**3. Principal responses to the most common stresses**

**Heat shock response:** The effect of heat shock and the induction of a stress response in *Lactobacillus* spp. have been studied for *Lactobacillus delbrueckii* subsp. *bulgaricus* [23] and *Lactobacillus paracasei* [24, 25], *Lactobacillus acidophilus*, *Lactobacillus casei* and *Lactobacillus helveticus* [26], *Lactobacillus collinoides* [27] , *Lactobacillus sakei* [28], *Lactobacillus johnsonii* [29], *Lactobacillus rhamnosus* [30], *Lactobacillus plantarum* [31-33] and *Lactobacillus salivarius* [34]. The heat resistance of LAB is a complex process involving proteins with different roles in cell physiology, including chaperone activity, ribosome stability, stringent response mediation, temperature sensing and control of ribosomal functions [31]. The time taken to initiate the stress response is different for different treatments and different strains. The major problem encountered by cells at high temperature is the denaturation of proteins and their subsequent aggregation. In addition Earnshaw et al. [35], , Texeira et al. [36] and Hansen et al. [37] described also as response to heat stress the destabilization of macromolecules as ribosomes and RNA as well as alterations of membrane fluidity.

Dynamic Stresses of Lactic Acid Bacteria Associated to Fermentation Processes 543

appeared to have different effects on different LAB as well as different effects on strains within the same genus. Moreover, the freezing response of the strains depends on the time of the cold shock process and the induction of cryotolerance appears to be dependent on the

Another interesting study regarding LAB response to sub-lethal cold stress was developed by Montanari et al.[14]. These Authors separated and quantified the cell cyclopropane fatty acids lactobacillic (C19cyc11) and dehydrosterculic (C19cyc9) to study the adaptive response to sub-lethal acid and cold stresses in *L. helveticus* and *Lactobacillus sanfranciscensis*. These microorganisms showed different fatty acids composition and environmental adaptation to short term cold and acidic stresses. In *L. helveticus* C19cyc11 dramatically increased after 2 h at 10°C and with the pH decrease, particularly in micro-aerobic conditions, in the presence of tween 80, and in anaerobic conditions. The increase of lactobacillic acid in *L. helveticus* is necessary to maintain the cell membrane in a suitable state of fluidity. Moreover, cyclopropane fatty acids confer resistance to ozonolysis, singlet oxygen and mild oxidative treatments [48, 49], suggesting a cross protection and response of LAB cell membrane to physicochemical stresses. A combined analysis of the genome-wide transcriptome and metabolism was performed with a dairy *Lactococcus lactis* subsp. *lactis* under dynamic conditions similar to the conditions encountered during the cheese-making process. Specific responses to acid and cold stresses were identified, but also the induction of unexpected pathways was determined. In

**Oxidative stress response:** LAB are facultative anaerobic microorganisms that have in common the reduction of part of pyruvate produced to lactate production in order to regenerate NAD+ from NADH formed during glycolysis. They do not require oxygen for growth and, in fact, a negative effect of oxygen on the development of these bacteria has often been observed. It was generally believed that these bacteria could under no condition use oxygen as the terminal electron acceptor [17]. However, many LAB have NADH oxidase and some can even express a functionally active respiratory chain in the presence of heme [51-57]. Respiration-competent LAB differ from the features of *Escherichia coli* and *Bacillus subtilis*, since they carry limited equipment for respiration. All respiring LAB carry genes encoding electron donor (NADH dehydrogenase) and a single electron acceptor (cytochrome bd oxidase) [58]. Addition of heme to the system activates respiration chain

When for some reasons the generation of free radicals is higher than the rate of their detoxification the cells are exposed to a constraint called "oxidative stress" [59]. For the food-associated LAB a still fragmented picture of the resistance mechanisms present emerges. Representatives of the different mechanisms have been described in different LAB [60-64]. Apart from the toxic effects of oxygen, aeration can induce important changes in the sugar metabolism of LAB. In fact, the presence of oxygen is a factor that greatly affects the outcome of a fermentation process. In general, LAB tolerate oxygen but grow better under nearly anaerobic conditions. However, in the presence of heme and oxygen LAB start respiration metabolism, by which the cell metabolism is reprogrammed so that pH, oxygen status, growth capacity and survival are markedly altered [56]. In the presence of oxygen

growth phase in which the cold shock took place [43-47].

particular, the induction of purine biosynthesis and prophage [50].

NADH oxidase activity, but none of the tested LAB synthesize heme [01].

Heat stress response is characterized by the transient induction of general and specific proteins and by physiological changes. In every strain tested the involvement of Heat Shock Proteins (HSPs such as DnaK, GroEL and GroES during the heat stress was clear) [23-38]. The role of these stress proteins is complex; in fact, the bind substrate proteins in a transient non-covalent manner prevent premature folding and promote the attainment to the correct state *in vivo*. The resistance to heat stress is higher when the cells were previously exposed and adapted to this type of stress in the stationary phase, otherwise, when pre-adapted in exponential phase, the cells are more sensitive. In particular, the storage stability of the culture that was heat shocked after stationary phase was superior to that of culture heat shocked after log phase [34, 23, 30].

**Cold shock response:** It is very important to improve knowledge about LAB behavior in cold environment. In fact, during industrial processes, like in cheese ripening and refrigerated storage of fermented products, these microorganisms are subjected to different temperatures far below the optimal growth temperature. When LAB living cells are exposed to these cold environments, important physiological changes occur, such as decrease in membrane fluidity and stabilization of secondary structures of RNA and DNA, resulting in a reduced efficiency of translation, transcription and DNA replication. The response of microorganisms to these effects is termed cold-shock response during which a number of Cold Induced Proteins (CIPs) are synthesized. The roles of these proteins are at the levels of membrane fluidity, DNA supercoiling and transcription and translation. Few papers have described cold shock proteins and mechanisms in LAB, in particular they have focused on *Lactococcus lactis* and *L. plantarum* [39-42]. Kim et al. [39, 40] tested different LAB to evaluate cold shock effects on cryotolerance. Improved understanding of cold-shock-induced cryotolerance may contribute to the development of environmental conditions that allow improved viability/activity of frozen or freeze-dried commercial LAB starter cultures. The results showed that, as with heat stress, there is also an improvement of the viability of the tested strains as concerning the cryotolerance after a cold shock. The process of freezing appeared to have different effects on different LAB as well as different effects on strains within the same genus. Moreover, the freezing response of the strains depends on the time of the cold shock process and the induction of cryotolerance appears to be dependent on the growth phase in which the cold shock took place [43-47].

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

shocked after log phase [34, 23, 30].

**3. Principal responses to the most common stresses** 

**Heat shock response:** The effect of heat shock and the induction of a stress response in *Lactobacillus* spp. have been studied for *Lactobacillus delbrueckii* subsp. *bulgaricus* [23] and *Lactobacillus paracasei* [24, 25], *Lactobacillus acidophilus*, *Lactobacillus casei* and *Lactobacillus helveticus* [26], *Lactobacillus collinoides* [27] , *Lactobacillus sakei* [28], *Lactobacillus johnsonii* [29], *Lactobacillus rhamnosus* [30], *Lactobacillus plantarum* [31-33] and *Lactobacillus salivarius* [34]. The heat resistance of LAB is a complex process involving proteins with different roles in cell physiology, including chaperone activity, ribosome stability, stringent response mediation, temperature sensing and control of ribosomal functions [31]. The time taken to initiate the stress response is different for different treatments and different strains. The major problem encountered by cells at high temperature is the denaturation of proteins and their subsequent aggregation. In addition Earnshaw et al. [35], , Texeira et al. [36] and Hansen et al. [37] described also as response to heat stress the destabilization of

macromolecules as ribosomes and RNA as well as alterations of membrane fluidity.

Heat stress response is characterized by the transient induction of general and specific proteins and by physiological changes. In every strain tested the involvement of Heat Shock Proteins (HSPs such as DnaK, GroEL and GroES during the heat stress was clear) [23-38]. The role of these stress proteins is complex; in fact, the bind substrate proteins in a transient non-covalent manner prevent premature folding and promote the attainment to the correct state *in vivo*. The resistance to heat stress is higher when the cells were previously exposed and adapted to this type of stress in the stationary phase, otherwise, when pre-adapted in exponential phase, the cells are more sensitive. In particular, the storage stability of the culture that was heat shocked after stationary phase was superior to that of culture heat

**Cold shock response:** It is very important to improve knowledge about LAB behavior in cold environment. In fact, during industrial processes, like in cheese ripening and refrigerated storage of fermented products, these microorganisms are subjected to different temperatures far below the optimal growth temperature. When LAB living cells are exposed to these cold environments, important physiological changes occur, such as decrease in membrane fluidity and stabilization of secondary structures of RNA and DNA, resulting in a reduced efficiency of translation, transcription and DNA replication. The response of microorganisms to these effects is termed cold-shock response during which a number of Cold Induced Proteins (CIPs) are synthesized. The roles of these proteins are at the levels of membrane fluidity, DNA supercoiling and transcription and translation. Few papers have described cold shock proteins and mechanisms in LAB, in particular they have focused on *Lactococcus lactis* and *L. plantarum* [39-42]. Kim et al. [39, 40] tested different LAB to evaluate cold shock effects on cryotolerance. Improved understanding of cold-shock-induced cryotolerance may contribute to the development of environmental conditions that allow improved viability/activity of frozen or freeze-dried commercial LAB starter cultures. The results showed that, as with heat stress, there is also an improvement of the viability of the tested strains as concerning the cryotolerance after a cold shock. The process of freezing Another interesting study regarding LAB response to sub-lethal cold stress was developed by Montanari et al.[14]. These Authors separated and quantified the cell cyclopropane fatty acids lactobacillic (C19cyc11) and dehydrosterculic (C19cyc9) to study the adaptive response to sub-lethal acid and cold stresses in *L. helveticus* and *Lactobacillus sanfranciscensis*. These microorganisms showed different fatty acids composition and environmental adaptation to short term cold and acidic stresses. In *L. helveticus* C19cyc11 dramatically increased after 2 h at 10°C and with the pH decrease, particularly in micro-aerobic conditions, in the presence of tween 80, and in anaerobic conditions. The increase of lactobacillic acid in *L. helveticus* is necessary to maintain the cell membrane in a suitable state of fluidity. Moreover, cyclopropane fatty acids confer resistance to ozonolysis, singlet oxygen and mild oxidative treatments [48, 49], suggesting a cross protection and response of LAB cell membrane to physicochemical stresses. A combined analysis of the genome-wide transcriptome and metabolism was performed with a dairy *Lactococcus lactis* subsp. *lactis* under dynamic conditions similar to the conditions encountered during the cheese-making process. Specific responses to acid and cold stresses were identified, but also the induction of unexpected pathways was determined. In particular, the induction of purine biosynthesis and prophage [50].

**Oxidative stress response:** LAB are facultative anaerobic microorganisms that have in common the reduction of part of pyruvate produced to lactate production in order to regenerate NAD+ from NADH formed during glycolysis. They do not require oxygen for growth and, in fact, a negative effect of oxygen on the development of these bacteria has often been observed. It was generally believed that these bacteria could under no condition use oxygen as the terminal electron acceptor [17]. However, many LAB have NADH oxidase and some can even express a functionally active respiratory chain in the presence of heme [51-57]. Respiration-competent LAB differ from the features of *Escherichia coli* and *Bacillus subtilis*, since they carry limited equipment for respiration. All respiring LAB carry genes encoding electron donor (NADH dehydrogenase) and a single electron acceptor (cytochrome bd oxidase) [58]. Addition of heme to the system activates respiration chain NADH oxidase activity, but none of the tested LAB synthesize heme [01].

When for some reasons the generation of free radicals is higher than the rate of their detoxification the cells are exposed to a constraint called "oxidative stress" [59]. For the food-associated LAB a still fragmented picture of the resistance mechanisms present emerges. Representatives of the different mechanisms have been described in different LAB [60-64]. Apart from the toxic effects of oxygen, aeration can induce important changes in the sugar metabolism of LAB. In fact, the presence of oxygen is a factor that greatly affects the outcome of a fermentation process. In general, LAB tolerate oxygen but grow better under nearly anaerobic conditions. However, in the presence of heme and oxygen LAB start respiration metabolism, by which the cell metabolism is reprogrammed so that pH, oxygen status, growth capacity and survival are markedly altered [56]. In the presence of oxygen

and during the fermentation metabolism, H2O2 is formed. Numerous species of LAB contain peroxidase and/or catalase to prevent and eliminate these deleterious effects [17]. Concerning the prevention of reactive oxygen species (ROS) formation, the scope of the reactions is the eliminations of free oxygen. In a study on *L. helveticus* the fatty acids composition in the cell membrane changed in response to oxidative stress. In fact, the activity of oxygen consuming desaturase system increased to reduce the free radical damage to the cell [19]. Generally, the response to oxidative stress of LAB is similar, but also depends on the species, on the strains and, with regard to catalase action, on the bacterial density [4]. In *L. lactis* several genes have been identified and the respective encoded proteins have been shown to contribute to oxidative stress resistance. Moreover, the induction of these genes is growth phase-dependent (exponential or stationary) and their products confer multi-stress resistance [52]. General stress resistance mechanisms may also confer resistance to oxidative stress. In fact, in a model system several acid resistant mutants of *L. lactis* that appeared also more resistant to oxidative stress were isolated [64].

Dynamic Stresses of Lactic Acid Bacteria Associated to Fermentation Processes 545

maintain redox balance, the rationale for the production of 2-methylbutanoic acid from leucine can be found in a newly proposed biosynthetic pathway leading to 2 methylbutanoic acid and 3 mol of ATP per mol of leucine. Leucine catabolism to 3 methylbutanoic and 2-methylbutanoic acids suggests that the switch from sugar to amino acid catabolism supports growth of *L. sanfranciscensis* in restricted environments such as

**Osmotic stress response:** In the various applications in food and feed industry LAB can be exposed to osmotic stress when important amounts of salts or sugars are added to the product [17]. In fact, in most of the food habitats where lactobacilli live, they are confronted with salt [67] and sugar stress [68]. Study on the differences between salt and sugar osmotic stress revealed that the hyperosmotic conditions imposed by sugar stress are much less detrimental and only transient (transient osmotic stress), because the cells are able to balance the extra and the intracellular concentrations of lactose and sucrose [17]. Bacteria need to adapt to this change in their environment in order to survive [69], and they can do it by accumulating (by uptake or synthesis) compatible solutes, generally of organic origin, under hyperosmotic conditions [17]. The compatible solutes are defined as osmoprotectants. The main strategy to adapt to high osmolarity of non-halophilic bacteria is associated with the enhancement of the osmotolerance [68]. Moreover, the osmoprotectants can also stabilize enzymes and provide protection not only against osmotic stress but also against other type of stresses (high temperature, freezing and drying). The intracellular accumulation of compatible solutes prevents the loss of water caused by high external osmolarity and allows the maintenance of turgor [68]. The accumulation of carnitin, betain and proline was determined in LAB grown in MRS and complex diluted MRS medium (DMRS medium) [70]. Moreover, a specific response mechanism to osmotic stress was identified in a sourdough model system [13]. In particular, the growth of *L. sanfranciscensis* under osmotic stress resulted in a relevant accumulation of 3-methylbutanoic acid. Its synthesis is associated with the BCCAs., is NAD+ dependent and produces NADH during the reaction [71]. The accumulation of 3-methylbutanoic acid as predominant metabolite has been also observed in model systems simulating sourdough as a consequence of osmotic,

**High pressure stress response:** High-pressure processing (HPP) or high pressure homogenization (HPH) are non-thermal processes capable of inactivating and eliminating pathogenic and food spoilage microorganisms in specific foods [11, 72], and it represents an exceptional stimulus for most mesophilic bacteria. Several proteins are induced after high pressure treatment and some of these have also been involved in the response to other various stresses [8]. The responses to HHP stress have been studied in particular on *L. sakei* and *L. sanfranciscensis* [73, 18]. These Authors suggested the presence of *de novo* protein synthesis as a consequence of HHP stress [73]. As concerning HPH several interesting studies on the responses on *Lactobacillus* spp., at the level of proteolytic and metabolic activities point of view have been conducted [11, 21, 22, 74]. HPH treatment positively affects the proteolytic activity of some of *Lactobacillus* strains, but the activation and the quantitative and qualitative changes of the metabolic activity appear to be the most promising results. The pre-treatment at different pressure was able to induce relevant

sourdough, characterized by acid stress and recurrent carbon starvation.

acid or oxidative stress [12, 15].

**Acid stress response:** Understanding the acid resistance mechanism used by LAB to survive to by-products of their own metabolism (i.e. homofermentative *L. lactis* converts 90% of metabolized sugar to lactic acid) and the response available in low-pH foods is of great importance. In LAB one of the most effective mechanisms for resistance in acid stress environment is the glutamate decarboxylase (GAD). In fact, few years ago, it was proposed that amino acid decarboxylase functions to control the pH of the bacterial environment by consuming hydrogen ions as part of carboxylation reaction [65]. LAB are also capable of inducing an Acid Tolerance Response (ATR) in response to mild acid treatments. The system induced includes pH homeostatis, protection and repair mechanisms. Genes and proteins, involved in pH homeostasis and cell protection or repair, play a role in acid adaptation, but this role can also extend to more general acid tolerance mechanisms. A more specific study was developed on the effects of lactic acid stress on *L. plantarum* by transcription profiling [66]. The difference, in terms of stress response, into the dissociated or undissociated forms of lactic acid has been highlighted. The toxicity of organic acids depends on their degree of dissociation and thus on the pH. For LAB end product inhibition by lactic acid could result in a disturbance of the regeneration of cofactor NAD+, especially under anaerobic conditions, in which the cell does not have the possibility of NAD+ regeneration by NADH oxidase. The response at membrane fatty acids level to acid stress was studied in *L. helveticus* and *L. sanfranciscensis* [14]. The relevant proportion of dodecanoic acid in the latter species under acid stress suggests that carbon chain shortening is the principal strategy of *L. sanfranciscensis* to modulate fluidity or chemico-physical properties of the membranes in the presence of acid stress. Moreover, a specific shift in leucine catabolic pathway at pH 3.6 was identified in *L. sanfranciscensis* [15]. In fact, the acid stress induced a metabolic shift toward overproduction of 3-methylbutanoic and 2 methylbutanoic acids, accompanied by sugar reduced consumption and primary carbohydrate metabolite production. The metabolites coming from branched chain amino acids (BCAAs) catabolism increased up to seven times under acid stress. While the overproduction of 3-methylbutanoic acid under acid stress can be attributed to the need to maintain redox balance, the rationale for the production of 2-methylbutanoic acid from leucine can be found in a newly proposed biosynthetic pathway leading to 2 methylbutanoic acid and 3 mol of ATP per mol of leucine. Leucine catabolism to 3 methylbutanoic and 2-methylbutanoic acids suggests that the switch from sugar to amino acid catabolism supports growth of *L. sanfranciscensis* in restricted environments such as sourdough, characterized by acid stress and recurrent carbon starvation.

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

and during the fermentation metabolism, H2O2 is formed. Numerous species of LAB contain peroxidase and/or catalase to prevent and eliminate these deleterious effects [17]. Concerning the prevention of reactive oxygen species (ROS) formation, the scope of the reactions is the eliminations of free oxygen. In a study on *L. helveticus* the fatty acids composition in the cell membrane changed in response to oxidative stress. In fact, the activity of oxygen consuming desaturase system increased to reduce the free radical damage to the cell [19]. Generally, the response to oxidative stress of LAB is similar, but also depends on the species, on the strains and, with regard to catalase action, on the bacterial density [4]. In *L. lactis* several genes have been identified and the respective encoded proteins have been shown to contribute to oxidative stress resistance. Moreover, the induction of these genes is growth phase-dependent (exponential or stationary) and their products confer multi-stress resistance [52]. General stress resistance mechanisms may also confer resistance to oxidative stress. In fact, in a model system several acid resistant mutants

of *L. lactis* that appeared also more resistant to oxidative stress were isolated [64].

**Acid stress response:** Understanding the acid resistance mechanism used by LAB to survive to by-products of their own metabolism (i.e. homofermentative *L. lactis* converts 90% of metabolized sugar to lactic acid) and the response available in low-pH foods is of great importance. In LAB one of the most effective mechanisms for resistance in acid stress environment is the glutamate decarboxylase (GAD). In fact, few years ago, it was proposed that amino acid decarboxylase functions to control the pH of the bacterial environment by consuming hydrogen ions as part of carboxylation reaction [65]. LAB are also capable of inducing an Acid Tolerance Response (ATR) in response to mild acid treatments. The system induced includes pH homeostatis, protection and repair mechanisms. Genes and proteins, involved in pH homeostasis and cell protection or repair, play a role in acid adaptation, but this role can also extend to more general acid tolerance mechanisms. A more specific study was developed on the effects of lactic acid stress on *L. plantarum* by transcription profiling [66]. The difference, in terms of stress response, into the dissociated or undissociated forms of lactic acid has been highlighted. The toxicity of organic acids depends on their degree of dissociation and thus on the pH. For LAB end product inhibition by lactic acid could result in a disturbance of the regeneration of cofactor NAD+, especially under anaerobic conditions, in which the cell does not have the possibility of NAD+ regeneration by NADH oxidase. The response at membrane fatty acids level to acid stress was studied in *L. helveticus* and *L. sanfranciscensis* [14]. The relevant proportion of dodecanoic acid in the latter species under acid stress suggests that carbon chain shortening is the principal strategy of *L. sanfranciscensis* to modulate fluidity or chemico-physical properties of the membranes in the presence of acid stress. Moreover, a specific shift in leucine catabolic pathway at pH 3.6 was identified in *L. sanfranciscensis* [15]. In fact, the acid stress induced a metabolic shift toward overproduction of 3-methylbutanoic and 2 methylbutanoic acids, accompanied by sugar reduced consumption and primary carbohydrate metabolite production. The metabolites coming from branched chain amino acids (BCAAs) catabolism increased up to seven times under acid stress. While the overproduction of 3-methylbutanoic acid under acid stress can be attributed to the need to **Osmotic stress response:** In the various applications in food and feed industry LAB can be exposed to osmotic stress when important amounts of salts or sugars are added to the product [17]. In fact, in most of the food habitats where lactobacilli live, they are confronted with salt [67] and sugar stress [68]. Study on the differences between salt and sugar osmotic stress revealed that the hyperosmotic conditions imposed by sugar stress are much less detrimental and only transient (transient osmotic stress), because the cells are able to balance the extra and the intracellular concentrations of lactose and sucrose [17]. Bacteria need to adapt to this change in their environment in order to survive [69], and they can do it by accumulating (by uptake or synthesis) compatible solutes, generally of organic origin, under hyperosmotic conditions [17]. The compatible solutes are defined as osmoprotectants. The main strategy to adapt to high osmolarity of non-halophilic bacteria is associated with the enhancement of the osmotolerance [68]. Moreover, the osmoprotectants can also stabilize enzymes and provide protection not only against osmotic stress but also against other type of stresses (high temperature, freezing and drying). The intracellular accumulation of compatible solutes prevents the loss of water caused by high external osmolarity and allows the maintenance of turgor [68]. The accumulation of carnitin, betain and proline was determined in LAB grown in MRS and complex diluted MRS medium (DMRS medium) [70]. Moreover, a specific response mechanism to osmotic stress was identified in a sourdough model system [13]. In particular, the growth of *L. sanfranciscensis* under osmotic stress resulted in a relevant accumulation of 3-methylbutanoic acid. Its synthesis is associated with the BCCAs., is NAD+ dependent and produces NADH during the reaction [71]. The accumulation of 3-methylbutanoic acid as predominant metabolite has been also observed in model systems simulating sourdough as a consequence of osmotic, acid or oxidative stress [12, 15].

**High pressure stress response:** High-pressure processing (HPP) or high pressure homogenization (HPH) are non-thermal processes capable of inactivating and eliminating pathogenic and food spoilage microorganisms in specific foods [11, 72], and it represents an exceptional stimulus for most mesophilic bacteria. Several proteins are induced after high pressure treatment and some of these have also been involved in the response to other various stresses [8]. The responses to HHP stress have been studied in particular on *L. sakei* and *L. sanfranciscensis* [73, 18]. These Authors suggested the presence of *de novo* protein synthesis as a consequence of HHP stress [73]. As concerning HPH several interesting studies on the responses on *Lactobacillus* spp., at the level of proteolytic and metabolic activities point of view have been conducted [11, 21, 22, 74]. HPH treatment positively affects the proteolytic activity of some of *Lactobacillus* strains, but the activation and the quantitative and qualitative changes of the metabolic activity appear to be the most promising results. The pre-treatment at different pressure was able to induce relevant

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

Dynamic Stresses of Lactic Acid Bacteria Associated to Fermentation Processes 547

inducers for gene expression when concentrations of these molecules exceed a certain threshold value [79]. These changes might eventually lead to competitive advantages for the population, more effective adaptation and responses to changing environmental conditions, or the co-ordination of interactions between bacteria and their abiotic and biotic environments [7]. In fact, microorganisms produce diffusible chemicals for the purpose of communication and it has been reported that the stress caused by the exposure of microbial cells to their own cell free conditioned media, containing metabolites and bioactive compounds including ''quorum sensing" molecules, including 2(5H)-furanones, promotes cell differentiation, autolysis and overproduction of specific metabolites [12, 80, 9, 10]. In this way the microbial cultures used in food fermentations can also contribute (by "secondary"

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

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

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.

reactions and relations) to the formation of flavor and texture [81].

fluctuation and LAB fermentation.

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

real food system, where different sources of stresses are often present.

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

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 certain microorganisms by other members of the habitat.

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


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 multiple mechanisms [77].

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

In a cell-density-dependent quorum-sensing system, bacteria produce extracellular signaling molecules such as peptides or post-translationally modified peptides that act as inducers for gene expression when concentrations of these molecules exceed a certain threshold value [79]. These changes might eventually lead to competitive advantages for the population, more effective adaptation and responses to changing environmental conditions, or the co-ordination of interactions between bacteria and their abiotic and biotic environments [7]. In fact, microorganisms produce diffusible chemicals for the purpose of communication and it has been reported that the stress caused by the exposure of microbial cells to their own cell free conditioned media, containing metabolites and bioactive compounds including ''quorum sensing" molecules, including 2(5H)-furanones, promotes cell differentiation, autolysis and overproduction of specific metabolites [12, 80, 9, 10]. In this way the microbial cultures used in food fermentations can also contribute (by "secondary" reactions and relations) to the formation of flavor and texture [81].
