THE QUALITY OF METHODOLOGY USED FOR
SELECTION
Many in vitro tests are performed when screening for potential
probiotic strains. The ability to adhere to the intestinal mucosa is
one of the more important selection criteria for probiotics
because adhesion to the intestinal mucosa is considered to be a
prerequisite for colonization (5). As substrata, enterocyte-like
Caco-2 tissue culture cells and intestinal mucus are currently
used. However, these represent only a distinct part of the intestinal
mucosa. In this respect, mucus-secreting HT29-MTX tissue
culture cells would come closer to the true situation in the intestine.
In addition to these models, human ileostomy glycoproteins
have been used to study adhesion to the small-intestinal mucosa
(6). All of these in vitro systems provide valuable information on
the ability of probiotics to adhere and colonize the intestine.
Adhesion to colonic or intestinal biopsy samples, if possible,
should be considered as a final in vitro adhesion test that would
be most like the in vivo situation. Not only would this be a better
approximation of the in vivo situation, it would allow for the
study of adhesion to different parts of the intestine. This is especially
important regarding immune stimulation by oral administration
of probiotics.
Adhesion is also considered important for stimulation of the
immune system. Adhesion to M cells or Peyer’s patches may therefore be an important determinant of possible immunestimulating
properties of probiotic microorganisms.
Fecal samples have been used in most colonization studies with
probiotic bacteria. These, however, reflect only the bacteriologic
situation in the fecal material and do not give an accurate picture
of the different parts of the gastrointestinal tract or the mucosal
layer of the gut. The use of biopsies from the intestinal mucosa is
a more accurate means of determining colonization. Lactobacillus
strains were found to adhere to rectal mucosa obtained from volunteers
who had consumed a fermented oatmeal soup (7).
When tested in vitro, probiotics are usually grown in laboratory
media. With many probiotics, the aim is at least transient
colonization, in which case the probiotics may need to grow or
at least metabolize in the intestine. The adhesive properties,
metabolism, and morphology of probiotics grown in intestinal
contents or intestinal mucus have been shown to be different
from those of probiotics grown in laboratory media. These differences
may affect the health effects of the probiotics. By using
culture media more resembling the nutrients available in the
intestine, one may obtain a more accurate understanding of the
properties of probiotics in vivo.
One of the selection criteria for probiotics is the production of
antimicrobial substances, and many probiotic bacteria have been
shown to produce them (8). Among these substances are not only
growth-inhibiting metabolites, eg, organic acids and hydrogen
peroxide, but also bacteriocins, adhesion inhibitors, and a range
of small antimicrobial substances. These substances have been
shown to be produced in laboratory media but their production
and efficacy in vivo remain uncertain (8). It has not been tested
whether administration of purified bacteriocins alone has effects,
eg, on diarrheal disease. Nor has it been tested whether bacteriocins
are produced in vivo. If bacteriocins are produced and
active in vivo, it may be necessary to assess their effects on the
indigenous microflora. There is the potential risk that beneficial
strains in the indigenous microflora are also affected by the presence
of a bacteriocin and that the bacteriocins may thereby alter
the natural resistance of the indigenous microflora to colonization.
Because the production of antimicrobial substances is
regarded as an important selection criterion for probiotics, it
must be confirmed whether these substances are indeed produced
in vivo and exert a beneficial effect. Intestinal or fecal
microflora studies are needed to confirm these properties.