in β-turns, form β-hairpin structures in apolar solvents as well in polar organic solvents such as dimethyl sulfoxide (DMSO), methanol and varying percentages of DMSO in chloroform (CHCl3). Presumably due to limited solubility, their conformations have not been investigated by experimental methods in water. We have examined the conformations of such designed peptides that fold into well-defined β-hairpin structures facilitated by β-turns, in the crystalline state and in solution, by Molecular Dynamics Simulations (MDS). The peptides fold into β-hairpin structures in water, starting from extended conformation. In DMSO, folding into β-hairpin structures was not observed, starting from extended conformation. However, when the starting structure is in β-hairpin conformation, unfolding is not observed during MDS in DMSO. Water clearly favours folding of short, hydrophobic peptides into β-turn and β-hairpin conformations from extended structures. DMSO does not have a denaturing effect on short, hydrophobic peptides.

Bacterial Type I Toxins: Folding and Membrane Interactions

Bacterial type I toxin-antitoxin systems are two-component genetic modules that encode a stable toxic protein whose ectopic overexpression can lead to growth arrest or cell death, and an unstable RNA antitoxin that inhibits toxin translation during growth. These systems are widely spread among bacterial species. Type I antitoxins are cis- or trans-encoded antisense small RNAs that interact with toxin-encoding mRNAs by pairing, thereby inhibiting toxin mRNA translation and/or inducing its degradation. Under environmental stress conditions, the up-regulation of the toxin and/or the antitoxin degradation by specific RNases promote toxin translation. Most type I toxins are small hydrophobic peptides with a predicted α-helical transmembrane domain that induces membrane depolarization and/or permeabilization followed by a decrease of intracellular ATP, leading to plasmid maintenance, growth adaptation to environmental stresses, or persister cell formation. In this review, we describe the current state of the art on the folding and the membrane interactions of these membrane-associated type I toxins from either Gram-negative or Gram-positive bacteria and establish a chronology of their toxic effects on the bacterial cell. This review also includes novel structural results obtained by NMR concerning the sprG1-encoded membrane peptides that belong to the sprG1/SprF1 type I TA system expressed in Staphylococcus aureus and discusses the putative membrane interactions allowing the lysis of competing bacteria and host cells.

Influence of the Breed of Sheep on the Characteristics of Zamorano Cheese

This work aimed to study the effects of using ewe’s milk from Churra, Assaf, or both breeds on the physicochemical and sensory characteristics of Zamorano cheese at the end of ripening. Zamorano cheese is a hard variety with protected designation of origin (PDO) produced in the province of Zamora (Spain) with raw or pasteurized ewe’s milk. Five batches of Zamorano cheese were produced with pasteurized ewe’s milk. One batch was elaborated using milk from the Churra breed, the other using milk from the Assaf breed, and the remaining three employed milk mixtures of Churra and Assaf breeds in the proportions 75:25, 50:50 and, 25:75, respectively. Cheeses made with a higher proportion of Churra milk showed a predominance of hydrophilic peptides, while hydrophobic peptides predominated in cheeses with a greater percentage of milk from the Assaf breed. The largest content of most free amino acids was found in cheeses produced with the highest percentage of Churra milk. These cheeses presented the highest values for fat acidity index and free fatty acids content and showed greater elasticity and adhesiveness, as well as lower granularity and hardness. In the sensory evaluation, aftertaste and persistence were higher in these cheeses, being scored with the best overall values.

Occurrence of Peptide-Peptide Interactions during the Purification of Self-Assembling Peptide f1-8 from a β-Lactoglobulin Tryptic Hydrolysate

Self-assembling peptides have gained attention because of their nanotechnological applications. Previous work demonstrated that the self-assembling peptide f1-8 (Pf1-8) that is generated from the tryptic hydrolysis of β-lactoglobulin can form a hydrogel after several purification steps, including membrane filtration and consecutive washes. This study evaluates the impact of each processing step on peptide profile, purity, and gelation capacity of each fraction to understand the purification process of Pf1-8 and the peptide-peptide interactions involved. We showed that peptide-peptide interactions mainly occurred through electrostatic and hydrophobic interactions, influencing the fraction compositions. Indeed, the purity of Pf1-8 did not correlate with the number of wash steps. In addition to Pf1-8, two other hydrophobic peptides were identified, peptide f15-20, and peptide f41-60. The gelation observed could be induced either through peptide-peptide interactions or through self-assembling, both being driven by non-covalent bond and more specifically hydrophobic interactions.

Masking terminal neo-epitopes of linear peptides through glycosylation favours immune responses towards core epitopes producing parental protein bound antibodies

Glycosylation of hydrophobic peptides at one terminus effectively increases their water-solubility, and conjugation through the opposing end to a carrier protein, renders them more immunogenic. Moreover, the glycosylation minimizes antibody responses to potentially deleterious, non-productive terminal neo-epitope regions of the peptides, and consequently shifts peptide immunogenicity towards the core amino acid residues. As proof of concept, glycopeptide-protein conjugates related to influenza hemagglutinin (HA), neuraminidase (NA), and the dimerization loop region of human epidermal growth factor receptor 2 (Her2), demonstrated a favorable production of core peptide specific antibodies as determined by ELISA studies. Furthermore, glycosylated Her2 peptide conjugate antisera were also shown to recognize full length Her2 protein by ELISA and at the cell surface through flow cytometry analysis. In contrast, unmasked peptide conjugates generated significant antibody populations that were specific to the terminal neo-epitope of the peptide immunogen that are notably absent in parental proteins. Antibodies generated in this manner to peptides in the dimerization loop of Her2 are also functional as demonstrated by the growth inhibition of Her2 expressing SKBR3 carcinoma cells. This method provides a technique to tailor-make epitope-specific antibodies that may facilitate vaccine, therapeutic and diagnostic antibody development.

Export of Rgg Quorum Sensing Peptides is Mediated by the PptAB ABC Transporter in Streptococcus Thermophilus Strain LMD-9

In streptococci, intracellular quorum sensing pathways are based on quorum-sensing systems that are responsible for peptide secretion, maturation, and reimport. These peptides then interact with Rgg or ComR transcriptional regulators in the Rap, Rgg, NprR, PlcR, and PrgX (RRNPP) family, whose members are found in Gram-positive bacteria. Short hydrophobic peptides (SHP) interact with Rgg whereas ComS peptides interact with ComR regulators. To date, in Streptococcus thermophilus, peptide secretion, maturation, and extracellular fate have received little attention, even though this species has several (at least five) genes encoding Rgg regulators and one encoding a ComR regulator. We studied pheromone export in this species, focusing our attention on PptAB, which is an exporter of signaling peptides previously identified in Enterococcus faecalis, pathogenic streptococci and Staphylococcus aureus. In the S. thermophilus strain LMD-9, we showed that PptAB controlled three regulation systems, two SHP/Rgg systems (SHP/Rgg1358 and SHP/Rgg1299), and the ComS/ComR system, while using transcriptional fusions and that PptAB helped to produce and export at least three different mature SHPs (SHP1358, SHP1299, and SHP279) peptides while using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using a deep sequencing approach (RNAseq), we showed that the exporter PptAB, the membrane protease Eep, and the oligopeptide importer Ami controlled the transcription of the genes that were located downstream from the five non-truncated rgg genes as well as few distal genes. This led us to propose that the five non-truncated shp/rgg loci were functional. Only three shp genes were expressed in our experimental condition. Thus, this transcriptome analysis also highlighted the complex interconnected network that exists between SHP/Rgg systems, where a few homologous signaling peptides likely interact with different regulators.