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The aim of this research was to purify and characterize the mode of action of enterocin S37, a bacteriocin produced by Enterococcus faecalis S37, a strain recently isolated from the chicken feces. Enterocin S37 has a molecular weight comprised between 4 and 5 kDa. It remained active after 1 h at 80oC and at pH values ranging from 4.0 to 9.0. Furthermore, cell free supernatant of Enterococcus faecalis S37 and purified enterocin S37 were active against Gram-positive bacteria including Listeria monocytogenes EGDe, L. innocua F, Enterococcus faecalis JH2-2, and Lactobacillus brevis F145. The purification of enterocin S37 was performed by ammonium sulfate precipitation followed up by hydrophobic interaction chromatography procedures. Treatment of enterocin S37 with proteinase K, α-chymotrypsin, and papain confirmed its proteinaceous nature, while its treatment with lysozyme and lipase resulted in no alteration of activity. Enterocin S37 is hydrophobic, anti-Listeria and likely acting by depletion of intracellular K+ ions upon action on KATP channels. This study contributed to gain more insights into the mode of action of enterocins. [ABSTRACT FROM AUTHOR]

A strain of Enterococcus faecium isolated from Bulgarian yellow cheese “kashkaval” produced a bacteriocin-like substance named enterococcin A 2000. The antibacterial substance had a low molar mass (<2 kDa), was relatively stable toward heat but was sensitive to selected proteolytic enzymes. It was active against Gram-positive bacteria including enterococci, such as Listeria, Bacillus and Streptococcus, and also against Gram-negative E. coli. Production of enterococcin A 2000 has a maximum near the end of the exponential phase of producer growth. The peptide was purified by ammonium sulfate precipitation, butanol extraction, followed by cation-exchange chromatography and reversed-phase chromatography. A partial sequence of purified enterococcin A 2000 indicated that this substance does not belong to the class IIa of bacteriocins presenting the consensus anti- Listeria motif YGNGV. [ABSTRACT FROM AUTHOR]

Two sites in Northeastern of Bulgaria were investigated as a source of Streptomyces populations to screen for production of heat-resistant collagenase. The distribution and diversity of collagenolytic activity in Streptomyces populations from protein-rich residue were studied. Collagenolytic activity was found in 14% of the 350 tested strains. Poor collagenase producers generally dominated (70%). Highly active collagenase producers were rarely found (12%) and they were mainly from Albus and Roseus groups. The isolated Streptomyces cultures developed a constitutive collagenase activity. Two promising, highly active, producers of heat-resistant collagenase were selected and identified as Streptomyces candidus 91 and S. cremeus 3. The investigated Streptomyces collagenases were active over a broad range of temperature (40-80°C) and a narrow range of pH values (6.5-8.5), with optima at 40°C and pH 7.5. [ABSTRACT FROM AUTHOR]

Bioactive peptides and free amino acids obtained from Bulgarian goat, sheep and cow white brined cheeses, produced with same starter culture, during ripening were evaluated. The concentration of total free amino acids was increasing in all tested cheeses in the first 30 days of ripening. In the next 30 days in sheep cheeses, the concentration increased as recorded for most of the amino acids. Amino acids with highest levels detected throughout the whole ripening period in goat, sheep and cow cheese types were leucine, phenylalanine, arginine, valine and lysine. MALDI-TOF analysis of evaluated cheeses resulted in detection of production of bioactive peptide derivates from milk proteins: 51 peptides in cow, 31 peptides in sheep and 22 peptides in goat cheeses. Peptide αs1-CN (f35-40) was found only in cow cheese. In cow cheese, higher intensity was detected for αs1-CN (f1-9) and β-CN (f194-203 and f203-219) peptides. In goat cheese was recorded αs1-CN peptides, and there was a tendency to increase the peptides released from β-CN, with the highest intensity of fragments αs1-CN (f1-9 and f24-30) and β-CN (f194-209 and f203-219). In sheep cheese, the recorded primarily peptides were αs1-CN and peptides released from β-CN. Different bioactive peptides, derivate from casein, were detected as follows: 6 peptides were ACE inhibitory peptides, 3 peptides were αS1-casokinins, 1 peptide was caseinophopeptide, 1 peptide was immunopeptide. Twelve bioactive peptides were recorded to be derivates from β-casein: 1 peptide was ACE peptide, 4 peptides were caseino-phosphopeptides, 1 peptide was immunopeptide, 1 peptide β-casokinin, 1 antibacterial peptide and 4 multifunctional peptides. Of peptides released by proteolysis of αS2-CN was found 1 bioactive peptide with antimicrobial activity. On our best knowledge, this paper contributes new data about free amino acids and bioactive peptides in the connection between type of milk and period for cheese ripening in the Bulgarian goat, sheep and cow white brined cheeses.

Grain hardness is an important quality trait of cereal crops. In wheat, it is mainly determined by the Hardness locus that harbors genes encoding puroindoline A (PINA) and puroindoline B (PINB). Any deletion or mutation of these genes leading to the absence of PINA or to single amino acid changes in PINB leads to hard endosperms. Although it is generally acknowledged that hardness is controlled by adhesion strength between the protein matrix and starch granules, the physicochemical mechanisms connecting puroindolines and the starch-protein interactions are unknown as of this time. To explore these mechanisms, we focused on PINA. The overexpression in a hard wheat cultivar (cv. Courtot with the Pina-D1a and Pinb-D1d alleles) decreased grain hardness in a dose-related effect, suggesting an interactive process. When PINA was added to gliadins in solution, large aggregates of up to 13 μm in diameter were formed. Turbidimetry measurements showed that the PINA-gliadin interaction displayed a high cooperativity that increased with a decrease in pH from neutral to acid (pH 4) media, mimicking the pH change during endosperm development. No turbidity was observed in the presence of isolated α- and γ-gliadins, but non-cooperative interactions of PINA with these proteins could be confirmed by surface plasmon resonance. A significant higher interaction of PINA with γ-gliadins than with α-gliadins was observed. Similar binding behavior was observed with a recombinant repeated polypeptide that mimics the repeat domain of gliadins, i.e., (Pro-Gln-Gln-Pro-Tyr)8. Taken together, these results suggest that the interaction of PINA with a monomeric gliadin creates a nucleation point leading to the aggregation of other gliadins, a phenomenon that could prevent further interaction of the storage prolamins with starch granules. Consequently, the role of puroindoline-prolamin interactions on grain hardness should be addressed on the basis of previous observations that highlight the similar subcellular routing of storage prolamins and puroindolines., Competing Interests: The authors have declared that no competing interests exist.

The presence of lipids within starch granules is specific to cereal endosperm starches. These starch lipids are composed of lysophospholipids, especially lysophosphatidylcholine (LysoPC) and free fatty acids that strongly impact the assembly and properties of cereal starches. However, the molecular mechanisms associated with this specific lipid routing have never been investigated. In this study, matrix-assisted laser desorption ionization mass spectrometry imaging revealed decreasing gradients in starch LysoPC concentrations from the periphery to the center of developing maize endosperms. This spatiotemporal deposition of starch LysoPC was similar to that previously observed for endoplasmic reticulum (ER)-synthesized storage proteins, i.e. zeins, suggesting that LysoPC might originate in the ER, as already reported for chloroplasts. Furthermore, a decrease of the palmitate concentration of amyloplast galactolipids was observed during endosperm development, correlated with the preferential trapping of palmitoyl-LysoPC by starch carbohydrates, suggesting a link between LysoPC and galactolipid synthesis. Using microarray, the homologous genes of the Arabidopsis ER-chloroplast lipid trafficking and galactolipid synthesis pathways were also expressed in maize endosperm. These strong similarities suggest that the encoded enzymes and transporters are adapted to managing the differences between chloroplast and amyloplast lipid homeostasis. Altogether, our results led us to propose a model where ER-amyloplast lipid trafficking directs the LysoPC towards one of two routes, the first towards the stroma and starch granules and the other towards galactolipid synthesis.