CRITERIA FOR FUNCTIONAL QUALITY ASSURANCE
The criteria currently used to select probiotics define the optimal
quality control of probiotic strains in industrial practice. Probiotics
are often used in fermented foods and fermentation acts to
retain and optimize microbial viability and productivity while
simultaneously preserving the probiotic’s properties. During fermentation,
several metabolic products appear in the food product,
including acetic acid, lactic acid, and possibly bacteriocins, and
the pH of the product is lowered. Such changes may affect the stability
of probiotic bacteria and may alter the bacteria’s functional
properties. Also, long-term industrial use of the starter culture for
production purposes may influence both viability and functional
properties. Important quality-control properties that must be constantly
controlled and optimized are the following: adhesive properties;
bile and acid stability; viability and survival throughout the
manufacturing process; effects on carbohydrate, protein, and fat
utilization; and, especially, colonization properties and immunogenicity.
Most of these properties are related to the physiologic
properties of the strain, but long-term industrial processing and
storage conditions may influence probiotic properties. Thus, in
addition to technologic properties, functional properties should
be considered in quality-control measures.
Acid and bile stability
To survive passage through the stomach and small intestine,
probiotic strains must tolerate the acidic and protease-rich conditions
of the stomach, and survive and grow in the presence of bile
acids. Acid tolerance is also important for the probiotics’ survival
in food (3). The dominant food vehicles for probiotics remain to
be yogurts and fermented milks, both of which provide a relatively
low-pH environment in which the probiotic bacteria must
survive. Hence, acid tolerance is one of the first properties
screened for when selecting probiotic strains. Simple in vitro tests
can be used to assess acid tolerance. Such tests have been applied
to lactic acid bacteria and Bifidobacterium strains used in the
dairy industry and proposed as probiotics. As shown in Figure 1,
the results of these tests can predict the ability of the strains to
survive in acidic products. In vivo validation of survival through
the human stomach is more difficult to obtain. In vitro assays
examining the inhibitory effect of bile acids on the growth of probiotic
strains are also relatively simple to perform, although
again, quantitative extrapolation to probiotic performance in vivo
is difficult. Intraspecies variation in the ability to grow in the
presence of bile is often observed between potential probiotic
strains (Figure 2), and in vitro tests can be used to select the best
strains on a relative basis.
These in vitro tests for selection of acid- and bile-tolerant
strains can readily be applied to ensuring the quality of probiotic
cultures during manufacture and storage and throughout the shelf
life of the product. Both short-term environmental factors affecting
gene regulation (eg, culture growth phase and stress leading
to the production of shock proteins), and selection of variants
through long-term subculturing may produce changes in culture
performance (3, 9). The former of these is the most likely to have
the greatest influence on probiotic performance (potentially positively)
because appropriate culture maintenance procedures limiting
the number of passages should prevent the selection of
genetic variants in industrial processes. Acid tolerance is likely to
be a relatively intrinsic property of bacteria and acidification of
culture broth during fermentation would also make selection of
less acid-tolerant variants unlikely if serial subculturing was practiced.
However, data showing the long-term stability of acid and
bile tolerance in probiotics during subculturing are lacking.
Adhesion stability
Adhesion characterization may be an important quality-control
method for assessing the surface structure of probiotic bacteria and
related gut barrier effects. In several studies, adhesion was related
to a shortening of duration of diarrhea, immunogenic effects, competitive
exclusion, and other health effects (2, 5, 10–12).
Adhesion of probiotic strains is variable. Adhesion in different
in vitro models varies even within the same strain and differences
between strains can be significant (13–15). Adhesion
of some common probiotic strains was studied by using a
human colon carcinoma cell line (Caco-2) and human ileostomy
glycoproteins as in vitro models for intestinal epithelium and
mucus, respectively (Figure 3). Of 6 probiotic strains tested,
only Lactobacillus johnsonii LJ-1 and Lactobacillus GG were