1. Introduction
Despite their negligible mass the microbial agents, starters and non starters, play a profound role in the characterization of the fermented foods in terms of chemical and sensorial properties. In fact, fermented foods may be defined as foods processed through the activity of microorganisms. Fermentation processes take a special place in the evolution of human cuisine, by altering the taste experience of food products, as well as extending the storage period. In particular, foods fermented with lactic acid bacteria (LAB) have constituted an important part of human diet and of fermentation processes (involving various foods, including milk, meat, vegetables and fruits) [1] since ancient times. They have played an essential role in the preservation of agricultural resources and in the improvement of nutritional and organoleptic properties of human foods and animal feed. Moreover, these organisms nowadays are increasingly used as health promoting probiotics, enzyme and metabolite factories and vaccine delivery vehicles [2].
It is interesting to outline how the changes of food characteristics during the fermentation process can be described as dynamic fluctuations of the food environment itself and, at the same time, stress source for the microorganisms involved [3, 4], such as LAB. In fact, whenever autochthonous bacteria are adapted and competitive in their respective environment, the environment can be described as stressful for LAB [5, 4]. The fermentation parameters, including temperature, water activity (Aw), oxygen, pH, as well as the concentration of starter cultures, affect the regulatory mechanism and the response mechanisms of LAB, as well as their effects on the final products properties [4].
When LAB are added to food formulations, several factors that may influence the ability of those microorganisms to survive, growth and become active in the new matrix have to be considered [6]. These factors include: 1) the physiological state of the LAB used as starters (whether the cells are from the logarithmic or the stationary growth phase); 2) the physical conditions of product ripening and storage (eg. temperature); 3) the chemical composition of the matrix (eg. acidity, available carbohydrates content, nitrogen source, mineral content, water activity and oxygen concentration); 4) possible interactions of the starter cultures with probiotics and other microorganisms naturally occurring or added to the system [6].
In figure 1 the main factors affecting the viability and the responses of LAB from production to storage are described [7].
To better elucidate what happens to LAB during fermentation processes, we decided to use a model (defined “virtual food”) that mimics various steps occurring during processing and that can affect LAB performances or viability.
2. Lactic acid bacteria and stress: Basic concepts
“Stress results from interactions between subjects and their environment that are perceived as straining or exceeding their adaptive capacities and threatening their well-being. The element of perception indicates that human stress responses reflect differences in personality, as well as differences in physical strength or general health” [8].
Stress has driven evolutionary changes (the development and natural selection of species over time). Thus, the species that adapted best to the causes of stress (stressors) have survived and evolved into the plant and animal kingdoms we now observe. The same evolutionary process regarded microorganisms. In fact, bacteria, irrespective of natural habitat, are exposed to constant fluctuations in their growth conditions. Consequently they have developed sophisticated responses, modulated by the re-modelling of protein complexes and by phosphorylation dependent signal transduction systems, to adapt and to survive to a variety of insults. To ensure survival to environmental adversities, bacteria may adapt to changes in their immediate vicinity by responding to the imposed stress. These responses are different and vast and depend on the microorganism nature and on the environmental stress and are accomplished by changes in the patterns of gene expression for those genes whose products are required to combat the deleterious [3]. In particular, cellular metabolic pathways are closely related to stress responses and the flux of particular metabolites to understand the hypothetically shifts and implications in the food systems has been studied in LAB [9-13, 4, 14, 15].
LAB are a functionally related group of organisms known primarily for their bioprocessing roles in food and beverages [16]. LAB play a crucial role in the development of the organoleptic and hygienic quality of fermented products. These microorganisms are used as starter cultures in many fermented products (i.e. beer, milk, dough, sausages and wine). Therefore, the reliability of starter cultures in terms of quality and functional properties (important for the development of aroma and texture), but also in terms of growth performance and robustness, has become essential for successful fermentations [17]. There have been some reports describing the physiological stress responses in LAB, particularly
LAB evolved specific mechanisms to respond and to survive to environmental stresses and changes (stress-sensing system and defences). In fact, microorganisms could have specific regulators tailored to each of their regulated genes and adapt their expression according to environment. Stress defences are good examples of such integrated regulation systems. Bacterial stress responses rely on the coordinated expression of genes that alter different cellular processes (cell division, DNA metabolism, housekeeping, membrane composition, transport, etc.) and act in concert to improve the bacterial stress tolerance. The integration of these stress responses is accomplished by networks of regulators that allow the cells to react to various and complex environmental shifts. LAB respond to stress in a very specific way dependent on the species, on the strains and on the type of stress. The best-studied stresses are acid, heat, oxidative and cold stresses, although for the latter most of the studies focused on a specific family of proteins instead of analyzing the whole response [4].
Despite the extensive use of LAB, there is a paucity of information concerning the stress-induced mechanisms studied
3. Principal responses to the most common stresses
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
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
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
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]:
Nutrient competition,
Generation of unfavorable environment,
Competition for attachment/adhesion sites.
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].
4. General steps regarding a virtual fermented food process
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.
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:
Antioxidants,
Anti-browning agents
Antimicrobials.
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
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
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
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
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.
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
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
In
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
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
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
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
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
5. Stress resistance of probiotic LAB
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
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.
5.1. Maintaining integrity of the cell envelope
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
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
5.2. Repair and protection of DNA and proteins
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 denatured proteins, and removal of damaged proteins.
5.3. Two-component and other regulatory systems
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 transmembrane sensing domains of the histidine protein kinase (HPK).
6. Methodological approaches to study the effects of stress on LAB
The study of stress responses by LAB is getting closer and closer to the different "omic" fields: genomic, proteomic and metabolomic. Other traditional approaches regarding the membrane cells composition and modifications, both from a structural (cellular fatty acids composition by gas-chromatographic method) and morphological (membrane and wall modification by electronic microscopy) point of view are still used.
Genes implicated in LAB stress responses are numerous and the levels of characterization of their actual role and regulation differ widely between species. The studies concerning stress responses in LAB sometimes benefit from the knowledge already acquired in other bacteria. For example, parts of the studies on heat response have been focused on specific genes because of their major role demonstrated in other microorganisms [17]. The cheapest and easiest way to study a stress response in LAB is to follow some specific genes related to stresses such as heat shock, salts and acids [114, 115]. This type of study is useful especially if the entire genome sequence of some LAB is still unknown. However, nowadays the study of whole trascriptome (the total set of RNAs) is one of the most exhaustive ways to study modifications of gene expression as a result of a stress condition. The transcriptome of a cell contains information about the biological state of the cell and the genes that play a role under specific circumstances. The principal technique used to study the trascriptome is microarray [116].
DNA microarray technology has been used in numerous experiments to analyze gene expression: one example is the evaluation of the general stress response of
The gene expression dynamics of
The study of trascriptome is a good approach that gives a good overview of the changes that can occur inside a stressed bacterium. A limitation of this technique is that it is expensive and requires that the genome sequences of the organisms under study should be available for designing the oligonucleotides for the microarray [119].
A different but, at the same time, related point of view regards the study of proteins and proteome. The most common method to obtain this information is to extract total proteins and separate them by a sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by a western blotting (in the first case) or a two dimesional electrophoresis (2D-E) analysis (in the second case). Also in this case if the study is focused on a single protein, it is necessary to know before the characteristic of the target protein to optimize the analytical conditions. 2D-Electrophoresis can provide more than 10000 detectable protein spots in a single gel run. Thus, proteins with post-translational modifications (PTMs), such as processing, phosphorylation and glycosylation, can be easily detected as separate spots. A spot separated by 2D-E theoretically consists of an almost homogeneous protein, and thus can be identified following digestion with a sequence-specific protease by peptide mass fingerprinting (PMF) approaches, typically using matrix-assisted laser desorption ionization (MALDI)- time-of-flight (TOF) mass spectrometers. The same level of automation is also available for proteomic approaches involving tandem mass spectrometry (MS-MS) analysis, extremely useful when studying organisms with incomplete or partial genomic information [120].
This kind of approach was used to investigate the cell surface proteins of a typical strain of
Due to increasingly available bacterial genomes in databases, proteomic tools have recently been used to screen proteins expressed by microorganisms in food, in order to better understand their metabolism
The global identification of stress-induced proteins in a given organism has technical limitations. Membrane proteins, for example, are rarely detected by this method. Secondly, it may be that changes in membrane proteins composition result from long-term adaptation processes, while short-term responses may primarily be accounted for the activation (and/or stabilization) of proteins already present. The latter hypothesis is valid especially in the case of transport systems, although for some of the systems studied a transcriptional induction has also been observed [17]. The use of this technique is not as widespread as that of DNA microarrays due to the challenges associated with the purification and separation of complex mixtures of proteins found in cell extracts. At the same time the study of the only transcriptome should take into consideration that a lot of post-transcriptional processes may act on RNA (ex. RNA interference, polyadenilation ecc) [125].
As reported above, the stress responses of LAB are studied also through the analysis of membrane composition, structure and integrity. Not unexpectedly, in fact, the cell membrane plays an important role in stress resistance. First of all, the membrane itself can change in adaptation to environmental conditions and these changes contribute to the protection of the bacteria [17]. The adaptive response to sub-lethal acid and cold stresses in
The last but not least approach used to study the stress response of LAB is the metabolic one. The study of the metabolites released, as a consequence of the stress exposure, can contribute to the understanding of the mechanisms that regulate the microbial interactions and the metabolic alterations induced by stress conditions. Moreover, these approaches can be exploited to identify which technological conditions induce microorganisms to produced desirable metabolites [4, 15].
With this perspective the use of GC-MS-SPME as a potent and easy tool to study the generation of volatile metabolite compounds such as flavoring molecules or aroma precursors was widely adopted [9,11-13, 15] and contributed to rationalize the process and optimize the products. In particular, the effects of HPH on different species of
Since all the techniques described above, if used alone, do not allow a total comprehension of stress responses, a lot of studies are trying to combine two or more approaches together. Combined transcriptomic and proteomic analyses were used to evaluate the glucose-limited chemo-stat in
Therefore it is possible to understand, from the references above, that techniques used to study the stress responses of LAB are taking more and more "omic" approach. This comports an accumulation of a huge number of data that it is not easy to manage and to compare. For this reason the use of new programs of data analysis is required. One of these approaches could be the use of heat maps, a technique born as a tool to understand microarray results [66]. Nowadays it could be useful also to manage the data from other fields: in fact, a heat maps was used to show the correlation between metabolites produced, the relative gene expression of specific genes and stress conditions [15]. The same useful tool, combined with other statistical analyses, has been also applied [132].
7. Conclusion
It is known that LAB can adapt to stress with different mechanisms widely studied in model and real systems. An overview of those responses has been described and reported in this chapter.
Stress not only induces changes enabling better survival, but also different performances in a system. In fermented food, the knowledge of the mechanisms that regulate LAB metabolic changes and their effects gain importance especially when those responses can be exploited in order to improve the food properties [4]. In particular, fermented foods are dynamic systems subjected to continuous evolution of their physico-chemical characteristics. The complex fluctuation of the food environment itself, during processing, is stress source for every microorganism involved and the changes that affect the fermented food habitats, can be perceived by LAB as stress.
In this chapter examples of the dynamic fluctuation effect on LAB metabolism have been described in order to outline that every reaction can cause a waterfall of metabolic events influencing the sensorial quality, the shelf-life and the bioactive compounds production of fermented foods.
The subjects of those events are LAB, indicating the importance of metabolism of these microorganisms in food. The cell physiology is crucial to ensure that cells are well suited to survival during downstream processes and that they exhibit high performances.
The production and exploitation of naturally adapted strains can be interesting for companies because of the absence of ethical and legal concerns. The adapted strains are not considered genetically modified microorganisms (GMOs) and therefore they can be applied in food processing without legal restrictions and, more important, without affecting the consumer perception, currently (in Europe) not ready to introduce in his diet foods produced with GMOs.
Individual stresses used in food processing and preservation may render probiotic LAB more resistant to further and different stresses, including those encountered in the human body, e.g. those encountered during gastro-intestinal passage (pH of the stomach, exposure to bile salts in small intestine etc.). A positive correlation has been recently observed between EPS production and resistance to bile salt and low pH stress in
This knowledge can open interesting perspectives to improve at the same time the performances of LAB, the quality of fermented food and the health-promoting properties of the LAB used.
Moreover, it will be interesting to identify the gastrointestinal tract also as a complex and dynamic system in which LAB need to adapt to adverse conditions, responding with metabolic shifts provided with interesting technological an healthy features.
The “omics” technologies could be particularly useful for identifying the mechanism leading to LAB stress responses. These approaches could also help to identify the mechanisms for cell fitness and stress adaptation that will be needed to develop more generic and science based technologies [7].
Acknowledgement
We thank Prof.ssa Maria Elisabetta Guerzoni for her enormous scientific support and Luca Vagnini for his graphic abilities (http://www.lucavagnini.com).
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