Effect of the fermentation pH on the storage stability of Lactobacillus rhamnosus preparations and suitability of in vitro analyses of cell physiological functions to predict it.

AIMS: To investigate how cell physiological functions can predict the stability of freeze-dried probiotics. In addition, the effect of the fermentation pH on the stability of probiotics was investigated. METHODS AND RESULTS: Fermenter-grown (pH 5.8 or 5.0) Lactobacillus rhamnosus cells were freeze-dried and their survival was evaluated during storage at 37 degrees C, in apple juice and during acid [hydrochloric acid (HCl) and malic acid] and bile exposure. Cells grown at pH 5.0 were generally coping better with acid-stress than cells grown at pH 5.8. Cells were more sensitive to malic acid compared with HCl. Short-term stability results of Lact. rhamnosus cells in malic acid correlated well with the long-term stability results in apple juice, whereas the results of cell membrane integrity studies were in accordance with bile exposure results. CONCLUSIONS: Malic acid exposure can prove useful in evaluating the long-term stability of probiotic preparations in apple juice. Fermentation at reduced pH may ensure a better performance of Lact. rhamnosus cells during the subsequent acid-stress. SIGNIFICANCE AND IMPACT OF THE STUDY: The beneficial effect of lowered fermentation pH to Lact. rhamnosus stability during storage in apple juice and the usefulness of malic acid test in predicting the stability were shown.

Persistence of oral colonization by the same Actinobacillus actinomycetemcomitans strain(s).

BACKGROUND: The Gram-negative facultatively anaerobic coccobacillus Actinobacillus actinomycetemcomitans is the major pathogen in localized juvenile periodontitis (LJP) and some forms of adult periodontitis (AP). A. actinomycetemcomitans can be grouped into 5 serotypes (a through e) based on differences in the carbohydrate moiety of cell surface lipopolysaccharide. The A. actinomycetemcomitans population is genetically heterogeneous. Since the studies on A. actinomycetemcomitans colonization have mostly applied only culture techniques, the clonality of the follow-up isolates has not been established. Thus, it is possible that, although A. actinomycetemcomitans could be repeatedly isolated from an individual, the initial colonizing strain was replaced by another strain. The aim of the study was to determine whether oral A. actinomycetemcomitans strains change spontaneously over time or after periodontal treatment. METHODS: A total of 922 A. actinomycetemcomitans isolates were recovered from 115 subjects. From each subject A. actinomycetemcomitans isolates were obtained from 2 to 9 follow-up samples 0.5 to 11.5 years apart. After the first sampling occasion, 99 subjects were treated for either LJP or AP, whereas the 16 non-periodontitis subjects received no treatment. All A. actinomycetemcomitans isolates were serotyped and 235 isolates from 52 subjects genotyped with AP-PCR and/or with ribotyping. RESULTS: Isolates of only one serotype, or non-serotypeable isolates alone, were repeatedly found in 104 subjects; serotype a occurred in 25%, b in 33%, c in 23%, d in 5%, e in 7%, and non-serotypeable isolates in 8% of these subjects. Two serotypes (or serotypeable isolates together with non-serotypeable isolates) occurred simultaneously in 9 subjects and in each of these subjects at least one of the serotypes was detected at each sampling occasion. In one subject the initial serotype reappeared although a different serotype was once seen alone, whereas in another subject the initial serotype could not be recovered later. Identical genotypes of A. actinomycetemcomitans were repeatedly detected in each of 52 subjects with follow-up isolates of the same serotype. CONCLUSIONS: The results showed that spontaneous or treatment-induced change in the oral A. actinomycetemcomitans strain(s) is extremely rare and that colonization with the same strain(s) seems to be remarkably persistent.

Actinobacillus actinomycetemcomitans serotype e--biotypes, genetic diversity and distribution in relation to periodontal status.

Actinobacillus actinomycetemcomitans isolates from 356 individuals were screened for identification of serotype e in order to investigate its distribution in relation to periodontal status. From subjects with serotype e, 1-6 isolates per subject (n = 61) were genotyped using arbitrarily primed-polymerase chain reaction (AP-PCR) and apaH gene polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) analysis to determine the genetic heterogeneity within the serotype. Furthermore, one serotype e strain per subject was tested for fermentation of 8 carbohydrates for biotyping. Among patients with adult periodontitis (n = 219), localized juvenile periodontitis (n = 55) and other forms of early-onset periodontitis (n = 18) serotypes b, a and c, respectively, were the most frequently detected serotypes. Non-periodontitis subjects (n = 64) were predominantly colonized with serotype c. Serotype e was found in 30 (14%) adult periodontitis patients, 2 (11%) early-onset periodontitis patients and in 5 (8%) non-periodontitis individuals, but in none of the 55 localized juvenile periodontitis patients. AP-PCR distinguished 3 and apaH gene PCR-RFLP analysis 2 genotypes among the 61 A. actinomycetemcomitans serotype e isolates, one genotype per subject. The AP-PCR genotypes 1 and 3 represented the apaH genotype 1 and the AP-PCR genotype 2 the apaH genotype 2. On the basis of variable fermentation of galactose and xylose, 3 biotypes among A. actinomycetemcomitans serotype e were established. Contrary to the absence of A. actinomycetemcomitans serotype e in localized juvenile periodontitis patients, its detection frequency was comparable among other forms of periodontitis and periodontal health. Clinical serotype e isolates form at least 2 genetic types and 3 biotypes.