**4. General steps regarding a virtual fermented food process**

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

free fatty acids such as oleic acid [21, 22].

1. Nutrient competition,

multiple mechanisms [77].

certain microorganisms by other members of the habitat.

peptides released by the actions of proteolytic enzymes [77].

2. Generation of unfavorable environment, 3. Competition for attachment/adhesion sites.

The mechanisms involved are common to all genera and include [78]:

changes in term of fermentation dynamics and metabolism with respect to the untreated cells [11]. The same approach was applied on *L. acidophilus* and *L. paracasei* to improve the technological performances of probiotic strains [21, 22, 74]. The sub-lethal treatment with HPH enhanced the capacity of some *in vitro* probiotic features (i.e. hydrophobicity and tolerance to simulated gastric acidity) in a strain dependant way. *L. paracasei* A13 enhanced cellular hydrophobicity and auto-aggregation capacity after HPH treatment at 50 MPa. On the contrary, the HPH treatment decreased these features in the other strains considered. Highest values of hydrophobicity were found for *L. acidophilus* DRU and its bile-resistant derivative *L. acidophilus* DRU+, while lower values were obtained for *L. paracasei* strain [74]. Moreover, the stress responses enable survival under more severe conditions, enhancing resistance to subsequent processing conditions [75]. HPH treatment at 50 MPa can favour the maintenance of cell viability during a refrigerated storage in buttermilk, a suitable medium to maintain the cell viability during refrigeration [76]. The increased viability can be attributed to the increased precocious availability of low molecular weight peptides and

**Competition and communication:** Food fermentations are typically carried out by mixed cultures consisting of multiple strains or species [77]. Mixed-culture food fermentations are of primary economic importance. The performance of these cultures, consisting of LAB, yeasts, and/or filamentous fungi, is not the simple result of "adding up" the individual single-strain functionalities, but is largely determined by interactions at the level of

General microbial interference is an effective non-specific control mechanism common to all populations and environments including foods. It represents the inhibition of the growth of

Most substrates for food fermentations have a highly heterogeneous physicochemical composition, which offers the possibility for the simultaneous occupation of multiple niches by "specialized" strains, for instance, through the utilization of different carbon sources. In these substrates, coexisting strains often interact through trophic or nutritional relations via

Carbon sources are often present at high concentrations in food substrates, and therefore competition concerns the rapid uptake of nutrients and conversion into biomass. In dairy fermentations nitrogen is limiting, and initially organisms compete for the free amino acids and small peptides available. While in the later stages of fermentation, they compete for the

In a cell-density-dependent quorum-sensing system, bacteria produce extracellular signaling molecules such as peptides or post-translationally modified peptides that act as

substrates, exchange of metabolites and growth factors or inhibiting compounds [77].

In the figure 2, the steps that mainly interest food fermentation are reported. A model virtual fermented food was identified to resume the common denominator of the fermented foods dynamics, particularly focused on the reciprocal influences between environmental fluctuation and LAB fermentation.

Whatever kind of food we want to produce, fermented or not, the first step of the process is the formulation: in this phase the main raw materials (meat, milk, fruit and vegetables or their derivatives) are mixed with other ingredients, that have different roles: salts or sugars to improve taste, spices to give specific sensorial quality and as antimicrobials, additives or other substances able to affect physical and structural properties, preservatives to improve microbial stability and shelf life. The addition of those ingredients can be perceived as stress. In fermented products, proper microorganisms, mainly yeasts and LAB, are also added as starter cultures, in order to start and lead the fermentation and to obtain a stable and standard final product. As a consequence, the microorganisms, naturally occurring or added as starter cultures, have to cope with a completely different system: in particular, naturally occurring microflora have to face the changes induced by the ingredients, while the starter cultures, deriving from growth media or added as lyophilized cultures, have to adapt to a real food system, where different sources of stresses are often present.

In particular, the first sub-lethal stress, which LAB face, regards the difference between the growth medium composition and the real food. Generally, LAB lyophilized cultures can be added to the ingredients after a reactivation and subsequently added to the product. This procedure identify the presence of a stress for the LAB cells. Starter cultures are added to the raw materials in large numbers and incubated under optimal conditions, but the adaptation to substrate or raw material is always necessary [82]. It is very important to consider the physiological state of the LAB before the inoculum. This state strongly depends on the time of harvesting of the culture (whether during the logarithmic or stationary phase of growth), on the conditions leading to transition to the stationary phase, on the treatment of the culture during and after harvesting and on the chemical composition of the environment. Therefore it is important during formulation and technological processes to consider also these factors, mainly for those products where microorganisms are added as starter cultures.

Dynamic Stresses of Lactic Acid Bacteria Associated to Fermentation Processes 549

The interaction between the starters and the ingredients and between the starters and the naturally present microbial population can trigger few important mechanisms that will influence the quality and the characteristics of the fermented product. Analogously, many food processes and formulations have been tested for safety by challenge test inoculating pathogen bacterial cells at different growth phases, and the results proved that cells grown to the stationary phase or adapted to various stresses have greater resistance than

Other ingredients usually added to obtain safe and stable products are food preservatives,

These latter are arbitrarily classified into two groups: traditional or "regulatory approved" and naturally occurring [84]. The former includes acidifiers such as acetic acid, lactic acid and citric acid and antimicrobials such as benzoic acid and benzoates, propionate, nitrites and nitrates, sorbic acid and sorbates and sulfites. The latter includes compounds from microbial, plant and animal sources that are, for the most part, only proposed for use in foods as antimicrobials (e.g. lactoferrin, lysozyme, nisin). Throughout the ages, food antimicrobials have been used primarily to prolong shelf-life and preserve quality of foods through inhibition of spoilage microorganisms, while only few are used exclusively to control the growth of specific foodborne pathogens (e.g. nitrite, used for hundreds of years to inhibit growth and toxin production of *Clostridium botulinum* in cured meats). In food formulation antimicrobials are part of a multiple intervention system that involves the chemical along with environmental (extrinsic) and food related (intrinsic) stresses and processing steps. Some of these substances (for example lactic acid and citric acid) provoke a direct acidification of a food or food ingredient, and therefore challenge the microflora inducing and increase of acid resistance of the microflora itself. In fermented food the situation can be somewhat different, because the pH is gradually lowered by LAB creating a

pH gradient, more likely than a sharp alteration in the pH due to direct acidification.

A good model describing the shock related to the inoculum of LAB in the raw complex material has been described during the production of fermented sausages [85]. The relatively high pH of raw meat rapidly decreases during the initial fermentation phase because organic acids, mainly lactate, are formed by LAB and the water activity is reduced during ripening, because of the addition of salt as well as drying. Furthermore, adjuvants, such as potassium or sodium nitrite and/or nitrate, are mostly added to optimize the

Generally strains used as starter cultures must tolerate these kinds of stresses and exhibit a high ecologic performance in the stressful food environment. Genes related to stress response are induced when *L. sakei* is inoculated in the raw meat system [86]. In fact, ctsR, a gene that coded for a class III heat shock proteins repressor associated with the environmental stress response of Gram positive bacteria, increased its expression when *L.* 

exponential cells [83].

a. Antioxidants,

fermentation process.

b. Anti-browning agents c. Antimicrobials.

including:

The interaction between the starters and the ingredients and between the starters and the naturally present microbial population can trigger few important mechanisms that will influence the quality and the characteristics of the fermented product. Analogously, many food processes and formulations have been tested for safety by challenge test inoculating pathogen bacterial cells at different growth phases, and the results proved that cells grown to the stationary phase or adapted to various stresses have greater resistance than exponential cells [83].

Other ingredients usually added to obtain safe and stable products are food preservatives, including:

a. Antioxidants,

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

**Figure 2.** Fermented food model: reciprocal influences between environmental fluctuation and lactic

acid bacteria fermentation.


These latter are arbitrarily classified into two groups: traditional or "regulatory approved" and naturally occurring [84]. The former includes acidifiers such as acetic acid, lactic acid and citric acid and antimicrobials such as benzoic acid and benzoates, propionate, nitrites and nitrates, sorbic acid and sorbates and sulfites. The latter includes compounds from microbial, plant and animal sources that are, for the most part, only proposed for use in foods as antimicrobials (e.g. lactoferrin, lysozyme, nisin). Throughout the ages, food antimicrobials have been used primarily to prolong shelf-life and preserve quality of foods through inhibition of spoilage microorganisms, while only few are used exclusively to control the growth of specific foodborne pathogens (e.g. nitrite, used for hundreds of years to inhibit growth and toxin production of *Clostridium botulinum* in cured meats). In food formulation antimicrobials are part of a multiple intervention system that involves the chemical along with environmental (extrinsic) and food related (intrinsic) stresses and processing steps. Some of these substances (for example lactic acid and citric acid) provoke a direct acidification of a food or food ingredient, and therefore challenge the microflora inducing and increase of acid resistance of the microflora itself. In fermented food the situation can be somewhat different, because the pH is gradually lowered by LAB creating a pH gradient, more likely than a sharp alteration in the pH due to direct acidification.

A good model describing the shock related to the inoculum of LAB in the raw complex material has been described during the production of fermented sausages [85]. The relatively high pH of raw meat rapidly decreases during the initial fermentation phase because organic acids, mainly lactate, are formed by LAB and the water activity is reduced during ripening, because of the addition of salt as well as drying. Furthermore, adjuvants, such as potassium or sodium nitrite and/or nitrate, are mostly added to optimize the fermentation process.

Generally strains used as starter cultures must tolerate these kinds of stresses and exhibit a high ecologic performance in the stressful food environment. Genes related to stress response are induced when *L. sakei* is inoculated in the raw meat system [86]. In fact, ctsR, a gene that coded for a class III heat shock proteins repressor associated with the environmental stress response of Gram positive bacteria, increased its expression when *L.* 

*sakei* starts to adapt to the raw environment. This mechanism demonstrated that the sudden changes in the environment conditions are perceived as stress by *Lactobacillus* species. In particular, in the case of *L. sakei*, added to raw meat and spices, the principal stress response regarded high osmolarity and temperature shifts. Moreover, the presence of curing salt is regarded as one of the major hurdles in the initial phase of sausages fermentation. Because nitrite was found to be the effective for growth inhibition of pathogens, nitrite was also hypothesized as a stressor for *L. sakei* [85] and the exposure of this strain to stresses can induce changes in metabolic activities in a food environment [4]. The metabolic changes in *L. sakei* resulted in enhanced exploitation of available nutrients or increased activity of glycolytic enzymes, leading to the accelerated production of lactic acid by stress-treated *L. sakei* cells [85]. However, the exposition of *L. sakei* to low temperature and high osmolarity gives rise to the repression of phosphofructokinase and consequently to a decreased flux through the glycolytic pathway [87].

Dynamic Stresses of Lactic Acid Bacteria Associated to Fermentation Processes 551

that can be performed during the processing and increase the initial pH, reducing also the nutrients content on the olive surface. The species able to face these kind of stresses usually belong to the genera *Pediococcus*, *Leuconostoc* and *Lactococcus*; after the first stage of fermentation, when the pH reaches 6, *L. plantarum* rapidly grows and dominate the fermentation, that goes on until the fermentable sugars are depleted. The viability and vigor of *L. plantarum* can be encouraged also by yeasts that are still present in this stage of

Moreover, the presence of some gases can modify the growth performances of LAB. That is also influenced by the mixing step of the ingredients in some food processes (e.g. dough mixing). In fact, in bread making process, the continuous agitation of the dough can increase the microbes exposure to oxygen, and this can be a source of oxidative stress, mainly for LAB that are usually anaerobic or facultative anaerobic. Also in these cases the bacteria can react in different ways, activating metabolic and transcriptional responses in order to

For the fermented vegetables ,above reported, the rapid consumption of oxygen due to the presence of yeasts and aerobic bacteria in the first stage of fermentation has a positive effect on LAB. In fact, they are exposed only for a short time to oxidative stress and, due to their

After formulation, the technological processes involving LAB include a fermentation

It is reported that various beneficial phenotypic traits of LAB in food fermentations such as rapid acidification, selective proteolysis, tolerance of osmotic and stresses, resistance to ROS, and ability to thrive in nutrient poor conditions and at low temperatures are influenced by stress responses in various species of LAB [95, 96]. The knowledge of these mechanisms, and mainly of the stress responses activated by the fermentation process parameters can be

The first metabolic reaction regards the oxidation of carbohydrates (this reaction depends on the hetero-fermentative or homo-fermentative species involved) that give rise to acids, alcohols and CO2. These metabolites are directly involved in flavor, aroma and texture of the product and in a second time can influence the production and the availability of other metabolites such as vitamins and antioxidant compounds [78]. Moreover, the LAB interactions with the ingredients increase also the digestibility and decrease the glycemic

At the same time with carbohydrates oxidation, other metabolic mechanisms interest LAB cells such as proteolysis and lipolysis. The first reaction produces polypeptides with interesting characteristics as antimicrobial compounds, salt substitutes (the oligopeptides are able to increase the palatability of the system), and amino acids deriving aromatic compounds. On the other hand lipolysis produces medium chain fatty acids, with important antimicrobial properties. All these reactions (carbohydrates oxidation, lipolysis and proteolysis) generate precursors for other mechanisms in the cells and in the food matrix

competitive advantage, they rapidly and intensively grow in the food system.

useful in order to develop strains with optimal fermentation characteristics [83].

index, enhancing the healthy features of the fermented foods [97].

fermentation and can produce vitamins [94].

detoxify ROS, as previously described.

process.

Moreover, it is important to consider that some ingredients can be also antimicrobials because of their own characteristics: in fact, if the recipe includes herbs and spices (aromatic plants, pepper), garlic and onions, an effect on microorganisms can be exerted by specific compounds characterizing these products, such as essential oils, terpenes and sulfur compounds [88].

Another essential aspect affecting the performances and metabolism of LAB are the intrinsic characteristics of raw materials that sometimes act in a synergic way with other ingredients. Considering for example fermented vegetables, the microflora of the starting fresh vegetables is typically dominated by Gram negative aerobic bacteria and yeasts, while LAB make up a minor portion of the initial population [89] and therefore they would not be able to start and lead a fermentation process. However, if anaerobic conditions are settled and salts are added, LAB can have a competitive advantage and induce spontaneous lactic acid fermentation. The growth of specific LAB is dependent on the chemical (substrate, salt concentration, pH) and physical (vegetable type, temperature) environments. As the environments change during fermentation, so can the dominant organisms, often leading to a specific and reproducible succession of bacteria.

In sauerkraut [89, 90] the presence of 1.8-2.2% of NaCl and a temperature of 18°C inhibits many strains of LAB, with the exception of *Leuconostoc mesenteroides* that initiates the fermentation; however this species is sensitive to acid conditions, so after a few days, when the concentration of lactic acid increases, *L. mesenteroides* is replaced by more acid resistant LAB such as *Lactobacillus brevis* and *L. plantarum*, able to further lower the pH up to 3-3.5, stabilizing the final product.

Considering olives fermentation is possible to outline the characteristics of the product affecting LAB: while the brine provides a good environment for LAB growth, with glucose, fructose and mannitol as the main source of fermentable sugars, the presence of high levels phenols (such as oleuropein) exert an antimicrobial activity, inhibiting some strains and selecting the types of organisms that predominate during the fermentation [91-93]. These LAB have to be resistant not only to phenols, but also to lye treatments and water washes, that can be performed during the processing and increase the initial pH, reducing also the nutrients content on the olive surface. The species able to face these kind of stresses usually belong to the genera *Pediococcus*, *Leuconostoc* and *Lactococcus*; after the first stage of fermentation, when the pH reaches 6, *L. plantarum* rapidly grows and dominate the fermentation, that goes on until the fermentable sugars are depleted. The viability and vigor of *L. plantarum* can be encouraged also by yeasts that are still present in this stage of fermentation and can produce vitamins [94].

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

through the glycolytic pathway [87].

a specific and reproducible succession of bacteria.

stabilizing the final product.

compounds [88].

*sakei* starts to adapt to the raw environment. This mechanism demonstrated that the sudden changes in the environment conditions are perceived as stress by *Lactobacillus* species. In particular, in the case of *L. sakei*, added to raw meat and spices, the principal stress response regarded high osmolarity and temperature shifts. Moreover, the presence of curing salt is regarded as one of the major hurdles in the initial phase of sausages fermentation. Because nitrite was found to be the effective for growth inhibition of pathogens, nitrite was also hypothesized as a stressor for *L. sakei* [85] and the exposure of this strain to stresses can induce changes in metabolic activities in a food environment [4]. The metabolic changes in *L. sakei* resulted in enhanced exploitation of available nutrients or increased activity of glycolytic enzymes, leading to the accelerated production of lactic acid by stress-treated *L. sakei* cells [85]. However, the exposition of *L. sakei* to low temperature and high osmolarity gives rise to the repression of phosphofructokinase and consequently to a decreased flux

Moreover, it is important to consider that some ingredients can be also antimicrobials because of their own characteristics: in fact, if the recipe includes herbs and spices (aromatic plants, pepper), garlic and onions, an effect on microorganisms can be exerted by specific compounds characterizing these products, such as essential oils, terpenes and sulfur

Another essential aspect affecting the performances and metabolism of LAB are the intrinsic characteristics of raw materials that sometimes act in a synergic way with other ingredients. Considering for example fermented vegetables, the microflora of the starting fresh vegetables is typically dominated by Gram negative aerobic bacteria and yeasts, while LAB make up a minor portion of the initial population [89] and therefore they would not be able to start and lead a fermentation process. However, if anaerobic conditions are settled and salts are added, LAB can have a competitive advantage and induce spontaneous lactic acid fermentation. The growth of specific LAB is dependent on the chemical (substrate, salt concentration, pH) and physical (vegetable type, temperature) environments. As the environments change during fermentation, so can the dominant organisms, often leading to

In sauerkraut [89, 90] the presence of 1.8-2.2% of NaCl and a temperature of 18°C inhibits many strains of LAB, with the exception of *Leuconostoc mesenteroides* that initiates the fermentation; however this species is sensitive to acid conditions, so after a few days, when the concentration of lactic acid increases, *L. mesenteroides* is replaced by more acid resistant LAB such as *Lactobacillus brevis* and *L. plantarum*, able to further lower the pH up to 3-3.5,

Considering olives fermentation is possible to outline the characteristics of the product affecting LAB: while the brine provides a good environment for LAB growth, with glucose, fructose and mannitol as the main source of fermentable sugars, the presence of high levels phenols (such as oleuropein) exert an antimicrobial activity, inhibiting some strains and selecting the types of organisms that predominate during the fermentation [91-93]. These LAB have to be resistant not only to phenols, but also to lye treatments and water washes, Moreover, the presence of some gases can modify the growth performances of LAB. That is also influenced by the mixing step of the ingredients in some food processes (e.g. dough mixing). In fact, in bread making process, the continuous agitation of the dough can increase the microbes exposure to oxygen, and this can be a source of oxidative stress, mainly for LAB that are usually anaerobic or facultative anaerobic. Also in these cases the bacteria can react in different ways, activating metabolic and transcriptional responses in order to detoxify ROS, as previously described.

For the fermented vegetables ,above reported, the rapid consumption of oxygen due to the presence of yeasts and aerobic bacteria in the first stage of fermentation has a positive effect on LAB. In fact, they are exposed only for a short time to oxidative stress and, due to their competitive advantage, they rapidly and intensively grow in the food system.

After formulation, the technological processes involving LAB include a fermentation process.

It is reported that various beneficial phenotypic traits of LAB in food fermentations such as rapid acidification, selective proteolysis, tolerance of osmotic and stresses, resistance to ROS, and ability to thrive in nutrient poor conditions and at low temperatures are influenced by stress responses in various species of LAB [95, 96]. The knowledge of these mechanisms, and mainly of the stress responses activated by the fermentation process parameters can be useful in order to develop strains with optimal fermentation characteristics [83].

The first metabolic reaction regards the oxidation of carbohydrates (this reaction depends on the hetero-fermentative or homo-fermentative species involved) that give rise to acids, alcohols and CO2. These metabolites are directly involved in flavor, aroma and texture of the product and in a second time can influence the production and the availability of other metabolites such as vitamins and antioxidant compounds [78]. Moreover, the LAB interactions with the ingredients increase also the digestibility and decrease the glycemic index, enhancing the healthy features of the fermented foods [97].

At the same time with carbohydrates oxidation, other metabolic mechanisms interest LAB cells such as proteolysis and lipolysis. The first reaction produces polypeptides with interesting characteristics as antimicrobial compounds, salt substitutes (the oligopeptides are able to increase the palatability of the system), and amino acids deriving aromatic compounds. On the other hand lipolysis produces medium chain fatty acids, with important antimicrobial properties. All these reactions (carbohydrates oxidation, lipolysis and proteolysis) generate precursors for other mechanisms in the cells and in the food matrix

that give rise to the dynamic environment characteristics of fermented foods. It is important to outline that the compounds produced by the cells, metabolizing the substrate, can modify the system, producing also compounds that can stimulate the growth of symbiotic species or inhibit the growth of antagonistic microorganisms.

Dynamic Stresses of Lactic Acid Bacteria Associated to Fermentation Processes 553

reported for *L. lactis* [104, 66]. The response of *L. sanfranciscensis* to osmotic stress (saccarose 40%) gives rise to the overproduction of 3-methylbutanoic acid and gamma-decalactones when *L. sanfranciscensis* was co-inoculated with yeasts, simulating a sourdough environment. The production of lactones can be indicated as unfavourable environment for microbial growth and metabolism. In fact, these compounds have both particular aromatic

The ability of the target strains to dominate the fermentation is related not only to the ingredients (as above described), but also to the fermentation conditions, mainly temperature and atmosphere. If the fermentation is not performed at the optimal growth temperature for the microorganisms, they could be unable to compete with naturally occurring microflora, and consequently the whole process could be compromised. On the contrary, some microbial species have developed specific thermal resistance mechanisms, and they can easily adapt to these unfavorable conditions without implications for the fermentation processes. Moreover, the adaptation to thermal stresses often leads to tolerance to other stresses, in a mechanism usually define "cross protection", as reported for *L. lactis* [105]. The ability of commercial *L. lactis* ssp. *lactis* and *L. lactis* ssp. *cremoris* to withstand freezing at –60°C for 24 h was significantly improved by a prior 25 min heat shock at ~40°C or by a 2 h cold shock at 10°C, opening interesting perspectives for the production on

Other Authors with regard to different stresses reported the "cross protection" mechanism: for example the mechanisms of multiple adaptations to hops of two different strains of *L. brevis* have been characterized [106]. Hop resistance of lactobacilli requires multiple resistance mechanisms. This is consistent with the stress conditions acting on bacteria in beer, which mainly consist of acid stress and the antimicrobial effect of the hop compounds, in addition to ethanol stress and starvation. The effect of interaction of acid stress and presence/absence of oxygen in the system on *L. helveticus* and *L. sanfranciscensis*, in particular on their cell membrane composition, has been reported [14]. Upon acid stress the level of cyclopropane fatty acids increased at the expense of the level of long-chain unsaturated fatty acids. *L. helveticus* and *L. sanfranciscensis*, exposed to acid sub lethal stress demonstrated the same increase in cyclopropane fatty acids. In particular, *L. helveticus* presented higher concentration of C19cyc11 at pH 4 and pH 3, while *L. sanfranciscensis* presented more C19cyc9 at pH 3 in microaerophilic condition without tween 80, at pH 3.6 in anaerobiosis with tween 80, and at pH 4 in anaerobiosis without tween 80. These results demonstrated

Consider the atmosphere, i.e. the presence or not of oxygen, as another important variable during fermentation, it is known that oxygen can inhibit the growth of LAB, especially in the first stages. However, the food system is usually a consortium of different microorganisms: for example in bakery products and in fermented sausages the fermentation is carried out both by yeasts and LAB; the formers can therefore consume the amount of oxygen present in the mix, allowing the growth of LAB. The same thing happens for fermented vegetables, where naturally occurring Gram negative bacteria and yeast

the same behavior in front of multiple stresses by LAB membrane [106, 14]

rapidly remove the oxygen, promoting the rapid predominance of Lactobacilli.

and antimicrobial features [13].

resistant starter cultures, both frozen or lyophilized [105].

The conversion of carbohydrates to metabolites as acetic acid, lactic acid or CO2 implies the acidification of the system. The contemporary pH decrease and the presence of sugar (osmotic stress) stimulate the exopolysaccharides (EPSs) production. In fact, in sourdough EPSs can be involved in acid tolerance of sourdough LAB [98]. EPSs are long-chain polysaccharides consisting of branched, repeating units of sugars or sugar derivatives. These sugar units are mainly glucose, galactose and rhamnose, in different ratios [99]. The presence of EPSs in the system can create a novel stress to the cells. The inclusion of cells within biofilm can increase their resistance to unfavorable environmental factors such as extreme temperature, low pH and osmolarity, the changes in the texture can induce in LAB also specific stress responses.

For example in yogurt production, the acidification by LAB implies proteins coagulation and thereby changes in the viscosity of the milk. In *L. bulgaricus*, during the acid adaptation present in the fermentation milk to obtain yogurt, some cellular changes were observed: the chaperones GroES, GroEL, HrcA, GrpE, DnaK, DnaJ, ClpE, ClpP and ClpL were induced and ClpC was repressed [100]. Some genes involved in the biosynthesis of fatty acids were induced (*fabH*, *accC*, *fabI*), while the genes involved in the mevalonate pathway of isoprenoid synthesis (*mvaC*, *mvaS*) were repressed [101, 102]. The changes in Aw value are depending not only on EPSs production by LAB after the exposition to acidic and osmotic stress, but also on the ingredients composition and on the step of fermentation.

Considering cheese, the Aw decreases during manufacture and ripening as a result of dehydration, salting, and production of water-soluble solutes from glycolysis, proteolysis, and lipolysis; the cheese Aw values range from 0.70 for extra hard cheeses to 0.99 for fresh, soft cheeses, such as cottage cheese, while semi-hard cheeses have Aw values of around 0.90. The cheese pH also decreases during manufacture and ripening [103]. The effects of different Aw and pH on *L. lactis* simulating cheese ripening have been analyzed [103]. The results evidenced that at low Aw, particularly at low pH, the growth and lactose utilization rates decreased and lactose fermentation to L-(1)-lactate switched to a pathway involving nontraditional saccharide products rather than the traditional lactococcal heterofermentative products.

In *L. plantarum* WCFS1 the addition of 300 mM and 800 mM of NaCl induced mild osmotic stress and osmotic stress respectively. In the presence of 800 mM of NaCl several genes showed an increased expression with respect to the control culture. In particular, those genes were associated with various stress responses in prokariotes, i.e. genes encoding Clp protease, an excinuclease, catalase (peroxide stress) and Dpr-like protein (peroxide stress). These differences in the gene expression were also identified in the presence of acid stress. These results suggest that lactic acid stress in *L. plantarum* WCFS1 also induces a more general stress response (as above described for different *Lactobacillus* species). An overlap between the stimulus for lactic acid and those for peroxide and UV radiation has also been reported for *L. lactis* [104, 66]. The response of *L. sanfranciscensis* to osmotic stress (saccarose 40%) gives rise to the overproduction of 3-methylbutanoic acid and gamma-decalactones when *L. sanfranciscensis* was co-inoculated with yeasts, simulating a sourdough environment. The production of lactones can be indicated as unfavourable environment for microbial growth and metabolism. In fact, these compounds have both particular aromatic and antimicrobial features [13].

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

inhibit the growth of antagonistic microorganisms.

also specific stress responses.

that give rise to the dynamic environment characteristics of fermented foods. It is important to outline that the compounds produced by the cells, metabolizing the substrate, can modify the system, producing also compounds that can stimulate the growth of symbiotic species or

The conversion of carbohydrates to metabolites as acetic acid, lactic acid or CO2 implies the acidification of the system. The contemporary pH decrease and the presence of sugar (osmotic stress) stimulate the exopolysaccharides (EPSs) production. In fact, in sourdough EPSs can be involved in acid tolerance of sourdough LAB [98]. EPSs are long-chain polysaccharides consisting of branched, repeating units of sugars or sugar derivatives. These sugar units are mainly glucose, galactose and rhamnose, in different ratios [99]. The presence of EPSs in the system can create a novel stress to the cells. The inclusion of cells within biofilm can increase their resistance to unfavorable environmental factors such as extreme temperature, low pH and osmolarity, the changes in the texture can induce in LAB

For example in yogurt production, the acidification by LAB implies proteins coagulation and thereby changes in the viscosity of the milk. In *L. bulgaricus*, during the acid adaptation present in the fermentation milk to obtain yogurt, some cellular changes were observed: the chaperones GroES, GroEL, HrcA, GrpE, DnaK, DnaJ, ClpE, ClpP and ClpL were induced and ClpC was repressed [100]. Some genes involved in the biosynthesis of fatty acids were induced (*fabH*, *accC*, *fabI*), while the genes involved in the mevalonate pathway of isoprenoid synthesis (*mvaC*, *mvaS*) were repressed [101, 102]. The changes in Aw value are depending not only on EPSs production by LAB after the exposition to acidic and osmotic

Considering cheese, the Aw decreases during manufacture and ripening as a result of dehydration, salting, and production of water-soluble solutes from glycolysis, proteolysis, and lipolysis; the cheese Aw values range from 0.70 for extra hard cheeses to 0.99 for fresh, soft cheeses, such as cottage cheese, while semi-hard cheeses have Aw values of around 0.90. The cheese pH also decreases during manufacture and ripening [103]. The effects of different Aw and pH on *L. lactis* simulating cheese ripening have been analyzed [103]. The results evidenced that at low Aw, particularly at low pH, the growth and lactose utilization rates decreased and lactose fermentation to L-(1)-lactate switched to a pathway involving nontraditional saccharide

In *L. plantarum* WCFS1 the addition of 300 mM and 800 mM of NaCl induced mild osmotic stress and osmotic stress respectively. In the presence of 800 mM of NaCl several genes showed an increased expression with respect to the control culture. In particular, those genes were associated with various stress responses in prokariotes, i.e. genes encoding Clp protease, an excinuclease, catalase (peroxide stress) and Dpr-like protein (peroxide stress). These differences in the gene expression were also identified in the presence of acid stress. These results suggest that lactic acid stress in *L. plantarum* WCFS1 also induces a more general stress response (as above described for different *Lactobacillus* species). An overlap between the stimulus for lactic acid and those for peroxide and UV radiation has also been

stress, but also on the ingredients composition and on the step of fermentation.

products rather than the traditional lactococcal heterofermentative products.

The ability of the target strains to dominate the fermentation is related not only to the ingredients (as above described), but also to the fermentation conditions, mainly temperature and atmosphere. If the fermentation is not performed at the optimal growth temperature for the microorganisms, they could be unable to compete with naturally occurring microflora, and consequently the whole process could be compromised. On the contrary, some microbial species have developed specific thermal resistance mechanisms, and they can easily adapt to these unfavorable conditions without implications for the fermentation processes. Moreover, the adaptation to thermal stresses often leads to tolerance to other stresses, in a mechanism usually define "cross protection", as reported for *L. lactis* [105]. The ability of commercial *L. lactis* ssp. *lactis* and *L. lactis* ssp. *cremoris* to withstand freezing at –60°C for 24 h was significantly improved by a prior 25 min heat shock at ~40°C or by a 2 h cold shock at 10°C, opening interesting perspectives for the production on resistant starter cultures, both frozen or lyophilized [105].

Other Authors with regard to different stresses reported the "cross protection" mechanism: for example the mechanisms of multiple adaptations to hops of two different strains of *L. brevis* have been characterized [106]. Hop resistance of lactobacilli requires multiple resistance mechanisms. This is consistent with the stress conditions acting on bacteria in beer, which mainly consist of acid stress and the antimicrobial effect of the hop compounds, in addition to ethanol stress and starvation. The effect of interaction of acid stress and presence/absence of oxygen in the system on *L. helveticus* and *L. sanfranciscensis*, in particular on their cell membrane composition, has been reported [14]. Upon acid stress the level of cyclopropane fatty acids increased at the expense of the level of long-chain unsaturated fatty acids. *L. helveticus* and *L. sanfranciscensis*, exposed to acid sub lethal stress demonstrated the same increase in cyclopropane fatty acids. In particular, *L. helveticus* presented higher concentration of C19cyc11 at pH 4 and pH 3, while *L. sanfranciscensis* presented more C19cyc9 at pH 3 in microaerophilic condition without tween 80, at pH 3.6 in anaerobiosis with tween 80, and at pH 4 in anaerobiosis without tween 80. These results demonstrated the same behavior in front of multiple stresses by LAB membrane [106, 14]

Consider the atmosphere, i.e. the presence or not of oxygen, as another important variable during fermentation, it is known that oxygen can inhibit the growth of LAB, especially in the first stages. However, the food system is usually a consortium of different microorganisms: for example in bakery products and in fermented sausages the fermentation is carried out both by yeasts and LAB; the formers can therefore consume the amount of oxygen present in the mix, allowing the growth of LAB. The same thing happens for fermented vegetables, where naturally occurring Gram negative bacteria and yeast rapidly remove the oxygen, promoting the rapid predominance of Lactobacilli.

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*.

Dynamic Stresses of Lactic Acid Bacteria Associated to Fermentation Processes 555

depending on the microbial partner. In fact, *L. sanfranciscensis* DPPMA174 induced the highest synthesis of PlnA, which, in turn, determined lethal conditions for it. The proteome of *L. sanfranciscensis* DPPMA174 responded to the presence of PlnA. The up-regulation of 31 proteins related to stress response, amino acid metabolism, energy metabolism, membrane transport, nucleotide metabolism, regulation of transcription and cell redox homeostasis was found. At the same time, other proteins such as cell division protein (FtsZ), glutathione reductase (LRH\_11212) and response regulator (rrp11) were down-regulated. These results demonstrated a hypothetically and interesting waterfall of events all related with stresses response and with the typical fermentation products dynamics (Figure 3). At the same time, the low pH values implied a poor expression of the genes involved in carbohydrate degradation in *L. plantarum* IMDO 130201. The bacterium was directed toward survival at low pH by amino acid conversions rather than by relying on growth [112]. The same behavior was identified in *L. sanfranciscensis* LSCE1 response to pH 3.6 [15]. Under the adopted experimental conditions, which did not produce any decrease in viability of *L. sanfranciscensis* LSCE1, the acid stress, within 2 h, was accompanied by a reduction of the carbohydrate metabolism, as shown by the decrease of ethanol, acetate, and lactate. This mechanism suggests the existence of a switch from sugar to amino acid catabolism that supports survival and growth also in specific and restricted environments, such as sourdoughs, characterized by acid stress and recurrent carbon starvation. Under the acid conditions (pH 3.6) and in the presence of specific nutrients 3-methylbutanoic acid was the predominant metabolite among those detected by solid phase micro-extraction gas chromatographic analysis and mass spectrometry (GC-MS-SPME), released after 2 h of acid stress exposure [15]. The acid stress implied less carbohydrate utilization and ethanol, lactate, and acetate production, but high amino acids catabolism that confers a different and characteristic metabolites pattern. Stress resistance assume great importance as one of the adaptation factors to gastrointestinal tract of

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

Adaptation factors include stress resistance, active metabolism adapted to the host

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.

probiotic strains as reported in a detailed review [113].

the health-promoting effects (probiotic factors) [113].

environment, and adherence to the intestinal mucosa and mucus.

**5. Stress resistance of probiotic LAB** 

Indeed, the protonated form of

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 sensing mechanism [79].

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 depending on the microbial partner. In fact, *L. sanfranciscensis* DPPMA174 induced the highest synthesis of PlnA, which, in turn, determined lethal conditions for it. The proteome of *L. sanfranciscensis* DPPMA174 responded to the presence of PlnA. The up-regulation of 31 proteins related to stress response, amino acid metabolism, energy metabolism, membrane transport, nucleotide metabolism, regulation of transcription and cell redox homeostasis was found. At the same time, other proteins such as cell division protein (FtsZ), glutathione reductase (LRH\_11212) and response regulator (rrp11) were down-regulated. These results demonstrated a hypothetically and interesting waterfall of events all related with stresses response and with the typical fermentation products dynamics (Figure 3). At the same time, the low pH values implied a poor expression of the genes involved in carbohydrate degradation in *L. plantarum* IMDO 130201. The bacterium was directed toward survival at low pH by amino acid conversions rather than by relying on growth [112]. The same behavior was identified in *L. sanfranciscensis* LSCE1 response to pH 3.6 [15]. Under the adopted experimental conditions, which did not produce any decrease in viability of *L. sanfranciscensis* LSCE1, the acid stress, within 2 h, was accompanied by a reduction of the carbohydrate metabolism, as shown by the decrease of ethanol, acetate, and lactate. This mechanism suggests the existence of a switch from sugar to amino acid catabolism that supports survival and growth also in specific and restricted environments, such as sourdoughs, characterized by acid stress and recurrent carbon starvation. Under the acid conditions (pH 3.6) and in the presence of specific nutrients 3-methylbutanoic acid was the predominant metabolite among those detected by solid phase micro-extraction gas chromatographic analysis and mass spectrometry (GC-MS-SPME), released after 2 h of acid stress exposure [15]. The acid stress implied less carbohydrate utilization and ethanol, lactate, and acetate production, but high amino acids catabolism that confers a different and characteristic metabolites pattern. Stress resistance assume great importance as one of the adaptation factors to gastrointestinal tract of probiotic strains as reported in a detailed review [113].
