Microbial Interactions in the Yoghurt Consortium:
Current Status and Product Implications
Research Scientist, Arla Foods Strategic Innovation Centre, Rørdrumvej 2, 8220 Brabrand, Denmark
Sander Sieuwerts, Research Scientist, Arla Foods Strategic Innovation Centre, Rørdrumvej 2, 8220 Brabrand, Denmark,
Tel: +4587466703; Fax: +4586281691; E-mail:
Received: May 25, 2016; Accepted: July 11, 2016; Published: July 14, 2016
The metabolic potential of the fermenting microbial culture
influences the characteristics of fermented products, but the extent
to which this occurs depends on environmental factors and the
microorganism's interactions with its environment. It is therefore not
possible to simply translate gene content to product characteristics
and this is particularly the case when cultures are consortia of
microorganisms influencing each other. The yoghurt consortium,
consisting of Streptococcus thermophilus and Lactobacillus delbrueckii
subsp. bulgaricus, is mainly characterized by mutualistic interactions
between the two species. This review provides an updated overview
of these interactions, based on the latest insights, and their impact
on fermentation and product characteristics. Finally, some leads are
given on how to use modulation of interactions as means to alter
Keywords: Streptococcus thermophilus; Lactobacillus delbrueckii
subsp. bulgaricus; Mutualism; Yoghurt; Proteolysis; Exopolysaccharides
Microbial interactions in food fermentations
Fermentation is a process in which a carbon source is
dissimilated by microorganisms yielding energy and without
net oxidation. The major end products of this fermentation by
microorganisms are generally alcohols and organic acids, such
as lactic acid, acetic acid and propionic acid. Many food products
are fermented or contain a component of fermentation in the
production process. Food is fermented for many reasons. These
reasons include improved microbial stability, resulting in shelflife
extension and higher safety, improved sensoric properties,
and increased availability of essential nutrients either as result of
production by the microorganisms or by improved digestibility
of the raw material . The metabolic potential of the fermenting
microbial culture will thus have a huge impact on the exact
characteristics of the fermented product. This metabolic potential
is captured in the microorganism's genes, but to what extent the
metabolism affects the product properties very much depends
on environmental factors such as temperature, micronutrient
availability, oxygen pressure and pH and on the microorganism's
interactions with its environment. It is therefore not possible
to simply translate gene content to product characteristics. Moreover, by far most cultures applied in food fermentations
are microbial consortia consisting of multiple species and/or
strains, where the consortium members affect each other by
various modes of interaction. The intermicrobial interactions
may be (i) direct communication via signaling molecules, (ii)
labor division by growth factor exchange, or (iii) effects induced
by changed physico-chemical properties of the environment or
production of inhibiting compounds [2,3]. These interactions
can have a positive, neutral, or negative influence on both the
effector and the target, making it possible to classify them based
on their mutually beneficial and detrimental effects on fitness .
There are six main classes: neutralism, amensalism, competition,
commensalism, parasitism and mutualism (Table 1). It must be
noted that in a competitive and mutualistic relationship both
microorganisms are effector and target at the same time, where
in the others there typically is one effector and one target.
Mutualism in the yoghurt consortium
Yoghurt is bovine milk typically fermented with a
combination of the two lactic acid bacteria species Streptococcus
thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.
Although there are reported cases of competition and
amensalism for some strain combinations [5,6], the latter mainly
as a result of bacteriocin production, this consortium is typically
characterized as one with mutualistic interactions (Figure 1).
The mutualistic nature of the below-explained interactions
is the basis behind the stability of the yoghurt consortium
. Where negative interactions such as competition tend to
lead to exclusion of one of the consortium members, mutual
dependency or stimulation generally leads to equilibrium. It has
been known for a long time that the yoghurt bacteria stimulate
Table 1: The six main classes of microbial interactions based on their
beneficial and detrimental effects on fitness of the effector and target
Effect on target
Effect on effector
each other's growth by the exchange of metabolites, a process
called protocooperation, but the presence and extent of these
interactions depends on the exact combination of strains as there
is strain to strain variation in metabolic potential. Typically, the
yoghurt fermentation contains two exponential growth phases
separated by a transition phase with lower growth  (Figure
2). The first exponential phase is characterized by growth of S.
thermophilus, which is more tolerant to neutral pH and more
effective in taking up Amino Acids (AA) and trace elements than
L. bulgaricus. During its growth, formic acid and folic acid are
produced. This can help purine biosynthesis in L. bulgaricus as
precursor and co-factor, respectively, because this bacterium
with its highly degraded genome  is missing genes for de novo
folic acid biosynthesis and is therewith impaired in effective
purine biosynthesis. Moreover, S. thermophilus consumes oxygen
and produces carbon dioxide, thereby benefitting the less oxygen
tolerant L. bulgaricus . In the transition phase, growth of S.
thermophilus slows down, mainly due to a lack of AA, notably
sulfur and branched-chain AA, as the free levels of these AA in
milk are low and most S. thermophilus strains do not express an
exoprotease to harvest oligopeptides and AA from milk proteins.
During this transition phase, growth of L. bulgaricus and its
expression of the protease gene prtB are initiated, increasing
the levels of oligopeptides that can be taken up by and support
a second exponential growth phase of S. thermophilus while also
supporting exponential growth of L. bulgaricus . The cell-wall
resident PrtB, however, does not release sufficient sulfur AA and
branched-chain AA as these are only present in minor fractions
of casein compared to the microorganisms' requirements.
Therefore genes in the pathways for these AA are the only de
novo AA biosynthesis genes that are upregulated during this
second exponential growth phase [8,11]. Other genes typically
upregulated in S. thermophilus during this phase are involved
in long-chain fatty acids production. L. bulgaricus, having an
incomplete pathway for de novo biosynthesis of these compounds,
may benefit of this. In addition, gene expression of pathways
for production of Exopolysaccharides (EPS) is elevated, as
evidenced by the increasing amount of EPS that is present in the
system, which continues in the stationary phase. This increased
expression of EPS genes may be a direct result of the increasing
availability of nitrogen source, i.e. casein hydrolysis products
[12,13]. It is hypothesized that EPS here play a role in ensuring
close proximities between the two species, thereby facilitating
the exchange of metabolites , additional to protection against
unfavorable conditions, such as high acidity. Cell aggregation, cell
to cell communication, protection against environmental factors
and accumulation of metal ions are all reported as functions of EPS
. A recent study also links S. thermophilus urease activity to
increased growth of L. bulgaricus . Not only the relation of this
reaction – from urea to ammonia and carbamate – with aspartate,
glutamine, and arginine and for the interaction relevant carbon
dioxide metabolism provides a benefit, but also the released
ammonia increases the pH both outside and inside cells, allowing
a higher growth and acid production. This deacidification
effect only occurs locally, indicating the importance of small
distance between the two bacteria. Another recently discovered
metabolite exchange involves glutathione, a widely distributed
antioxidant that can be produced by S. thermophilus but not L.
Figure 1: representation of the interactions between S. thermophilus and L. bulgaricus during yoghurt fermentation and their effects on product characteristics.
The dotted lines indicate that EPS is hypothesized to facilitate the exchange of metabolites by establishing close proximities between the
two species →: production or enzymatic activity;→: positive effect of the component; &: negative effect; : neutral or yet to be confirmed
effect; EPS: Exopolysaccharides; LCFA: Long-Chain Fatty Acids.
Figure 2: Schematic representation of the growth phases in a typical co-fermentation of S. thermophilus (red) and L. bulgaricus (green) and the most
important factors that determine their growth behaviors. AA: Amino Acids; LCFA: Long-Chain Fatty Acids.
bulgaricus, which was shown to relieve acid stress in the latter,
resulting in improved growth . Finally, there has also been
reported an exchange of ornithine and putrescine between the
two species, but the function of this exchange is not elucidated
yet. Possible functions include the production of carbon dioxide
and the use of putrescine as co-factor in cell division or as metal
ion chelation agent . Hereby, it is noteworthy that metal
ions, in particular iron and manganese, are quite scarce in milk
and efficient systems to take up these ions may certainly benefit
growth of the microorganisms. That matches with reported
elevated expression of genes involved in iron chelation  in
S. thermophilus in co-culture with L. bulgaricus compared to its
Effects of interactions and strains on product
The extent and nature of the mutual interactions between
the yoghurt bacteria will largely determine the performances
of both species and this depends on the exact combinations of
strains, moving the equilibrium in the consortium more towards
one of the species rather than a one to one ratio. Combinations
in which the interactions between the two species are fine-tuned
promote the most efficient mutual growth with the least loss or
lack of exchanged metabolites. Poor interactions lead to reduced
fermentation rate or unbalanced growth as a result of lacking
nutrients as can be concluded from combining genome-scale
metabolic models of both species  or kinetic models . For
example, some strains of S. thermophilus express the exoprotease
PrtS, which makes them independent of L. bulgaricus for their
AA supply, even though it has been shown that PrtS has no
influence on acidification and microbial composition when PrtB
is present. In contrast, when combined with a non-proteolytic L.
bulgaricus strain, PrtS mainly supports growth of S. thermophilus,
leading to low counts of L. bulgaricus  and altered secondary
metabolite accumulation . What are the consequences of
these interactions on product characteristics? Affecting the
interactions will always result in a shift in microbial composition
and therewith impact species-specific contributions to the
environment. In the here mentioned example with reduced L.
bulgaricus counts, the yoghurt is more likely to be mild, because
L. bulgaricus is typically associated with lower pH as this species
is generally more acid tolerant than its counterpart. Moreover,
since S. thermophilus mostly is the largest contributor of EPS
and aroma compounds , notably of acetoin and diacetyl that
together with acetaldehyde make the typical yoghurt flavor, a
change in composition in the yoghurt culture will affect viscosity
and flavor, as was exemplified in a recent study . In a typical
yoghurt culture with a non-proteolytic S. thermophilus, high
levels of L. bulgaricus are not only associated with high acidity
but also with bitterness, as the activity of its exoprotease may
result in accumulation of bitter tasting peptides and amino acids
[23,24]. With the current trend in the dairy industry towards
milder yoghurts with a good mouthfeel (high viscosity), it is not
unsurprising that culture producers try to engineer combinations
of S. thermophilus and L. bulgaricus strains that result in high
counts of the former and low counts of the latter, i.e. cultures in
which the interactions between the two species work in such a
way that S. thermophilus sufficiently benefits from L. bulgaricus'
proteolysis while not very much promoting its growth. Using
only S. thermophilus is often not possible, because proteolysis
is required for optimal growth and acidification and because in
many countries it is obligatory to have both species, or at least
both cocci and bacilli, present and alive in order to be able to call
the product yoghurt . Moreover, an effective fermentation
with both species and concomitant proteolytic and peptidolytic
action is for example essential to decrease allergic reactivity
to β-lactoglobulin and α-lactalbumin among > 5% of the infant population and around 2% of the adults, as was showcased by Bu and coworkers . The extent of dissimilation of allergic
reaction causing proteins and reduction in allergic reactions
are, however, highly dependent on the used strains and their
proteolytic and peptidolytic capabilities [27,28]. Considering
such properties of effective fermentations are not in focus of
culture producers and dairy industry yet except in few cases
where specialized products are made, but with the rise in welfare
diseases, healthy and nutritious foods become more and more
There are multiple ways to change product characteristics by
modifying interactions with the environment and with consortium
members. As mentioned, by changing the strain combination,
it is possible to acquire different extents of the aforementioned
mutualistic interactions resulting in different employments of
the microorganisms' metabolic potentials thereby changing
the metabolite profile of the yoghurt . This will result in a
different taste and texture. As another example, extension of the
fermentation time, notably of the second exponential phase, by
lowering the fermentation temperature will lead to a final higher
production of aroma compounds and EPS . It is not possible
to simply assume that higher EPS levels naturally lead to higher
viscosity, though. The exact nature of EPS – e.g. capsular or free,
electric charge, monomer composition, extent of branching and
types of side-groups – together with the amount and composition
of milk proteins largely determine viscoelastic and structural
properties of the EPS-protein matrix and therewith organoleptic
properties such as creaminess and mouth thickness, as was
exemplified in two recent studies [29,30]. EPS produced by
some strains interact better with caseins and others with whey
proteins. It is therefore essential for starter culture producers
and yoghurt manufacturers to not only pick the right strain
combination for an optimal fermentation and flavor balance
, but also to accommodate the right strain combination with
the right protein composition and to use the right fermentation
conditions in order to make yoghurt with exactly the desired
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