The aim of the present study was to analyse the type of adhesive factors of selected probiotic strains. A large number of lactic acid bacteria with intestinal and dairy origin were collected and assessed for adhesion on Caco-2 cell line. From the best adherent bacteria, four strains were selected for further research: Lactobacillus gasseri G7, L. plantarum F1, L. helveticus AC and L. delbrueckii subsp. bulgaricus B14. The average number of adhered bacteria was 17 per one Caco-2 cell in the case of L. gasseri G7 and 21 per cell in the case of L. plantarum F1. Treatment with ethylenediaminetetraacetic acid (EDTA), trypsin and metaperiodic acid in separate assays revealed that cell-bonded extracellular proteins were responsible for the adhesion of the selected L. gasseri, L. plantarum and L. helveticus strains, in contrast to the L. delbrueckii subsp. bulgaricus strain, whose adhesive factors were identified as cell-bonded exopolysaccharides. The cell-wall proteins from the first three strains were isolated, fractionated and assessed for adhesion to Caco-2 cells. Based on the attachment properties of the purified proteins towards Caco-2 cells, it was clearly proved exactly which proteins are involved in the adherence. L. plantarum F1 strain contains two adhesive proteins in contrast to the other selected strains containing one adhesive protein each. The determination of the factors mediating the adhesive abilities of the selected strains provides important information about the possible ways to preserve and increase adhesive properties towards epithelium cells.
Some lactic acid bacteria strains in milk media are capable of releasing bioactive peptides. In this study, we evaluated the angiotensin-converting enzyme (ACE)inhibitory activity of 180 lactic acid bacteria and selected several Lactobacillus helveticus, L. delbrueckii subsp. bulgaricus and L. casei strains that demonstrated strong ACE-inhibitory activity. The aim was to carry out a molecular study on the bioactive peptides released by the strains with the best ACE-inhibitory properties and by the strains demonstrating a calcium-binding effect. To the best of our knowledge, this is the first study of bioactive peptides in Bulgarian white cheese. Peptides with the strongest ACE-inhibitory activity were purified and sequenced. The strains were assessed for production of peptides with calcium-binding properties. These peptides were isolated, purified and sequenced. Two strains releasing bioactive peptides with the strongest ACE-inhibitory and calcium-binding activities were selected for development of cheese starters. The strain with the best ACE-inhibitory activity was L. helveticus A1, which releases the peptide Ala-Leu-Pro-Met as a main contributor to the ACE inhibition. The strain with the best calcium-binding activity was L. casei C3 releasing the peptide SpLSpSpSpE (fraction 15–20 of ß-casein) as a main contributor to calcium binding. After pilot production of cheeses with the developed starters, the ACE-inhibitory and calcium-binding effects were confirmed during the cheese ripening. The addition of the two selected adjunct cultures led to increased production of bioactive peptides in the cheese. In this way, it is possible to increase the functional properties of Bulgarian white brined cheese.
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