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Fermentation technologies for the production of probiotics

with high viability and functionality


Christophe Lacroix and Selcuk Yildirim
There is growing scientic evidence supported by mechanistic
and clinical studies that probiotics can provide health benets.
As probiotics are highly sensitive to many environmental
factors, and because the propagation of many strains of
intestinal origin is not straightforward, most commercial strains
are selected on the basis of their technological properties
ruling out some strains with promising health properties. To
date, probiotic production has almost exclusively been carried
out using conventional batch fermentation and suspended
cultures, in some cases combined with the use of sublethal
stresses to enhance cell viability, the addition of protectants or
microencapsulation to provide cell protection. However, other
less conventional fermentation technologies, such as
continuous culture and immobilized cell systems, could have
potential for enhancing the performance of these fastidious
organisms. These technologies might be employed to develop
strains with improved physiology and functionality in the gut
and to enlarge the range of commercially available probiotics,
as well as expanding product applications.
Addresses
Laboratory of Food Biotechnology, Institute of Food Science and
Nutrition, ETH Zurich, Schmelzbergstrasse 7, CH-8092 Zurich,
Switzerland
Corresponding author: Lacroix, Christophe
(christophe.lacroix@ilw.agrl.ethz.ch)
Current Opinion in Biotechnology 2007, 18:176183
This review comes from a themed issue on
Food biotechnology
Edited by Christophe Lacroix and Beat Mollet
Available online 2nd March 2007
0958-1669/$ see front matter
# 2007 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2007.02.002
Introduction
With our rapidly increasing knowledge of intestinal micro-
biota and modulating factors, interest in supplementing
various types of food products with probiotic bacteria has
grown signicantly. The term probiotic refers to a prep-
aration of dened microorganisms, or a product containing
viable micoorganisms, (mainly lactobacilli and bidobac-
teria) in sufcient numbers to alter the microora in a
compartment of the host andbring benecial healtheffects
[1]. There is growing scientic evidence that maintenance
of healthy gut microbiota can provide several health
benets for the host and that probiotics might contribute
[2,3]. However, there are signicant technological chal-
lenges for probiotics which, being of intestinal origin, are
sensitive to many environmental stresses, such as acidity,
oxygen and heat. Before a probiotic can benet human
health it must fulll several criteria [4,5

]. Firstly, a pro-
biotic must have scientically validated health properties
and demonstrated safety. It should also have good tech-
nological properties so that it can be produced on a large
scale and incorporated into food products without loosing
viability and functionality or creating unpleasant avor or
texture. Additionally, a probiotic must exhibit highsurvival
rates in downstream processes (such as centrifugation and
drying) and in food products during storage. High survival
through the upper gastrointestinal tract and high viability
at its site of action are also a requirement, together with
high activity in the gut environment.
The viability of probiotics is a key parameter for devel-
oping probiotic foods. Although the amount of cells
required to produce therapeutic benets is not known
and might vary as a function of the strain and the health
effect desired, in general a minimum level of more than
10
6
viable probiotic bacteria per millilitre or gram of food
product is accepted [6]. Several factors illustrated in
Figure 1 affect the viability of probiotic bacteria until
they reach the target site of the host. Despite the import-
ance of viability, several surveys have shown large uctu-
ations and poor viability of probiotic bacteria in food
[7,8]. Currently commercial strains are largely selected
for their technological properties, ruling out some strains
with promising health properties. Consequently, indus-
trial demand for new technologies that enable high cell
yield at large scale and ensure probiotic stability in food
remains strong, because many strains of intestinal origin
are difcult to propagate and high survival is important
for both economic reasons and health effects. In addition,
more efcient technologies could lead to greater product
efcacy and strain diversication with the development of
technologically unsuitable strains into products.
This paper discusses the latest developments in fermenta-
tiontechnologies for producingprobiotic bacteria as well as
potential new approaches for enhancing the performance
of these fastidious organisms during fermentation, down-
stream processing, and utilization in commercial products,
and for improving functionality in the gut. Processes that
includesublethal stress applications duringcell production
and new fermentation technologies, such as immobilized
cell biolm-type fermentations, are promising in this
respect and could be used to prole cell physiology to
optimize survival and functionality in the gut.
Current Opinion in Biotechnology 2007, 18:176183 www.sciencedirect.com
Technologies currently used to enhance cell
viability
Industrial processes for food culture production, includ-
ing probiotics, almost exclusively use conventional batch
fermentation with suspended cells. Several approaches
have been investigated to enhance cell viability during
downstream processing, storage and eventually digestion
[9]. These include the application of sublethal stresses
during fermentation, the addition of protectants, includ-
ing compatible solutes (e.g. betaine which has been
extensively studied), and cell protection by microencap-
sulation (discussed by Champagne and Fustier in this
issue). The ability of microorganisms to grow and survive
depends largely on their capacity to adapt to changing
environments. Adaptation to adverse environments is
usually associated with the induction of a large number
of genes, the synthesis of stress-response proteins, and
the development of cross-resistance to various stresses
[10

]. On the basis of this knowledge, methods for


enhancing the long-term survival of lactobacilli and bi-
dobacteria by decreasing the lethal effects of environ-
mental stresses and process conditions have been recently
investigated to improve cell resistance to environmental
stresses occurring during production, storage or digestion.
Presently, such techniques mainly rely on the incubation
of free cells under starving or other stressful conditions,
such as heat, high concentrations of salt, bile salts, hydro-
gen peroxide or low pH [11

].
The response of different strains of bidobacteria to
increasing sublethal temperatures, salt or bile salt treat-
ments by synthesizing specic stress-protecting proteins
resulted in improved cell tolerance to further homologous
or heterologous stresses [12]. Heat adaptation with cross
protection (i.e. heat and salt stress conditioning) has been
shown to improve the survival of Lactobacillus paracasei
NFBC 338 during spray drying [13]. In another study, the
survival of Bidobacterium longum in growth medium at
6 8C signicantly increased when cells were submitted to
starving conditions for 30 or 60 min [14]. Likewise the
acid tolerance of Bidobacterium lactis (at pH 3.5 in syn-
thetic gastric uid) increased signicantly after decreas-
ing the growth medium pH from 6.0 to 5.2 and under
conditions of starvation. Stationary-phase acid and heat
treatments of bidobacteria strains also produced differ-
ent adaptative and cross-protective responses [15]. Cell
responses to sublethal stresses depend on the strain and
applied stresses [16]. Therefore, in the absence of general
mechanisms for sublethal stress adaptation, fermentation
conditions must be determined for each bacterium. This
process is labour-intensive and costly. In addition, sub-
lethal stresses can lead to decreases in cell activity and
yield [13] and eventually to changes in the functionality
of probiotic cells in the gut. Clearly there is a need to
develop a more in-depth understanding of stress-response
mechanisms of probiotics.
New fermentation technologies using
suspended cells
Continuous fermentation
Until now, very few data have been reported on continu-
ous fermentations with probiotics, although this approach
could provide benets as recently reviewed by Doleyres
et al. [11

] for bidobacteria. Under carefully selected


conditions, continuous culture can lead to both high cell
yield and process volumetric productivity, as well as
Probiotic fermentation technology Lacroix and Yildirim 177
Figure 1
Main factors affecting the viability of probiotics from production to the gastrointestinal tract.
www.sciencedirect.com Current Opinion in Biotechnology 2007, 18:176183
decreasing the demand for downstream processing
capacity [17]. Under steady-state operation, cells pro-
duced during continuous culture are in a controlled
physiological state that can be manipulated by environ-
mental parameters, such as medium composition and
dilution rate, the latter determining the specic growth
rate of the microorganisms in the system. Although con-
tinuous fermentations can be more difcult to operate
under industrial conditions, because they are highly
susceptible to contamination and cell characteristics
can be lost over time, this technology is worth investi-
gation and could be used to produce cells with different
physiologies and to apply stresses under well-controlled
conditions. An example of a two-stage continuous fer-
mentation process for the production of stress-adapted
probiotics is illustrated in Figure 2a. This type of con-
tinuous system could provide a new tool for the more
efcient screening of sublethal stresses than conventional
batch cultures, because the system stabilizes rapidly to
changing conditions in the second reactor (after 5 to 7
residence times, corresponding to a fewhours). Currently,
no data are available on the potential and limitations of
continuous fermentations for probiotic production with
controlled physiology.
Membrane bioreactors
In a membrane system with continuous feeding of fresh
medium, cells are retained in the bioreactor by an ultra-
ltration or microltration membrane whereas small mol-
ecules diffuse through the pores of the membrane
according to their size. Therefore, inhibitory metabolic
products are eliminated in the permeate and cells are
concentrated on the retentate side. The concentrated cell
fraction can be harvested batch-wise or continuously with
no, or minimal, additional downstream treatment for cell
concentration before freezing or freeze-drying. Few data
have been reported on probiotic cell production in mem-
brane reactors, although these systems could result in
high cell yields and volumetric productivities. Taniguchi
et al. [18] reported nal B. longum concentrations in a
membrane bioreactor that were seven times higher than
those obtained during free-cell batch fermentations. Sim-
ilarly, Corre et al. [19] measured high cell yields and a
15-fold improvement of volumetric productivity com-
pared with free-cell batch cultures for Bidobacterium
bidum. However, cell physiology and functionality were
not studied. Indeed, cells are subjected to many stresses
in membrane bioreactors, such as low nutrient concen-
tration, oxygen, osmotic and mechanical stresses that
could affect sensitive bacteria, but might also lead to
cross-protection effects for other stresses. Finally, close
cell-to-cell contacts owing to high cell density in the
retentate section might induce quorum-sensing responses
affecting cell physiology. Research is needed on these
factors and their effects on physiology, tness and func-
tionality of probiotics to explore the potential of these
systems for the production of stress-adapted cells.
Probiotic fermentation with immobilized cell
technology
In nature, microorganisms often live as highly organized
communities enclosed in a self-produced polymeric
matrix (or biolm) that adheres to an inert or living surface
[20]. Biolms generally comprise a mixture of com-
ponents, such as proteins, nucleic acids and exopolysac-
charides. The exopolysaccharides are produced by most
bacteria either as cell-wall polysaccharides or as extra-
cellular excretions into the surrounding environment [21].
The formation of biolms is a major strategy for bacterial
survival. When biolms are produced above a certain
population density, bacterial cells undergo complex
cell-to-cell interactions; signals of quorum-sensing sys-
tems accumulate to threshold concentrations that control
the expression of various genes in different parts of
178 Food biotechnology
Figure 2
Two-stage continuous fermentation processes for the production of
stress-adapted probiotics, with a first reactor (R1) operated with free or
immobilized cells and a second reactor (R2) inoculated with free cells
produced in R1. (a) Reactor R1 operated with free cells (at low dilution
rate for producing cells in late exponential growth phase); the image on
the left shows a photomicrograph of Bifidobacterium longum produced
in this system. (b) Reactor R1 operated with immobilized cells at high
dilution rate and cell density; the image on the left shows an optical
micrograph of a section of bead-immobilized Lactococcus lactis
(1.4 10
11
colony-forming units/g bead, colonies stained with o-
toluidine). Cells produced in the first reactor R1 with defined physiology
are subjected to sublethal stresses (e.g. starvation, low pH, high
temperature, salt, etc.; stress illustrated with a lightening bolt) in the
second reactor R2 under controlled conditions. The system stabilizes
rapidly to changing conditions in the second reactor (after 5 to 7
residence times, corresponding to 2.5 to 3.5 h for a system designed for
a 30 min stress application), allowing for efficient screening of several
stresses during the same culture experiment.
Current Opinion in Biotechnology 2007, 18:176183 www.sciencedirect.com
biolms and at different stages of their development [22].
Such natural biolm structures are also found in the gut,
where bacteria grow on the gut mucosa and attached to
food particles [23] (also see the article by Macfarlane and
Macfarlane in this issue). The biolm phenotype has
been extensively described in Gram-negative bacteria,
but little is known about biolm formation in Gram-
positive bacteria.
Studies with Gram-positive food pathogens showed that
cells grown in biolms exhibit an altered phenotype with
respect to bacterial physiology, metabolism and gene
transcription, as recently reviewed for Listeria [24]. Infec-
tious bacteria in biolms are generally more resistant to
antibiotics and unfavourable environmental factors such
as extreme temperatures, low pH values and osmolarity
[25]. The increased resistance of bacteria to antibiotic
treatments in the host was dened as one of the main
reasons for biolm-associated infections [26]. However,
there are currently few data reported on the physiological
changes induced by growth in biolms in benecial food
bacteria, including probiotics.
Cell immobilization has been used to perform high cell
density fermentations for both cell and metabolite pro-
duction. For food applications, cell entrapment in food-
grade biopolymer gel matrices (e.g. k-carrageenan, algi-
nate and gellan) has been most widely used [27]. Several
advantages over free-cell fermentations have been
demonstrated: high cell densities, reuse of biocatalysts,
improved resistance to contamination and bacteriophage
attack, enhancement of plasmid stability, prevention
from washing-out during continuous cultures, and the
physical and chemical protection of cells [27]. The immo-
bilization and growth of cells in porous solid supports
during incubation in a nutritive medium results in the
formation of a high cell density region (cell concentrations
typically ranging from 5 10
10
to 5 10
11
colony-form-
ing units/ml, which are 10- to 50-fold higher than for
traditional batch cultures). This region extends from the
biocatalyst surface to a radial position where cell growth is
prevented by a lack of substrate, accumulation of inhibi-
tory products and/or other unfavourable environmental
conditions such as low pH [28,29]. Biolm-type cell
growth occurring in the peripheral layers of gel beads
has been shown to give a high rate of cell release into the
bulk fermentation broth, as a result of high pressure
owing to cell expansion, collisions and shearing forces
in the bioreactor, leading to an efcient inoculation of the
bulk medium [29,30]. Only a few studies have examined
the application of cell immobilization for probiotic pro-
duction. Continuous fermentation with B. longum
immobilized in gellan gum gel beads produced high cell
concentrations and fourfold increased volumetric pro-
ductivity at a dilution rate of 0.5 h
1
when compared
with free-cell batch cultures at optimal pH [17]. Doleyres
et al. [31] recently showed that immobilization of a mixed
culture containing a dominant Lactococcus lactis strain and
a less competitive B. longum strain allowed stable con-
tinuous production of concentrated mixed culture in a
two-reactor system similar to that illustrated in Figure 2b;
temperature was a crucial parameter for the strain ratio in
the mixed culture.
Several studies have shown that immobilized and
released bacteria exhibit changes in growth, morphology
and physiology compared with cells produced in conven-
tional free-cell cultures (summarized in Table 1 for lactic
Probiotic fermentation technology Lacroix and Yildirim 179
Table 1
Reported effects of cell immobilization on the physiology of potential probiotic bacteria.
Cultivation mode Species Changes in cell physiology Reference
FC batch
a
/IC batch Lactococcus and
Leuconostoc strains
Increased tolerance to quaternary ammonium sanitizers [46]
FC continuous
a
/IC
continuous
Lactococcus lactis Change of redox state [47]
Change of intracellular pH
Change in lactate dehydrogenase synthesis
IC continuous Lactococcus lactis Increased acidifying activity with culture time [48]
FC batch
a
/IC continuous Bidobacterium longum
Lactococcus lactis
Increased tolerance to hydrogen peroxide
b
[33

]
Increased survival during freeze-drying
b
Increased survival in gastrointestinal conditions
b
Decreased sensitivity to antibiotics
b
Decreased sensitivity to nisin Z
b
FC batch
a
/IC batch Lactobacillus rhamnosus Changed exopolysaccharide production from soluble to insoluble
b
[36]
Induced aggregation
b
FC batch
a
/IC continuous
fermentation
Lactobacillus rhamnosus Increased acidifying capacity
b
[34]
Increased acid tolerance
b
Increased nisin Z tolerance
b
Lactococcus lactis Increased acidifying capacity
b
IC continuous fermentation Lactobacillus delbueckii Increased acidifying capacity
b
[35]
Decreased autolytic activity
b
Increased survival during freeze-drying
b
a
Used as a reference for the comparison.
b
Time effect was shown. FC, free cell; IC, immobilised cell.
www.sciencedirect.com Current Opinion in Biotechnology 2007, 18:176183
acid bacteria and probiotics, and reviewed by Junter et al.
[32

] for a variety of other articially and naturally


immobilized microorganisms). However, the effects of
immobilization on the technological and physiological
characteristics of probiotic bacteria have only recently
been reported [33

]. In a two-stage continuous culture


system containing separately immobilized B. longum and
L. lactis, as shown in Figure 2b, free cells from both strains
in the efuent medium showed increased tolerance to
various stresses, including freeze-drying, hydrogen per-
oxide, simulated gastrointestinal conditions, nisin and
antibiotics [33

]. The tolerance markedly increased with


culture time and was generally higher after 6 days than
that of stationary-phase cells produced during free-cell
batch fermentations. Other recent studies with immobil-
ized lactobacilli also detected important physiological
changes, such as a large increase in cell tolerance to nisin
Z and low pH, enhanced acidication capacity [34,35],
induction of aggregation, and high production of insolu-
ble exopolysaccharides [36].
Several factors have been proposed to account for the
non-specic stress adaptation of immobilized cells: the
presence of a steep gradient of inhibitory products
(organic acids) and pH in the active biocatalysts, owing
to high cell concentrations and activity and diffusion
limitations; the selection of stress-resistant subpopu-
lations with time; and quorum sensing effects [28,33

].
The very high cell packing in peripheral layers of beads
and the close contacts between immobilized cells might
induce physiological characteristics that are typically con-
trolled by quorum sensing and regulate the expression of
certain genes. In a cell-density-dependent quorum-sen-
sing system, bacteria produce extracellular signaling mol-
ecules such as peptides or post-translationally modied
peptides that act as inducers for gene expression when
concentrations of these molecules exceed a certain
threshold value. These changes might eventually lead
to competitive advantages for the population, more effec-
tive adaptation and responses to changing environmental
conditions, or the co-ordination of interactions between
bacteria and their abiotic and biotic environments [37]. As
such, cells produced by immobilized cell technology
might exhibit physiology proles that are better suited
for adaptation to growth in the very competitive con-
ditions of the gastrointestinal tract, but this remains to be
demonstrated.
Current data suggest that cell immobilization combined
with continuous culture might be used to efciently
produce, in a one-step process, probiotic cells with
enhanced tolerance to environmental stresses and
improved technological and functional characteristics.
Moreover, a second fermentation stage can be added to
perform additional adaptation under conditions of
starvation or other sublethal stresses. However, additional
studies are needed to unravel the molecular and physio-
logical mechanisms of these effects and to develop indus-
trial applications based on this innovative technology.
Biomarkers for viability and functionality
assessment and for process monitoring
Because probiotics must maintain optimal viability in
both products and the gut, previous studies on technology
and stress adaptation of probiotic bacteria have almost
exclusively relied on cell viability measurements, mainly
with plate counts. However, culture-based methods pro-
vide only a biased estimate of viable cell numbers
because sublethally injured or dormant bacteria often
cannot be detected [38]. In addition, culture-based
analysis of viability is not an adequate predictor for
probiotic functionality, which should be the ultimate
marker for technological developments, as recently
shown by Matto et al. [39]. Therefore, in the eld of
probiotics, additional methods should be used to study
cell physiology for fundamental research, process devel-
opment and monitoring, and product assessment. Mol-
ecular methods such as ow cytometry and uorescence
in situ hybridization can be used to assess viability in
products, as recently shown for the quality assessment of
commercial probiotic capsules and non-dairy drinks [40

].
Flow cytometry also enables subpopulations to be dis-
tinguished and physically sorted for further characteriz-
ation [41

]; therefore, this technique can be particularly


useful for studying stress adaptation mechanisms of sub-
populations.
Currently, one important limitation for establishing
optimal conditions for probiotic production is the absence
of prole targets with respect to optimum cell physiology
leading to high functionality of probiotics in the gut. In a
recent study (unpublished work cited by de Vries et al.
[42

]), DNA microarrays were used to monitor the gene


expressionof Lactobacillus plantarumin surgically removed
intestinal segments. The specic expression of genes
encoding sugar uptake and metabolism, amino acid bio-
synthesis, cell division and stress-related genes was
observed, indicating survival, metabolic acitivity and
growth of probiotics in the gut. This approach could be
a powerful and high-throughput tool not only for providing
insight into in vivo hostmicrobe interactions [42

], but
also for developing physiological prole targets for pro-
biotic production. More generally, the omics technol-
ogies transcriptomics, proteomics and metabolomics
could be used to assess and compare the physiological
proles of probiotics in the gut, at different stages of
production, and following their addition to food. One
possible aim for process development and optimization
could be to obtain cells during and after production that
have similar proles to cells in the gut, in addition to high
cell yield and viability.
Flow cytometry and PCR methods might provide rapid
and efcient tools for the characterization of cell
180 Food biotechnology
Current Opinion in Biotechnology 2007, 18:176183 www.sciencedirect.com
physiology during and after production. After the identi-
cation of biomarkers for cell functionality by omics
methods, new uorescent stains for these biomarkers
could be developed and applied for process monitoring
and product quality control. For example ow cytometry
can quantify specic intracellular proteins and measure
membrane integrity [43]. Recently, automated ow cyto-
metry has been developed and used to monitor continu-
ous fermentations at the single-cell level [44,45].
Conclusions
Until now, technological developments for the pro-
duction of probiotics have focused on approaches to
obtain as many cells as technically and/or economically
possible. Clearly, in addition to high cell numbers, cell
physiology is crucial to ensure that cells are well-suited to
survival during downstream processes and that they exhi-
bit high functionality. Astrategy for future research aimed
at optimizing probiotic production is presented in
Figure 3. Furthermore innovative fermentation technol-
ogies to produce food cultures need to be investigated in
relation to generating cells with high viability and func-
tionality upon delivery. Reliable and convenient bio-
markers need to be developed for process monitoring
and product assessment. In this regard, the omics tech-
nnologies could be particularly useful for identifying such
functionality-relevant biomarkers. These approaches
could also help to identify the mechanisms for cell tness
and stress-adaptation that will be needed to develop more
generic and science-based technologies for the pro-
duction of sensitive probiotics, thereby enlarging the
range of commercial probiotics and product applications.
Moreover, these tools might facilitate screening
approaches to identify new probiotic strains that combine
suitable technological and functional qualities.
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have been highlighted as:
of special interest
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