High Adhesion Of Lactobacillus Screening, Identification And Surface Hydrophobic Properties

Lactic acid bacteria (LAB) has important physiological functions on inhibition of pathogen invasion and proliferation through adhesion to the intestinal mucosa and forming a stable biological barrier. Therefore, adhesion ability is one of the important indicators for evaluation of LAB probiotic properties. It was reported that the bacterial surface hydrophobic properties were correlated to the bacteria adhesion to the host, but also to the clearance of the pathogen, however, its relevance is still needed for further study to confirm. In this study, we determined the adhesion ability of47LAB strains to murine macrophagocyte, spleen cell, intestinal epthelial cell and mucus. The surface hydrophobicity, auto-aggregationg, and co-aggregation properties of LAB strains with different adhesion abilities were then analyzed as well as their inhibition of pathogenic bacteria in vitro. Seven strains of LAB with high adhesion ability were identified using physiological-biochemical test combined with16S rDNA sequencing, and were typed using molecular fingerprinting technique. The results of this study provided the theory basis and strain resources for further screening and application of highly adhesive strains of LAB. The main results are described as follows.(1) The in vitro adhesion ability of47strains of LAB was determined by adhesion model using murine macrophagocyte, spleen cell, intestinal epthelial cell and mucus. Results showed that LAB had selectivity on adhesion to different cell types. The average level of adhesion ability of47LAB strains to macrophage (19.2bacteria per cell) was significantly higher than those to spleen cell (9.1bacteria per cell) and intestinal epithelial cell (6.2bacteria per cell). Adhesion ability of47strains of LAB to mucus varied from1.7%to35.2%, and the average value was5.0%. The average level of adhesion ability for5cocci strains reached to14.4%, in which adhesion ability of strain F28-8was significantly higher than that of other strains.(2) Ten LAB strains with different adhesion ability were sleeted, and analyzed for their surface hydrophobicity, auto-aggregation and co-aggregation properties. Surface hydrophobicity ability of3LAB strains with high adhesion ability (F28-8, Y45-30and Y27-4) were higher than90%, which were significantly higher than those of strains with low adhesion ability (p<0.01). Two strains with different aggregation and adhesion properties were analyzed by scanning electron microscope analysis, and the results showed that the surface of F28-8was roughness, however H13was relativley smooth. Cells of strain F28-8can be aggregated together more tightly than that of strain H13. There were lots of Vibrio parahaemolyticus adhered to F28-8and forming dense cenobium, which may accelerate the coagulation and sedimentation of Vibrio parahaemolyticus in the matrix.(3) Seven strains of cocci isolates with different adhesion ability were identified by physiological-biochemical test and16S rDNA sequencing. Both results showed that they were all belonged to Pediococcus pentosaceus. The RAPD fingerprinting of7strains of Pediococcus pentosaceus showed that there were five different subtypes (type Aa、Bb、Cb、Da and Ea) in these strains, interestingly,2highly adhesive strains (F28-8and Y27-4) had the same fingerprint, and both belonged to the type Ea.(4) The antibacterial effects of7Pediococcus pentosaceus strains were tested. The fermentation supernatants of these strains had antibacterial effect against Vibrio parahaemolyticus ATCC33847. The diameters of inhibition zone varied from21.0mm to25.5mm, in which the highest strain was F3. The antibacterial effects were reduced when the fermentation supernatants were dealed with catalase, proteinase K and trypsin, which showed that more than half the antibacterial effect was caused by lactate, and in addition to this, there existed a small amount of hydrogen peroxide and protein in antibacterial compositions. The inhibitory effect remained more than75%when the fermentation supernatant dealed at70℃,100℃,121℃for30min, which showed that the antibacterial substances of7strains of LAB were stable thermal treament. We measured the viable Vibrio parahaemolyticus in the system of auto-aggregationg and co-aggregation, and the viable counts of Vibrio parahaemolyticus was reduced for1.7Log CFU/mL after Vibrio parahaemolyticus co-aggregation with F28-8at2h. The results showed that the clear effect of LAB against pathogenic Vibrio was due to the combined action of the co-aggregation effect and antibacterial effect.