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35 Biochemistry and Probiotics 677bifidobacteria (Desjardins et al. 1991). Indeed, someL. aci-
dophilusandBifidobacteriumcultures have proteolytic activities
as high as those of yogurt starters (Shihata and Shah 2000), but
still do not multiply as fast in milk. In some instances, mixing a
nonproteolyticBifidobacteriumstrain with a highly proteolytic
L. acidophilusculture will be helpful (Klaver et al. 1993), but if
the LAB grows too fast the fermentation time is shortened and
counts in probiotic bacteria can even be lowered (Shihata and
Shah 2002).
Proteolytic activities are not only crucial for the development
of pure cultures, but also they are also crucial in a symbiotic
relationship between the two strains that compose the yogurt
cultures. As for probiotics, yogurt starters require an exogenous
nitrogen source and utilize peptides and proteins in their growth
medium (milk) by more or less complete enzyme systems. Be-
sides the free amino acids, the enzymatic hydrolysis of milk
proteins results in the liberation of peptides of varying sizes
and soluble nitrogenous compounds. In yogurt, peptides, free
amino acid and other released components depends on the type
of milk (animal species, season), type of proteolytic enzyme and
bacterial strains, heat treatment, manufacturing techniques, and
storage conditions (Zourari et al. 1992, Tamime and Robinson
1999). In yogurt,L. bulgaricusis the main species responsible
for increasing free amino acid (Zourari et al. 1992, El-zahar
et al. 2004) that partially explains the associative growth rela-
tionship that exists betweenS. thermophilusandL. bulgaricus.
Therefore, the proteinase activity ofL. bulgaricushydrolyzes
the casein to yield polypeptides, which are broken down by the
peptidases ofS. thermophiluswith the liberation of amino acids
(Tamime and Robinson 1999). The proteinase ofL. bulgaricus
is more active onβ-casein than on whey proteins (Zourari et al.
1992) and has an optimum pH between 5.2 and 5.8 (Argyle
et al. 1976, Ezzat et al. 1987). The importance of peptides for
their growth stimulation and their acidification is now well es-
tablished, especially forS. thermophilus(Zourari et al. 1992).
According to Accolas et al. (1971), the stimulation ofS. ther-
mophilusby milk culture filtrate ofL. bulgaricuswas due to the
presence of valine, leucine, isoleucine, and histidine. Bracquartet al. (1978) as well as Bracquart and Lorient (1979) reported
that reducing the growth medium of valine, histidine, glutamic
acid, tryptophan, leucine, and isoleucine results in reduction in
stimulation ofS. thermophilusto 50% (Tamime and Robinson
1999).Soy and PulsesSoy beverages contain, by order of importance, sucrose,
stachyose, raffinose, glucose, and fructose. Therefore, to grow
in milk, the ability to use lactose is critical, but cultures have a
variety of options in soy substrates. The assimilation of sucrose
requires invertase, while the hydrolysis of stachyose and raf-
finose demandsα-galactosidase (α-gal). In soy substrates, the
lactobacilli and the streptococci mainly use sucrose, glucose,
and fructose, while the bifidobacteria tend to use stachyose and
raffinose (Champagne et al. 2009). However, the ability of pro-
biotic bacteria to synthesize these enzymes varies much between
strains andL. acidophilus, as well as yogurt starter cultures may
possessα-gal (Donkor et al. 2007). Therefore, strain selection
must be carried out (Champagne et al. 2009). Theα-gal ofB.
longumhas an optimal activity at pH 5.8 and between 40–45◦C
(Garro et al. 1994). In soy, numerous enzymatic substrates are
required to assimilate all carbohydrates. Thus,α-gal will hy-
drolyze stachyose and then raffinose to generate galactose and
sucrose (Connes et al. 2004). Invertase is then required for su-
crose hydrolysis. Therefore, complete assimilation of the soy
carbohydrates requires two sets of hydrolytic enzymes. There-
after, it is the ability to assimilate the monosaccharides that
modulates growth rates. Not all probiotic cultures can metab-
olize the products ofα-gal and invertase (galactose, glucose,
and fructose) (Table 35.2), and even then, growth rates vary
(Table 35.1).
It was observed thatB. longumR0175 acidified milk at a much
slower rate thanLactobacillus helveticusR052 (Fig. 35.1), and
that the bifidobacteria grew at half the rateof L. helveticuson
lactose (Table 35.1). These data suggest that acidification rates
of milk by two probiotic bacteria (Fig. 35.1) can potentiallyTable 35.2.Fermentation of Some Carbohydrates by Various Probiotic BacteriaFood Based PrebioticsSpecies Glu Fru Suc Lac Mal Sta Raf Man FOS GOS Inu LtlLactobacillus acidophilus + V ++V −−V + V +
Lactobacillus casei +++−V −+V + V +
Lactobacillus plantarum ++++ +++ + V
Lactobacillus reuteri ++++ + + −
Lactobacillus rhamnosus ++++ +−− −
Bifidobacterium lactis +++++
Bifidobacterium longum +−V +++++ + + ++Source: From Pokusaeva et al. 2011, Tungland 2000, Champagne et al. 2010, and Dellaglio et al. 1994.
Symbols:+=positive,−=negative, V=variable.
Sugars:Glu, glucose; Fru, fructose; Suc, sucrose; Lac, lactose; Mal, maltose; Sta, Stachyose; Raf, raffinose; Man, mannitol; Inu, inulin; Ltl,
lactulose; FOS, fructooligosaccharides; GOS, galactooligosaccharides.