Bioactive peptides (BPs) are molecules of paramount importance with great potential for the development of functional foods, nutraceuticals or therapeutics for the prevention or treatment of various ...diseases. A functional BP-rich dairy product was produced by lyophilisation of bovine milk fermented by the autochthonous strains
subsp.
ZGBP5-51,
ZGBP5-52 and
ZGBP5-53 isolated from the same artisanal fresh cheese. The efficiency of the proteolytic system of the implemented strains in the production of BPs was confirmed by a combined high-throughput mass spectrometry (MS)-based peptidome profiling and an in silico approach. First, peptides released by microbial fermentation were identified via a non-targeted peptide analysis (NTA) comprising reversed-phase nano-liquid chromatography (RP nano-LC) coupled with matrix-assisted laser desorption/ionisation-time-of-flight/time-of-flight (MALDI-TOF/TOF) MS, and then quantified by targeted peptide analysis (TA) involving RP ultrahigh-performance LC (RP-UHPLC) coupled with triple-quadrupole MS (QQQ-MS). A combined database and literature search revealed that 10 of the 25 peptides identified in this work have bioactive properties described in the literature. Finally, by combining the output of MS-based peptidome profiling with in silico bioactivity prediction tools, three peptides (
QFLPYPYYAKPA
,
VAPFPEVFGK
,
ARHPHPHLSF
), whose bioactive properties have not been previously reported in the literature, were identified as potential BP candidates.
Lactiplantibacillus plantarum D13 shows antistaphylococcal and antilisterial activity, probably due to the synthesis of a presumptive bacteriocin with antibiofilm capacity released in the cell-free ...supernatant (CFS), whose inhibitory effect is enhanced by cocultivation with susceptible strains. An in silico analysis of the genome of strain D13 confirmed the pln gene cluster. Genes associated with plantaricin biosynthesis, structure, transport, antimicrobial activity, and immunity of strain D13 were identified. Furthermore, the predicted homology-based 3D structures of the cyclic conformation of PlnE, PlnF, PlnJ, and PlnK revealed that PlnE and PlnK contain two helices, while PlnF and PlnJ contain one and two helices, respectively. The potential of the strain to modulate the intestinal microbiota in healthy or dextran sulphate sodium (DSS)-induced colitis mouse models was also investigated. Strain D13 decreased the disease activity index (DAI) and altered the gut microbiota of mice with DSS-induced colitis by increasing the ratio of beneficial microbial species (Allobaculum, Barnesiella) and decreasing those associated with inflammatory bowel disease (Candidatus Saccharimonas). This suggests that strain D13 helps to restore the gut microbiota after DSS-induced colitis, indicating its potential for further investigation as a probiotic strain for the prevention and treatment of colitis.
Human milk not only provides a perfect balance of nutrients to meet all the needs of the infant in the first months of life but also contains a variety of bacteria that play a key role in tailoring ...the neonatal faecal microbiome. Microbiome analysis of human milk and infant faeces from mother-breastfed infant pairs was performed by sequencing the V1-V3 region of the 16S rRNA gene using the Illumina MiSeq platform. According to the results, there is a connection in the composition of the microbiome in each mother-breastfed infant pair, supporting the hypothesis that the infant's gut is colonised with bacteria from human milk. MiSeq sequencing also revealed high biodiversity of the human milk microbiome and the infant faecal microbiome, whose composition changes during lactation and infant development, respectively. A total of 28 genetically distinct strains were selected by hierarchical cluster analysis of RAPD-PCR (Random Amplified Polymorphic DNA-Polymerase Chain Reaction) electrophoresis profiles of 100 strains isolated from human milk and identified by 16S RNA sequencing. Since certain cellular molecules may support their use as probiotics, the next focus was to detect (S)-layer proteins, bacteriocins and exopolysaccharides (EPSs) that have potential as therapeutic biomolecules. SDS-PAGE (Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis) coupled with LC-MS (liquid chromatography-mass spectrometry) analysis revealed that four
strains expressed S-layer proteins, which were identified for the first time in strains isolated from human milk. The potential biosynthesis of plantaricin was detected in six
strains by PCR analysis and
antibacterial studies.
H NMR (Proton Nuclear Magnetic Resonance) analysis confirmed EPS production in only one strain,
MC1. The overall microbiome analysis suggests that human milk contributes to the establishment of the intestinal microbiota of infants. In addition, it is a promising source of novel
strains expressing specific functional biomolecules.
This study aimed to define a consortium of lactic acid bacteria (LAB) that will bring added value to dried fresh cheese through specific probiotic properties and the synthesis of bioactive peptides ...(biopeptides). The designed LAB consortium consisted of three
strains: S-layer carrying
D6, exopolysaccharides producing
D12 and plantaricin expressing
D13, and one
strain,
ZGZA7-10. Chosen autochthonous LAB strains exhibited efficient adherence to the Caco-2 cell line and impacted faecal microbiota biodiversity. The cheese produced by the LAB consortium showed better physicochemical, textural and sensory properties than the cheese produced by a commercial starter culture. Liquid chromatography coupled with matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (LC-MALDI-TOF/TOF) showed the presence of 18 specific biopeptides in dried fresh cheeses. Their identification and relative quantification was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using multiple reaction monitoring (MRM). The results also showed that their synthesis resulted mainly from β-casein and also α-S1 casein degradation by proteolytic activities of the LAB consortium. The designed LAB consortium enhanced the functional value of the final product through impact on biopeptide concentrations and specific probiotic properties.
Increasing awareness of the importance of nutrition in health promotion and disease prevention has driven to the development of foods for special medical purposes (FSMPs). In this study, the ...probiotic strain
ssp.
(
431
) was incorporated into FSMPs to develop an innovative product. The aim was to investigate the influence of the FSMP matrix on the specific probiotic properties of
431
.
A series of
experiments were performed as part of the probiotic approach. After evaluation of antibiotic susceptibility profiles, functional properties such as survival under simulated gastrointestinal tract (GIT) conditions, bile salt deconjugation activities, cholesterol assimilation, antagonistic activity against spoilage bacteria and adhesion to Caco-2 cell line monolayers and extracellular matrix proteins were investigated.
The
431
strain, both the lyophilised strain and the strain isolated from the FSMP matrix, effectively survived the simulated adverse gastrointestinal conditions without significant effects of the food matrix. The effect of the FSMP matrix on the deconjugation activity of the bile salts of
431
was minimal; however, cholesterol assimilation was increased by 16.4 %.
431
had antibacterial activity against related lactic acid bacteria regardless of whether it was used in FSMPs or not. Conversely, the probiotic strain isolated from FSMP matrix had significantly higher inhibitory activity against six potential pathogens than the lyophilised culture. The autoaggregation ability of the
431
cells was not affected by the FSMP matrix. The adhesion of
431
bacterial cells to the extracellular matrix proteins was reduced after treatment with proteinase K, with the highest adhesion observed to laminin. The adhesion of
431
reduced the binding of
3014 by 1.81 log units and the binding of
. Typhimurium FP1 to Caco-2 cell lines by 1.85 log units, suggesting the potential for competitive exclusion of these pathogens.
The results support the positive effect of the FSMP matrix on the specific probiotic properties of
431
, such as antibacterial activity, bile salt deconjugation and cholesterol assimilation, while the incorporation of this probiotic strain adds functional value to the FSMPs. The synergistic effect achieved by the joint application of
431
and innovative FSMP matrix contributed to the development of the novel formulation of an improved functional food product with added value.
Proteases of Lactobacillus and Lactococcus strains catalyze casein degradation in fermented dairy products, which can result in the production of bioactive peptides. Proteolytic properties of a ...selection of lactic acid bacteria (LAB) strains previously isolated in Croatia, including Lactococcus lactis and Lactobacillus strains, are described. All strains of Lactobacillus and Lactococcus showed an Fmc+ phenotype that can be associated with efficient growth in milk. The significant caseinolytic effect, after incubation of culture supernatant or concentrated cell biomass, was observed for Levilactobacillus brevis D6 and Lactiplantilactobacillus plantarum D13 after growth in the optimal growth medium, while for Lc. lactis ZGBP5-32 and Levilactobacillus brevis SF9B strains after growth in skimmed milk. To assess the LAB growth in skimmed milk, the acidification rate was monitored. Statistically, significant acidification capacity was determined for L. plantarum D13 in the optimal medium and by the proteolytic strain L. helveticus M92 in skimmed milk. After extraction of proteinases from the strains with caseinolytic activity, protein samples were analysed by the SDS-PAGE method. The protein extract of the strains Lc. lactis ZGBP5-32 and ZGZA7-10, retained proteolytic activity even at very low concentrations. The ultrafiltration improved protein extraction which possibly contained putative protease, as a decrease in contaminating proteins was confirmed by SDS-PAGE in samples of L. brevis D6 and SF9B, L. fermentum D12 and L. plantarum D13.
Autori u radu donose preliminarne rezultate arheoloških istraživanja lokaliteta Stari
grad Ljubuški koje je Studij arheologije Filozofskog fakulteta Sveučilišta u Mostaru
proveo u dvije istraživačke ...kampanje, tijekom lipnja 2020. i ožujka 2021. godine. Arheološka
istraživanja i konzervatorsko-restauratorski radovi obavljeni su u okviru dva EU projekta prekograničnih suradnji: FORT-NET i Heritage REVIVED. Iako je riječ o manjim istraživanjima, ona su polučila značajne rezultate koji u mnogome već sada mijenjaju dosadašnje spoznaje, osobito one o vremenu izgradnje i korištenja prostora Staroga grada Ljubuški.
Lactic acid bacteria (LAB) produce a broad spectrum of exopolysaccharides (EPSs), commonly used as texturizers in food products. Due to their potential contribution to LAB probiotic properties, like ...adhesion to human epithelial cells and competitive exclusion of pathogens from human intestinal epithelial cells, this study was focussed on the structural and functional characterization of the EPSs produced by two Limosilactobacillus fermentum strains – MC1, originating from mother's milk, and D12, autochthonous from Croatian smoked fresh cheese. Whole-genome sequencing and functional annotation of both L. fermentum strains by RAST server revealed the genes involved in EPS production and transport, with some differences in functionally related genes. EPSs were extracted from the cell surface of both bacterial strains and purified by size-exclusion chromatography. Structural characterization of the EPSs, achieved by chemical analyses and 1D and 2D NMR spectroscopy, showed that both strains produce an identical mixture of three different EPSs containing galactofuranose and glucopyranose residues. However, a comparison of the functional properties showed that the MC1 strain adhered better to the Caco-2 cell line and exhibited stronger antimicrobial effect against Salmonella enterica serovar Typhimurium FP1 than the D12 strain, which may be attributed to the potential bacteriocin activity of the MC1 strain.
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•two EPS-producing L. fermentum strains were isolated from different sources.•WGS showed that the genomes of L. fermentum MC1 and D12 are slightly different.•both strains produce an identical mixture of three different EPSs.•in vitro experiments showed different functional properties of the two strains.
Lactiplantibacillus plantarum D13 shows antistaphylococcal and antilisterial activity, probably due to the synthesis of a presumptive bacteriocin with antibiofilm capacity released in the cell-free ...supernatant (CFS), whose inhibitory effect is enhanced by cocultivation with susceptible strains. An in silico analysis of the genome of strain D13 confirmed the pln gene cluster. Genes associated with plantaricin biosynthesis, structure, transport, antimicrobial activity, and immunity of strain D13 were identified. Furthermore, the predicted homology-based 3D structures of the cyclic conformation of PlnE, PlnF, PlnJ, and PlnK revealed that PlnE and PlnK contain two helices, while PlnF and PlnJ contain one and two helices, respectively. The potential of the strain to modulate the intestinal microbiota in healthy or dextran sulphate sodium (DSS)-induced colitis mouse models was also investigated. Strain D13 decreased the disease activity index (DAI) and altered the gut microbiota of mice with DSS-induced colitis by increasing the ratio of beneficial microbial species (Allobaculum, Barnesiella) and decreasing those associated with inflammatory bowel disease (Candidatus Saccharimonas). This suggests that strain D13 helps to restore the gut microbiota after DSS-induced colitis, indicating its potential for further investigation as a probiotic strain for the prevention and treatment of colitis.
The presence of surface (S)-layer proteins as a rare property of lactic acid bacteria plays an important role in the expression of the probiotic properties of the producer strain and protects the ...cells from unfavourable environmental conditions. Therefore, the aim of this work was to evaluate the role of S-layer proteins of four Levilactobacillus brevis MB1, MB2, MB13, and MB20 strains, previously isolated from human milk. The results showed better survival under simulated gastrointestinal tract (GIT) conditions and better adhesion to Caco-2 cells, achieved when S-proteins were present on the surface of the examined probiotic Levilactobacillus brevis strains. Free cells and cells microencapsulated in alginate, with or without galactooligosaccharides (GOS) and fructooligosaccharides (FOS), survived in high numbers during freeze-drying and one-month storage at 4 °C, as well as during exposure to simulated GIT conditions (>108 CFU/g), thus fulfilling the primary technological criterion for the selection of probiotic strains. Higher survival of freeze-drying of encapsulated cells than free cells, can be attributed to the protective effect of alginate, GOS and FOS as prebiotic agents. However, GOS was more protective than FOS in maintaining the viability of microencapsulated Levilactobacillus brevis MB1, MB2, MB13 and MB20 cells in alginate during storage and under the harsh conditions of GIT and freeze-drying.
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