**5. Stress resistance of probiotic LAB**

554 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

sensing mechanism [79].

Some secondary metabolites such as bacteriocins can play a role in LAB performances and metabolism, affecting also the total population and ecology of fermented foods [107, 108]. Bacteriocins are antimicrobial peptides or proteins produced by bacteria that can be active on different microorganisms, depending on their structure. LAB belonging to the genera *Lactococcus*, *Pediococcus*, *Lactobacillus*, *Leuconostoc*, *Carnobacterium*, *Propionibacterium* are known to produce bacteriocins with both narrow and broad inhibitory spectra [109]. The use of functional LAB starter cultures (eg. bacteriocinogenic starter cultures), well adapted to the environment and the process conditions applied, may contribute to the development of better controllable and more efficient production processes [110]. An example can be nisin, a peptide produced by *L. lactis* ssp. *lactis*, that has a narrow spectrum affecting primarily only Grampositive bacteria and their spores, including lactic acid bacteria, *Bacillus*, *Clostridium*, *Listeria*, and *Streptococcus*. However some LAB such as *Streptococcus thermophilus* and *L. plantarum* are able to produce the enzyme nisinase, which neutralizes the antimicrobial activity of the

peptide [111]. Therefore these LAB could be suitable for a co-fermentation with *L. lactis*.

Another interesting case of bacteriocin production, as a consequence of oxidative stress and carbon dioxide exposure, has been reported [110]: oxidative stress and carbon dioxide are involved in the production of a specific bacteriocin, amylovorin L, by *Lactobacillus amylovorus*, able to inhibit other LAB species. During traditional sourdough fermentation, a decrease in redox potential of the rather firm mixture occurs. The oxygen initially present is consumed by *Candida* spp. or converted into hydrogen peroxide or water, thereby creating microaerophilic or anaerobic environment in which the growth of the desired LAB is favored. While in a large-scale sourdough type II fermentation currently the use of dough mixture with high dough yield is exploited. This sourdough has to be stirred to liberate part of the carbon dioxide produced to prevent running over. During mixing, oxygen is incorporated into the dough. Also, the development of yeast and hence the production of carbon dioxide is favored in continuously stirred sough mixtures with high water content. Elevation of the airflow rates leading to oxidative stress conditions resulted in an enhanced specific amylovorin L production. Growth in the presence of carbon dioxide also increased the specific bacteriocin production. Mild aeration or a controlled supply of oxygen as well as growth in an environment containing high amounts of carbon dioxide might thus contribute to the competitiveness of *L. amylovorus* DCE471 in a sourdough ecosystem [110]. The production of plantaricin A by *L. plantarum* was also demonstrated in relation to a quorum

Another example of the influence of the process on LAB metabolism has been widely described [112]. These Authors monitored the evolution of the gene expression of *L. plantarum* IMDO 130201 during a sourdough process. In particular, the genes and the metabolites related to acidic stress were analyzed. It is interesting to highlight that during the pH decrease (production of lactic acid by *L. plantarum*) the genes coding for plantaricin production had higher levels of expression at low pH values, indicating that the bacteriocin production was activated under acid stress conditions by *L. plantarum* IMDO 130201 strain. The presence of the pheromone plantaricin A (PlnA) in a system inoculated with *L. plantarum* DC400 was also reported [79]. Biosynthesis of PlnA was variously stimulated There are two main categories of factors that contribute to the optimal functioning of probiotic lactobacilli: factors that allow optimal adaptation to the new niches that they temporarily encounter in the host (adaptation factors) and factors that directly contribute to the health-promoting effects (probiotic factors) [113].

Adaptation factors include stress resistance, active metabolism adapted to the host environment, and adherence to the intestinal mucosa and mucus.

In fact, probiotic lactobacilli encounter various environmental conditions upon ingestion by the host and during transit in the gastro intestinal tract (GIT). They need to survive to: 1) the harsh conditions of the stomach secretion generating a fasting pH of 1.5, increasing to pH 3 to 5 during food intake; 2) the bile excreted by liver in small intestine represents another challenge for bacteria entering the GIT. Bile salts also seem to induce an intracellular acidification so that many resistance mechanisms are common for bile and acid stress. Indeed, the protonated form of

Dynamic Stresses of Lactic Acid Bacteria Associated to Fermentation Processes 557

bile salts is thought to exhibit toxicity through intracellular acidification in a manner similar to those of organic acids like the lactic acid produced by the lactobacilli themselves. For a detailed overview of acid, bile, and other stress resistance mechanisms of lactobacilli, the reader is referred to more extensive review [113]. 3) In analogy to the stresses encountered by intestinal pathogens, they also encounter oxidative and osmotic stress in GI tract. 4) Interactions with other microbes and 5) Interactions with cells of the host immune system and the various antimicrobial products that they produce can also impose a serious threat for the probiotic microbes. Analogously to what described in food LAB, the phenomenon of cross-adaptation is often observed, i.e., that adaptation to one stress condition also protects against another stress factor, implying some common mechanisms. In this respect, also for probiotic LAB non-actively-growing stationary-phase cells are generally more resistant to

The different macromolecules constituting the cell membranes and cell walls of lactobacilli have been shown to contribute to maintaining cell integrity during stress to various degrees. For example, low pH caused a shift in the fatty acid composition of the cell membrane of an oral strain of *L. casei*. Similarly, bile salts have been shown to induce changes in the lipid cell

The role of EPS in acid and bile resistance is less clear. However, EPS production has not been studied in detail after exposure to bile. In fact, to our knowledge, phenotypic analyses of dedicated *Lactobacillus* mutants affected in EPS biosynthesis genes have not yet been performed. Homopolysaccharides (HoPSs) from *L. reuteri* have been reported to have a more established role in stress resistance by the maintenance of the cell membrane in the

A number of proteins that play a role in the protection or repair of macromolecules such as DNA and proteins also seem to be essential for acid and bile resistance. Intracellular acidification can result in a loss of purines and pyrimidines from DNA. Bile acids have also been shown to induce DNA damage and the activation of enzymes involved in DNA repair. Perhaps even more vital in the general stress response are chaperones that intervene in numerous stresses for important tasks such as protein folding, renaturation, protection of

Mechanisms to specifically sense the presence of certain stress factors and regulate gene expression in response to these stimuli are also crucial for bacterial survival under adverse conditions. Although these mechanisms are not well characterized for lactobacilli, they often involve two-component regulatory systems (2CRSs). 2CRSs allow bacteria to sense and respond to changes in their environment after receiving an environmental signal through

various stressors than early-log-phase cells.

membrane of *Lactobacillus reuteri* CRL1098.

**5.1. Maintaining integrity of the cell envelope** 

physiological liquid crystalline phase under adverse conditions.

**5.2. Repair and protection of DNA and proteins** 

denatured proteins, and removal of damaged proteins.

**5.3. Two-component and other regulatory systems** 

transmembrane sensing domains of the histidine protein kinase (HPK).

**Figure 3.** Sourdough fermentation dynamics. Case of possible parallel phenomena interesting acid and osmotic stress.

bile salts is thought to exhibit toxicity through intracellular acidification in a manner similar to those of organic acids like the lactic acid produced by the lactobacilli themselves. For a detailed overview of acid, bile, and other stress resistance mechanisms of lactobacilli, the reader is referred to more extensive review [113]. 3) In analogy to the stresses encountered by intestinal pathogens, they also encounter oxidative and osmotic stress in GI tract. 4) Interactions with other microbes and 5) Interactions with cells of the host immune system and the various antimicrobial products that they produce can also impose a serious threat for the probiotic microbes. Analogously to what described in food LAB, the phenomenon of cross-adaptation is often observed, i.e., that adaptation to one stress condition also protects against another stress factor, implying some common mechanisms. In this respect, also for probiotic LAB non-actively-growing stationary-phase cells are generally more resistant to various stressors than early-log-phase cells.
