Study of the physicochemical and biological stability of pediocin PA-1 in the upper gastrointestinal tract conditions using a dynamic in vitro model.

AIMS: To evaluate the survival of Pediococcus acidilactici UL5 and its ability to produce pediocin PA-1 during transit in an artificial gastrointestinal tract (GIT). To investigate the physicochemical and biological stability of purified pediocin PA-1 under GIT conditions. METHODS AND RESULTS: Skim milk culture of Ped. acidilactici UL5 was fed to a dynamic gastrointestinal (GI) model known as TIM-1, comprising four compartments connected by computer-controlled peristaltic valves and simulating the human stomach, duodenum, jejunum and ileum. This strain tolerated a pH of 2·7 in the gastric compartment, while lower pH reduced its viability. Bile salts in the duodenal compartment brought a further 4-log reduction after 180 min of digestion, while high viable counts (up to 5 × 10(7) CFU ml(-1) fermented milk) of Ped. acidilactici were found in both the jejunal and ileal compartments. Pediococcus acidilactici recovered from all four compartments was able to produce pediocin at the same level as unstressed cells. The activity of the purified pediocin in the gastric compartment was slightly reduced after 90 min of gastric digestion, while no detectable activity was found in the duodenal, jejunal and ileal compartments during 5 h of digestion. HPLC analysis showed partial degradation of the pediocin peptide in the duodenal compartment and massive breakdown in the jejunal and ileal compartments. CONCLUSIONS: Pediococcus acidilactici UL5 showed high resistance to GIT conditions, and its ability to produce pediocin was not affected, suggesting its potential as a probiotic candidate. The physicochemical and biological stability of pediocin was significantly poor under GIT conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Pediococcus acidilactici UL5 appears to be a potential probiotic candidate because its capacity to produce pediocin PA-1 is not affected by the GI conditions as well as the strain shows an acceptable survival rate. Meanwhile, purified pediocin PA-1 losses activity during GIT transit; microcapsules could be used to deliver it to the target site.

Improvement of texture and structure of reduced-fat Cheddar cheese by exopolysaccharide-producing lactococci.

The objective of this study was to evaluate the effect of capsular and ropy exopolysaccharide (EPS)-producing strains of Lactococcus lactis ssp. cremoris on textural and microstructural attributes during ripening of 50%-reduced-fat Cheddar cheese. Cheeses were manufactured with added capsule- or ropy-forming strains individually or in combination. For comparison, reduced-fat cheese with or without lecithin added at 0.2% (wt/vol) to cheese milk and full-fat cheeses were made using EPS-nonproducing starter, and all cheeses were ripened at 7 degrees C for 6 mo. Exopolysaccharide-producing strains increased cheese moisture retention by 3.6 to 4.8% and cheese yield by 0.28 to 1.19 kg/100 kg compared with control cheese, whereas lecithin-containing cheese retained 1.4% higher moisture and had 0.37 kg/100 kg higher yield over the control cheese. Texture profile analyses for 0-d-old cheeses revealed that cheeses with EPS-producing strains had less firm, springy, and cohesive texture but were more brittle than control cheeses. However, these effects became less pronounced after 6 mo of ripening. Using transmission electron microscopy, fresh and aged cheeses with added EPS-producing strains showed a less compact protein matrix through which larger whey pockets were dispersed compared with control cheese. The numerical analysis of transmission electron microscopy images showed that the area in the cheese matrix occupied by protein was smaller in cheeses with added EPS-producing strains than in control cheese. On the other hand, lecithin had little impact on both cheese texture and microstructure; after 6 mo, cheese containing lecithin showed a texture profile very close to that of control reduced-fat cheese. The protein-occupied area in the cheese matrix did not appear to be significantly affected by lecithin addition. Exopolysaccharide-producing strains could contribute to the modification of cheese texture and microstructure and thus modify the functional properties of reduced-fat Cheddar cheese.

Genetic and phenotypic diversity of Bifidobacterium thermacidophilum fecal isolates from newborns.

This study was undertaken to genetically identify and phenotypically characterize 14 bifidobacteria isolated from 20 breast-fed newborns. These isolates showed 98%-99% similarity to Bifidobacterium thermacidophilum subsp. suis based on 16S rDNA. Further analysis by pulsed-field gel electrophoresis of chromosomal DNA digested with XbaI revealed 4 distinct restriction patterns. The predominant pattern, shared by 8 (57%) isolates, produced a macro-restriction profile with about 13 large fragments ranging in size from >242.5 to 23.1 kb, whereas the other 6 displayed 3 distinct restriction profiles all characterized by more micro- than macro-restriction, with fragments ranging in size from 97 to 9.4 kb. Phenotypic characteristics, including carbohydrate fermentation profile, maximal growth temperature, and antibiotic susceptibility, varied widely even among strains showing the same restriction profile. The presence of B. thermacidophilum in stools of newborn infants may indicate the potential of these bacteria for aiding the development of the intestinal ecosystem.

Antibiotic susceptibility profile of bifidobacteria as affected by oxgall, acid, and hydrogen peroxide stress.

The effects of acid, oxgall, and H(2)O(2) on susceptibilities to antibiotics and nisin were examined for 13 strains of bifidobacteria. Susceptibilities to ampicillin, cloxacillin, penicillin, vancomycin, kanamycin, neomycin, paramomycin, streptomycin, chloramphenicol, erythromycin, tetracycline, and nisin A were assayed by a microdilution broth method. Acid-, oxgall- and H(2)O(2)-stressed variants were produced and assayed. Exposure to a pH of 2.0 for 60 min reduced susceptibilities to cloxacillin and nisin A but increased susceptibilities to ampicillin, vancomycin, aminoglycosides, chloramphenicol, and erythromycin in a strain-dependent manner. Exposure to oxgall (0.3%) for 90 min increased susceptibilities to cell wall-directed antibiotics and aminoglycosides but increased resistances to tetracycline and nisin A. Oxidative stress increased the susceptibilities of 70% of the strains to ampicillin and chloramphenicol, of 50% of the strains to cloxacillin and tetracycline, and of 40% of the strains to erythromycin but did not affect susceptibilities to vancomycin, kanamycin, and nisin A. This study shows that exposure of bifidobacteria to stressful conditions resembling those in the gastrointestinal tract may substantially modify their susceptibilities to antibiotics and may thus affect their probiotic capacities, especially when they are used for the management of intestinal infections and antibiotic-associated diarrhea.

Identification and molecular characterization of the chromosomal exopolysaccharide biosynthesis gene cluster from Lactococcus lactis subsp. cremoris SMQ-461.

The exopolysaccharide (EPS) capsule-forming strain SMQ-461 of Lactococcus lactis subsp. cremoris, isolated from raw milk, produces EPS with an apparent molecular mass of >1.6 x 10(6) Da. The EPS biosynthetic genes are located on the chromosome in a 13.2-kb region consisting of 15 open reading frames. This region is flanked by three IS1077-related tnp genes (L. lactis) at the 5' end and orfY, along with an IS981-related tnp gene, at the 3' end. The eps genes are organized in specific regions involved in regulation, chain length determination, biosynthesis of the repeat unit, polymerization, and export. Three (epsGIK) of the six predicted glycosyltransferase gene products showed low amino acid similarity with known glycosyltransferases. The structure of the repeat unit could thus be different from those known to date for Lactococcus. Reverse transcription-PCR analysis revealed that the eps locus is transcribed as a single mRNA. The function of the eps gene cluster was confirmed by disrupting the priming glycosyltransferase gene (epsD) in Lactococcus cremoris SMQ-461, generating non-EPS-producing reversible mutants. This is the first report of a chromosomal location for EPS genetic elements in Lactococcus cremoris, with novel glycosyltransferases not encountered before in lactic acid bacteria.