RESUMO
A selection of 36 commercial probiotic fermented dairy products from UK and Europe markets were evaluated for the numbers, types, and viability of Lactobacillus strains against the stated information on their packages. A comparative study was carried out on selectivity of MRS-Clindamycin, MRS-Sorbitol, and MRS-IM Maltose, to select the right medium for enumeration of probiotic Lactobacillus. Based on selectivity of medium for recovery of the targeted lactobacilli, and also simplicity of preparation, MRS-Clindamycin was chosen as the best medium for enumeration of probiotic Lactobacillus in fermented milks. The results of enumeration of lactobacilli showed that 22 out of a total 36 tested products contained more than 106 colony-forming units/g at the end of their shelf life, which comply with the recommended minimum therapeutic level for probiotics. Rep-PCR using primer GTG-5 was applied for initial discrimination of isolated strains, and isolates, which presented different band profile, were placed in different groups. The isolated Lactobacillus spp. were identified mainly as Lactobacillus acidophilus, Lactobacillus casei, and Lactobacillus paracasei by analysis of partial sequences of the 16S ribosomal RNA and rpoA genes.
RESUMO
Bikalga is a Hibiscus sabdariffa seed fermented condiment widely consumed in Burkina Faso and neighboring countries. The fermentation is dominated by Bacillus subtilis group species. Ten B. subtilis subsp. subtilis (six isolates) and Bacillus licheniformis (four isolates) isolated from traditional Bikalga were examined for their antimicrobial activity against a panel of 36 indicator organisms including Gram-positive and Gram-negative bacteria and yeasts. The Bacillus spp. isolates showed variable inhibitory abilities depending on the method used. Both Gram-positive and Gram-negative bacteria were inhibited in the agar spot assay while only Gram-positive pathogens were inhibited in the agar well diffusion assay. Cell free supernatants (CFS) of pure cultures of 3 B. subtilis subsp. subtilis (G2, H4 and F1) strains inhibited growth of Listeria monocytogenes, Micrococcus luteus, Staphylococcus aureus and Bacillus cereus, while CFS of 2 B. licheniformis (E3 and F9) strains only inhibited M. luteus. The antimicrobial substance(s) produced by B. subtilis subsp. subtilis H4 was further characterized. The antimicrobial substance(s) produced by H4 was detected from mid-exponential growth phase. The activity was sensitive to protease and trypsin, but resistant to the proteolytic action of proteinase K and papain. Treatment with α-amylase and lipase II resulted in a complete loss of antimicrobial effect, indicating that a sugar moiety and lipid moiety are necessary for the activity. Treatment with mercapto-ethanol resulted in a significant loss, indicative of the presence of disulfide bridges. The antimicrobial activity of H4 was heat resistant and active at pH3-10. PCR detection of yiwB, sboA, spoX, albA and spaS, etnS genes and genes coding for surfactins and plipastatins (fengycins) indicated a potential for subtilosin, subtilin and lipopeptide production, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out and a single band of approximately 4kDa had antimicrobial activity. Ultra high performance liquid chromatography-time of flight mass spectrometry (UHPLC-TOFMS) analysis of the 4kDa band allowed identification of surfactin and a protein with a monoisotopic mass of 3346.59Da, which is dissimilar in size to subtilosin and subtilin. Surfactin is a cyclic lipoheptapeptide, which contains a ß-hydroxy fatty acid, but no di-sulfide bridges or sugar residues. The complete loss of activity upon amylase treatment indicates that surfactin was not responsible for the observed antimicrobial effect. However, it cannot completely be ruled out that surfactin acts synergistically with the detected protein, though further investigations are needed to confirm this.