Modulation of human humoral immune response through orally administered bovine colostrum.

Eighteen healthy volunteers were randomized into two treatment groups and consumed liquid prepackaged bovine colostrum whey and placebo for 7 days. On days 1, 3 and 5, an attenuated Salmonella typhi Ty21a oral vaccine was given to all subjects to mimic an enteropathogenic infection. The circulating antibody secreting cells and the expression of phagocytosis receptors of the subjects before and after oral immunization were measured with the ELISPOT assay and flow cytometry. All subjects responded well to the vaccine. No significant differences were observed in ELISPOT values for IgA, IgG, IgM, Fcgamma and CR receptor expression on neutrophils and monocytes between the two groups. There was a trend towards greater increase in specific IgA among the subjects receiving their vaccine with bovine colostrum. These results suggest that bovine colostrum may possess some potential to enhance human special immune responses.

Lactobacillus crispatus and its nonaggregating mutant in human colonization trials.

A wild-type Lactobacillus crispatus, showing a cell aggregation phenotype and its spontaneous nonaggregating mutant were compared for their in vitro adhesion properties to human ileal mucus and to a cultured human colonic cell line (Caco2) and for their in vivo colonization and adhesion potential with colonoscopy patients as volunteers in feeding trials. The wild-type strain adhered better to mucus or to Caco2 cells than did the mutant. Altogether, three human trials with the wild type and two with the mutant strain were performed. In two of the trials, the wild type could be recovered from either fecal samples or biopsies taken from the colon, while the mutant strain could not be demonstrated in either of the trials where it was used. The L. crispatus colonies recovered from the trials were often mixed, and several enterococci and lactobacillus strains coaggregating with L. crispatus wild type could be isolated. The results indicate that the surface-mediated properties, such as aggregation, of lactobacilli can have a role in adhesion and colonization.

Assessment of adhesion properties of novel probiotic strains to human intestinal mucus.

Potential new probiotic strains Lactobacillus brevis PELI, L. reuteri ING1, L. rhamnosus VTT E-800 and L. rhamnosus LC-705 were assessed for their adhesion properties using the human intestinal mucus model. The effect on the adhesion of exposure to acid and pepsin and to milk were tested to simulate gastric and food processing conditions, and the effect of different growth media on adhesion was tested. The properties of the four strains were compared to the well-investigated probiotic L. rhamnosus strain GG. Three of the tested strains showed significant adhesion properties in the mucus model, while L. brevis PELI had intermediate adhesion and L. rhamnosus LC-705 adhered poorly. Pretreatment with different milks decreased the adhesion and low pH and pepsin treatment reduced the adhesion of all tested strains except L. rhamnosus LC-705. No competitive exclusion of pathogenic Salmonella typhimurium or Escherichia coli SfaII was observed. The results indicate that major differences exist between tested proposed probiotic strains. The growth media and the food matrix significantly affect the adhesive ability of the tested strains. This has previously not been taken into account when selecting novel probiotic strains.

Quality assurance criteria for probiotic bacteria.

Acid and bile stability and intestinal mucosal adhesion properties are among the criteria used to select probiotic microbes. The quality control of probiotic cultures in foods traditionally has relied solely on tests to ensure that an adequate number of viable bacteria are present in the products throughout their shelf lives. Viability is an important factor, but not the only criterion for quality assurance. To be effective, probiotic strains must retain the functional health characteristics for which they were originally selected. Such characteristics include the ability to survive transit through the stomach and small intestine and to colonize the human gastrointestinal tract. In vitro test protocols can be readily adopted to examine the maintenance of a strain's ability to tolerate acidic conditions, survive and grow in the presence of bile, and metabolize selective substrates. Molecular techniques are also available to examine strain stability. Adhesion characterization may be an important quality-control method for assessing gut barrier effects. Adhesion has been related to shortening the duration of diarrhea, immunogenic effects, competitive exclusion, and other health effects. Adhesion properties should be carefully monitored, including adhesion to intestinal cells (eg, Caco-2) and human intestinal mucus. This article outlines the types of in vitro testing that can be used to ensure quality control of functional probiotic strains.

Chemical, physical and enzymatic pre-treatments of probiotic lactobacilli alter their adhesion to human intestinal mucus glycoproteins.

Intestinal mucus glycoproteins extracted from faeces of healthy adult subjects were used as a substratum for bacterial adhesion to investigate the effects of physical, chemical and enzymatic pre-treatments of the bacteria on their adhesion. The strains studied were Lactobacillus acidophilus 1 (LCI, Nestlé), L. rhamnosus strain GG (ATCC 53103), L. rhamnosus LC-705, and L. casei strain Shirota (Yakult, Yakult Ltd). Hereafter the strains are referred to as LA1, LGG, LC-705, and Shirota, respectively. Strains LA1 and LGG adhered greatly whereas the adhesion of strains LC-705 and Shirota to intestinal mucus glycoproteins was low. Adhesion of LA1 and LGG was reduced by boiling, autoclaving and by pepsin and trypsin treatments suggesting that the bacterial protein structures are essential for their adhesion. Treatment in ethanol and in propanol prior to adhesion significantly increased the adhesion of LA1 and LC-705, respectively. Adhesion of Shirota strain was not altered by any of the treatments.

Quantitative approach in the study of adhesion of lactic acid bacteria to intestinal cells and their competition with enterobacteria.

To describe the phenomena of bacterial adhesion to intestinal cells and the competition for adhesion between bacteria, mathematical equations based on a simple dissociation process involving a finite number of bacterial receptors on intestinal cell surface were developed. The equations allow the estimation of the maximum number of Lactobacillus sp. and Escherichia coli cells that can adhere to Caco-2 cells and intestinal mucus; they also characterize the affinity of the bacteria to Caco-2 cells and intestinal and fecal mucus and the theoretical adhesion ratio of two bacteria present in a mixed suspension. The competition for adhesion between Lactobacillus rhamnosus GG and E. coli TG1 appeared to follow the proposed kinetics, whereas the competition between Lactobacillus casei Shirota and E. coli TG1 may involve multiple adhesion sites or a soluble factor in the culture medium of the former. The displacement of the adhered Lactobacillus by E. coli TG1 seemed to be a rapid process, whereas the displacement of E. coli TG1 by the Lactobacillus took more than an hour.

Binding of aflatoxin B1 alters the adhesion properties of Lactobacillus rhamnosus strain GG in a Caco-2 model.

Lactic acid bacteria have been previously reported to possess antimycotoxigenic activities both in vitro and in vivo. The objective of this study was to investigate the effect of aflatoxin B1 on adhesion capability of Lactobacillus rhamnosus strain GG using a Caco-2 adhesion model. Removal of aflatoxin B1 by L. rhamnosus strain GG reduced the adhesion capability of this strain from 30% to 5%. It is therefore concluded that aflatoxins may influence the adhesion properties of probiotics able to sequester them, and subsequently these bacteria may reduce the accumulation of aflatoxins in the intestine via increased excretion of an aflatoxin-bacteria complex.

The effect of probiotic bacteria on the adhesion of pathogens to human intestinal mucus.

Human intestinal glycoproteins extracted from faeces were used as a model for intestinal mucus to investigate adhesion of pathogenic Escherichia coli and Salmonella strains, and the effect of probiotics on this adhesion. S-fimbriated E. coli expressed relatively high adhesion in the mucus model, but the other tested pathogens adhered less effectively. Probiotic strains Lactobacillus GG and L. rhamnosus LC-705 as well as a L. rhamnosus isolated from human faeces were able to slightly reduce S-fimbria-mediated adhesion. Adhesion of S. typhimurium was significantly inhibited by probiotic L. johnsonii LJ1 and L. casei Shirota. Lactobacillus GG and L. rhamnosus (human isolate) increased the adhesion of S. typhimurium suggesting that the pathogen interacts with the probiotic.

In vitro adhesion and platelet aggregation properties of bacteremia-associated lactobacilli.

Eight bacteremia-associated Lactobacillus strains were evaluated in vitro for the ability to adhere to human intestinal mucosa and to aggregate platelets. Adherence varied significantly among the strains, and platelet aggregation was induced by three strains. In conclusion, strong binding ability does not appear to be a prerequisite for the involvement of lactobacilli in bacteremia or to their ability to aggregate platelets.

Human ileostomy glycoproteins as a model for small intestinal mucus to investigate adhesion of probiotics.

Human ileostomy glycoproteins were used as a model for small intestinal mucus to investigate the adhesion of 12 Lactobacillus strains, one Lactococcus strain and one Propionibacterium strain, Both probiotic and dairy strains were tested. Adhesive and non-adhesive Escherichia coli strains were used as controls. All the strains were also tested for their adhesion to polystyrene. Adhesion to ileostomy glycoproteins and to polystyrene varied significantly among the strains tested. Lactobacillus rhamnosus (human isolate), Lactobacillus GG, Lact. acidophilus 1 and P. freudenreichii adhered to ileostomy glycoproteins. Adhesion was concentration-dependent and the most adhesive strains were able to saturate the substratum. These results indicate that human ileostomy glycoproteins can be used as a model system to select potential probiotic strains to complement the adhesion test with intestinal cell lines.

Adhesion of some probiotic and dairy Lactobacillus strains to Caco-2 cell cultures.

The adhesion of 12 different Lactobacillus strains was studied using Caco-2 cell line as an in vitro model for intestinal epithelium. Some of the strains tested have been used as probiotics, and most of them are used in the dairy and food industry. Human and bovine enterotoxigenic Escherichia coli strains were used as positive and negative control, respectively. Bacterial adhesion to Caco-2 cell cultures was quantitated using radiolabelled bacteria. The adherence of bacteria was also observed microscopically after Gram staining. Viability of bacteria prior to adhesion was verified using flow cytometry. Among the tested strains, L. casei (Fyos) was the most adhesive strain and L. casei var. rhamnosus (Lactophilus) was the least adhesive strain, approximately 14 and 3% of the added bacteria adhered to Caco-2 cell cultures, respectively. The corresponding values for positive and negative control E. coli strains were 14 and 4%, respectively. The Lactobacillus strains tested could not be divided into distinctly adhesive or non-adhesive strains, since there was a continuation of adhesion rates. The four most adhesive strains were L. casei (Fyos), L. acidophilus 1 (LC1), L. rhamnosus LC-705 and Lactobacillus GG (ATCC 53103). No significant differences in the percentage adhesion were observed between these strains. Adhesion of all the strains was dependent on the number of bacteria used, since an approximately constant number of Caco-2 cells was used, indicating that the Caco-2 cell binding sites were not saturated. Viability of bacteria was high since approximately 90% of the bacteria were viable with the exception of L. acidophilus 1 which was 74% viable. Microscopic evaluations agreed with the radiolabelled binding as evidenced by observing more bacteria in Gram-stained preparations of good adhering strains compared to poorly adhering strains.