Introduction

Elie Metchnikoff, a Nobel Award winner, is credited with developing the initial notion of probiotics. In 1908, he postulated that the bacteria in fermented milk might be reason for the Balkan population’s high life expectancy. Metchnikoff’s theory, however, was ignored for almost a century. The potential health advantages of probiotic lactic acid bacteria have revived interest in them during the past two decades, and as a result, probiotics are now considered essential to human [1]. Probiotics are projected to have a USD 57.8 billion market value in 2022. With an expected CAGR of 8.1% by 2027, it will likely reach USD 85.4 billion [2]. Live microorganisms known as “probiotics” are said to have health advantage when taken in sufficient amounts [3], mainly includes lactic acid bacteria (LAB), Propionibacteria and Bifidobacteria. Many probiotics which belong to genera such as Enterococcus, Bacillus, Escherichia, etc., do not have GRAS status and not included in qualified presumption of safety (QPS), in addition to that they are comparatively less popular probiotic than LAB, Bifidobacteria and Propionibacteria. Hence, most of the studies discussed in this review are related to these three genera. LAB are rod or spherical, gram-positive, acid-tolerant, typically nonsporulating, low-GC bacteria that have similar physiological and metabolic traits [4]. Propionibacterium is a gram-positive, mesophilic, aerotolerant, non-motile, and non-spore-forming bacterium that has a high GC content. It has low nutritional needs and can persist in unfavorable conditions [5]. Bifidobacterium is a genus of gram-positive, nonmotile, often branched rod, anaerobic bacteria [6]. While Lactobacilli are widely regarded as helpful microbes and some strains, such as probiotics, are even thought to promote wellness, their broad historical use has helped them gain approval as generally recognized as safe (GRAS) for human [7].

The human digestive tract has been found to benefit greatly from probiotic microbes and milk-based foods recognized as an effective delivery system since long time. Probiotic products are also growing in popularity using other non-dairy alternatives, such as plant-based foods including grains, fruits and vegetables that have low allergic response rates [8]. Probiotics or products containing probiotics can be helpful in intestinal illnesses, inflammatory bowel disease, diabetes, allergy, inability to digest milk sugar [9], vitamin production [10, 11], tightening of gut barrier [12,13,14], boosts immunity [15, 16], intestinal motility [17, 18], gut microbiome [19,20,21], and lifestyle-related diseases by reducing their clinical symptoms [22,23,24,25]. To obtain the claimed health benefits from the commercial probiotic strain, they should be in the active physiological state because under stressful environment they utilize its energy toward the self-survival and do not provide mentioned health benefit to the host [26, 27].

Nowadays, commercially available probiotics are taken orally as nutritional supplements, such as sachets, pills, capsules, or as one of the ingredients of foods like milk-based products and/or plant-based products. Primary target site of probiotic bacteria is the human intestine; thus, they must reach there in active physiological state to obtain claimed therapeutic benefits. Probiotic bacteria differ in their ability to remain alive and multiply in various carrier matrices as a result of stressors associated with various food processing methods, storage environments and gastrointestinal tract (GIT) [22]. Any alteration to the bacterial DNA, protein, or vital component that reduces viability of cell is known as stress. A cell will strive to re-establish its metabolic pattern in an effort to either survive or grow more quickly as a result of such modifications [28]. As probiotics are live entity, they would face various stresses like presence of organic acids, heating injury, ice crystals piercing, high osmotic pressure in surrounding, antimicrobial compounds, dissolved oxygen in food, and low water activity, either in the carrier matrix or GIT [29]. The metabolic pathways of LAB may be greatly affected by certain stressful situations, hence, limiting their therapeutic efficacy in terms of inhibiting pathogen through secretion of antimicrobial compounds, competing with pathogens for adherence to the intestinal epithelium, boosts immunity by interacting with the intestinal cell line, enhance gut barrier function, produces bioactive compounds, etc. [12, 15, 22, 30]. In fact, a stressed carrier matrix alters the physiology of microbial cell, which may impair the effectiveness of probiotic bacteria [26, 27]. Probiotics may find it difficult to survive in the food, which could hinder their delivery to the target site. On the contrary, many LAB are equipped with adaptation mechanisms and counteract these stresses by multiple responses, e.g., acid neutralization under acid stress, secretion of heat shock proteins during heat stress, rerouting metabolic pathways during bile and acid stress, and modification of cell envelope under osmotic stress. They are safeguarded from severe environmental shocks by these adaption techniques.

It is crucial to have a strong knowledge of how probiotic bacteria respond to external factors to choose strains that will work better as starter cultures and probiotics [31, 32]. It is also crucial to fully comprehend the parameters that enable bacterial viability and the mechanism that enables them to endure inhospitable environments while maintaining a normal physiological state [33, 34]. The objectives of this review were to discuss the various response strategies used by the bacteria to combat the stressful conditions encountered during food preparation as well as the GIT transit and to give an insight to application of stress-adapted probiotics in a customized food preparation such as low-pH food (fermented milk or fruit juices), food stored at very low temperature (ice cream and frozen dessert), and freeze-dried probiotic cells.

Factors Affecting the Viability of Probiotics

Probiotics experiences the different stresses at different stages of food manufacturing, i.e., probiotic preservation (freeze dried, DVS), fermentation in food matrix, refrigerated storage, and human GIT (digestive enzymes, stomach pH, bile, osmotic constraints, commensal microbes, etc.). Following are the stress factors which hampers the growth of probiotic microorganisms [35] such as moisture/water activity, low pH, high bile concentration, oxygen permeability through packaging, osmotic stress (due to sugar and salt addition), metabolites of other bacteria, nutrient depletion/competition for nutrients, post-acidification, presence of harmful microbes, storage condition (temperature, humidity), mechanical stress (pressing, vigorous shaking/centrifuge), heat stress, and chilling injuries. For probiotics to remain alive, become widely distributed, and provide their health advantages, they must adapt to this challenging environment and be protected. When probiotic bacteria are subjected to different stressful environments, they are known to activate number of defense mechanisms in order to overcome the stress and remain viable.

General and Common Stress Responses

When probiotics are under stress, they try to safeguard themselves by expressing numerous coping strategies in the matrix. These coping strategies are more or less same and common for environmental stresses like acid stress, bile stress, heat stress, cold stress, osmotic stress, oxidative stress, etc. The most common defence mechanisms exhibited by probiotics in the stressful conditions are discussed below [36]. General and common stress responses exhibited by probiotic bacteria are shown in Fig. 1.

Fig. 1
figure 1

General stress responses exhibited by probiotic bacteria [36]

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Accumulation of Compatible Solutes and Energy Storage Compounds

A smaller organic molecule which is polar, water soluble, and having neutral isoelectric point is known as a compatible solute. It acts as an osmolyte and helps bacterial cells to acclimatize to osmotic stress [37]. To increase the proliferation of cells and re-establish original pressure during osmotic stress, bacteria increase the concentration of compatible solutes [37], which the cell either transports from the surrounding or produces itself [38]. There are two groups of compatible solutes: the first group includes sugars and polyols (trehalose, glycerol), while the second group includes amino acids [39].

Trehalose assists in refolding of protein, inhibits protein aggregation, and safeguards cellular proteins from reactive oxygen species (ROS) damage [40]. Trehalose may also help keep plasma membranes intact [41]. After the external carbon supply has been used up, trehalose can serve as an internal source of carbon [40]. Bacteria were known to accumulate trehalose in their surrounding medium when exposed to high sugar content. It was reported that trehalose concentration is increased by Propionibacterium freudenreichii and Lactobacillus casei under osmotic stress [40, 42]. In contrast to a chemically defined medium, the solutes accumulate more readily. Some challenging conditions may also cause P. freudenreichii to increase the concentration of trehalose such as very low temperature [43], presence of oxygen, and low pH [40, 41]. Moreover, trehalose also helps in reducing viability loss of freeze-dried cells during storage [44].

Glutamate and lysin accumulations help the lactobacilli cell under many adverse environment [34]. Propionibacterium acidopropionicii concentrated arginine and aspartate following acid stress [45]. Under acidic environments, L. plantarum activates the lysine degradation pathway [46]. Phosphates and glycogen are found to be used as energy storage compound by several probiotic bacteria.

Regulation of Energy Production

During stressful conditions, a cell regulates the various metabolic pathways and substrate conversion to counteract the stress and maintain the homeostasis. Under normal condition, ATPase synthesize ATP by proton motive force and stores energy. But under stressful conditions like acid and bile stress, this protein works in reversible fashion, it expulses the proton from the cytoplasm by hydrolysing ATP [47, 48]. Adenosine triphosphatase (ATPase) activity was regulated at transcriptional level [49] and ATPase expression was correlated with bile stress and acid tolerance [45]. When grown in MRS medium at 37 °C with constant pH 4.8 for 14 h, L. rhamnosus GG overexpressed F0F1-ATP synthase genes while proteins taking part in DNA and RNA synthesis were significantly reduced [50]. Similar results were observed in the following study. Increased production of F0F1-ATPase was reported at pH 4.8 than pH 7 by a probiotic strain, B. longum NCIMB 8809 [51].

Even substrate conversion is also redirected under stresses. For example, during acid stress Lactobacilli decreases lactic acid production by rerouting the glucose metabolic pathway [34]. Under cold stress, Propionibacteria restrict the production of propionate and acetate from lactate by diverting pyruvate toward other pathways [43]. The arginine deaminase (ADI) pathway is about five times more active in P. acidopropionici and Lactobacilli to prevent pHi from dropping too much. By this way, arginine can be broken down to produce ATP, ammonia, and carbon dioxide [45, 52, 53]. The pH homeostasis is achieved by the release of ammonia and carbon dioxide [45] and energy generated used by the ATPase to get rid of protons from the cell.

Impact on Bacterial Envelope

Probiotic bacteria experience cell envelope injury during harsh environment [34]. The cell membrane acts as a first line of protection shield against any hurdle. The stressors, such as presence of organic acids, freezing temperature, high temperature, and bile salts, can have an impact on the cell membrane. Moreover, cell envelop plays an extremely crucial role in maintaining cell intact and reducing the osmotic pressure under osmotic stress [34]. Different adaptive strategies are employed by bacteria to restore the integrity of the membrane and cell wall.

Variations in fluidity tend to be countered by modification of membrane constituents as a defense strategy that take place in stressful situations to maintain intact bilayer structure. As a result of acid stress, L. casei is found to produce more cyclopropane fatty acids and less unsaturated to saturated fat. It helps the cell to prevent inflow of proton by making cytoplasmic membrane stiffer and more compact [49]. It was also reported that unsaturated fatty acids in cytoplasmic membranes of L. helveticus increased when it was under heat stress, which reduced membrane fluidity [54]. P. freudenreichii produces branched chain fatty acids through degradation of branched amino acids using a variety of enzymes [55], helping to keep the membrane fluid to fend off cold stress.

Increase in hydrophobicity of cell wall is one of the response mechanisms adopted by many probiotics against different stresses. Many Lactobacilli were found to overproduce S-layer proteins during bile, acid, heat, and osmotic stress [56, 57]. Such S-layer proteins function as a coating of insulation, thereby, protecting the cell from any injury and lethal effect. It was also observed that exopolysaccharides (EPS) defend bacteria from stress like heat, bile, low pH, and osmotic. Such EPS can be either tightly or weakly linked to the cell surface [58,59,60].

Production of Chaperones and Stress Responsive Proteases

Under extreme stress, the expression of chaperones and proteases is rapidly accelerated by the bacteria. They either refold the denatured protein to correct configuration or degrade them. When damage is irreparable, proteases serve as the ultimate line of defense by promoting recycling of amino acids of denatured protein. By destroying proteins whose activities are no longer necessary as a result of changes to environment, the process of proteolysis of cellular proteins can significantly contribute to homeostasis [34]. Proteins that have been damaged were correctly folded by chaperones and DnaK (heat shock protein) is a widely recognized cellular chaperone which bring damaged proteins to correct configuration [34].

When L. rhamnosus is exposed to pH 4.8, the ClpE chaperone concentration was two times greater in the late lag phase of growth, demonstrating its defensive function in acidic stress [50]. In a study, S. thermophilus produced higher amount of elongation factor G (EF-G) and Tuf proteins after 3 h of exposure to acidic pH 5 than at normal physiological pH 6.8 [61]. EF-G promotes the translocation step in bacterial protein synthesis and Tuf involved in molecular chaperone activity. Various stress responsive proteins such as small heat shock proteins (HSP), cold shock proteins, antifreeze proteins, mRNA binding proteins, moonlight protein (possess adhesive property), etc., are overproduced during heat, cold, osmotic, bile, and acid stress. Main stress response mechanisms of bacteria under various stresses like osmotic, acid, oxidative, heat, cold and bile are shown in Fig. 2.

Fig. 2
figure 2

Main stress response mechanisms of bacteria under various stresses like osmotic, acid, oxidative, heat, cold, and bile [36]. Peptidoglycan is represented in blue. Membrane lipids under normal growth are represented in gray. Amounts of saturated (blue), unsaturated (red), and cyclic (yellow) fatty acids are modulated by treatments. S-layer proteins, which may be involved in adaptation, are represented in yellow and red outside the peptidoglycan. Lipoteichoic acids, whose length is modulated, are presented in green. Inducible transmembrane ATPase and Osmoprotectant uptake systems are represented in pink and blue, respectively. In the cytoplasm, general stress proteins are represented by different colors. Colored circles represent different osmoprotectant and energy storage compounds. Crosses on circles mean the conversion of the molecule. The chromosome is represented in black

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Individual Stress: Impact and Response by Probiotics

Acid Stress

Lactic acid production during fermentation by lactic acid bacteria causes acidification of carrier matrix. This undissociated organic acid enters the cell through simple diffusion and dissociate inside the cell due to high pH. This leads to cause acidic cytoplasm and damages DNA, proteins, and many vital biomolecules which are necessary for the cell’s viability [62]. Low pH in gastric conditions also prevents the microbial colonization in GIT [63]. Many bacteria activated the defense mechanism during acid stress; such studies are discussed below.

Neutralization of cytoplasm by metabolism of amino acid is one of the mechanisms activated during acid stress by the probiotic bacteria. Amino acid decarboxylation produces ATP as well as neutralize acid by producing alkaline metabolites. Overexpression of glutamic acid decarboxylase (GAD) genes of L. reuteri strain 100–23 in mouse stomach shows the activation of protection system against acid stress [64]. In a study, histidine decarboxylation pathway from Str. thermophilus CHCC1524 introduced to L. lactis NZ9000 showed tenfold higher acid stress survival at acid stress, 2.5 pH for 2 h compared to wild type [65]. Such Lactobacilli have potential to be utilized in low-pH carrier matrix like fermented foods.

Malate decarboxylation is called malolactic fermentation (MLF). It releases CO2 which neutralizes the protons [66]. When 30 mM malate added to the carrier matrix, it improved the low pH 2.5 survival of the L. casei ATCC 334 [49]. Many probiotic bacteria are found to produce EPS, which shield themselves against low pH. The L. helveticus ATCC 15807 secreted greater EPS at pH 4.5 than normal physiological pH. EPS prevent the penetration of free H+ into the cell cytoplasm [67].

Probiotics can boost the functioning of the F0F1 ATPase that utilizes ATP to propel the evacuation of H+ from the cell and so maintain pHi homeostasis [63]. L. rhamnosus GG increased F0F1-ATP synthase production, when grown in whey broth (5% hydrolyzed whey, 0.6% casein hydrolysate and 0.0015% MnSO4, and water, pH 5.8). Various other acid stress responses such as cell signaling by LuxS (involved in quorum sensing), ClpE (degradation of misfolded proteins during stress responses), and peptidoglycan biosynthesis along with F0F1-ATP synthase were upregulated in whey broth having pH 4.8 compared to pH 5.8 [50].

Probiotics fight against low pH stress through over production of common stress proteins and chaperones such as GroEL, GroES, DnaK, and Clp [68]. It was reported that the HSP and chaperones (DnaK, GrpE, GroEL, and GroES) were abundant in L. plantarum 423 under acid stress, i.e., in MRS at 2.5 pH for 2 h [46]. Alteration in cell membrane composition is another protection strategy in probiotics during acid stress. The composition of fatty acids in cell membrane of previously acid exposed cells of L. casei ATCC 334 was checked in a study. The higher contents of saturated fatty acid (SFA) and cyclopropane fatty acid (CFA) were reported [49]. These SFA and CFA play functional role in modulation of membrane features like fluidity, hydrophobicity, and proton permeability under adverse environment.

In one study, L. plantarum 423 was added to MRS broth with 2.5 pH for 2 h. By analyzing proteomic profile, it was found that proteins involved in transcription, translation, and cell division were decreased under stress conditions. These proteins are not vital for cell’s viability but they are involved in cell growth and divisions. Further they studied glucose consumed and lactic acid produced in MRS broth under low pH and normal physiological environment. Under control condition 12.9 g/L glucose was consumed and 2.7 g/L lactic acid was produced, whereas in stress condition 19.0 g/L glucose was consumed and 0.7 g/L lactic acid was produced [46]. Increased glucose consumption and reduced lactic acid production during acid stress mean that cell is utilizing glucose as a source of energy to cope with stressful condition. It channelizes the metabolic pathway of glucose in such a way that end product is not acid but some other neutral and /or alkaline substances under acid stress. Figure 3 presents an overview of the many modes of acid resistance in lactic acid bacteria. Acid stress response in various beneficial bacteria is given in Table 1.

Fig. 3
figure 3

Mechanisms of acid tolerance in lactic acid bacteria [adopted from 141]. ADP adenosine diphosphate, AI-2 auto-inducer 2, ATP adenosine triphosphate, CFA cyclopropane fatty acids, Dnak molecular chaperone protein, GABA γ-aminobutyrate, GAD glutamate decarboxylase, LuxS S-ribosylhomocysteinelyase, Nth endonuclease, RecA DNA repair protein, RecO DNA repair protein, Shsp small heat shock protein, SmnA AP endonuclease, TCS two-component signal system, and UvrA ultraviolet excinuclease

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Table 1 Acid stress response in various beneficial bacteria
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Bile Stress

In order to move through the host’ GI system, probiotic bacteria must overcome many key obstacles and one such obstacle is bile in the human intestine. The natural level of bile in the human gut is around 0.05 to 2%. Bile acid serves as a biological detergent and exhibits variety of negative consequences, such as DNA damage, protein misfolding or denaturation, formation of secondary structure of RNA, reducing pH of cell cytoplasm, and dissolving cellular lipids [69, 70]. Research findings have shown that probiotic bacteria respond to bile hurdle by increasing the level of chaperones, proteases, proteins involved in bile detoxification and export, redox enzymes, and cell wall– and membrane-bound constituents, which consistently alters cell envelope characteristics [71,72,73].

Many probiotics possess enzyme, bile salt hydrolase which provides protection against bile stress. Moreover, probiotic LAB cleans the cellular environment from bile employing export mechanisms. In both Lactobacilli and Bifidobacteria, many transporter proteins and potential bile efflux systems were characterized as well as located using transcriptome method [68, 71, 74,75,76]. When L. fermentum NCDC 605 was exposed to 1.2% bile in MRS broth for 6 h, the following changes were observed: alterations in the energy metabolism, such as a rise in ATP synthesis; alterations in glycolytic end product concentrations; alteration in shape and size of L. fermentum NCDC 605 cells; molecular chaperones and proteases genes upregulated. All these alterations in L. fermentum NCDC 605 tend to help the cell to survive under bile stress [77]. The morphological changes may be due to alteration in cell surface by (a) excretion of EPS, (b) modifications to the cell membrane’s fatty acid makeup, and (c) changes in surface-associated proteins.

One of the enzymes called bile salt hydrolase (BSH) is involved in deconjugation of primary bile salt and releases free insoluble cholic acid (unconjugated acid) and a residue of taurine or glycine. Other commensal microbes could then disintegrate the unconjugated acids or excreted out by host [78]. When L. plantarum Lp91 was exposed to 2% bile for 3 h in MRS broth, the expression of bsh gene increased six times compared to control [79]. The multidrug transporters and bile efflux pumps are the primary mechanisms in Bifidobacterium breve UCC2003 for bile acid detoxification during gastrointestinal transit. When bile efflux pump Bbr_0838 in B. breve UCC2003 has been inactivated, through insertional mutation, the capacity of the genetically modified cells to grow in the presence of cholic acid (0.05%) has been reduced significantly (p < 0.05) than the unmodified, original cells [76].

In another study, B. breve UCC2003 was allowed to form biofilms in reinforced clostridial medium for 24 h during many stressful conditions like acidic environment at pH 4, pH 4.5, pH 5, and pH 5.8; bile stress at porcine 0.05%, 0.1%, 0.5%, 1%, and 2%; salt stress at NaCl 94.5 mM, 103 mM, 171 mM, 256 mM, and 426 mM; osmotic stress at sucrose 1.46 mM, 2.92 mM, 14.6 mM, 29.2 mM, and 58 mM. They found that at 2% porcine concentration significantly (p < 0.05) increased the higher biofilm formation as compared to other. Also, among all the stress conditions, bile stress at various concentration produced highest biofilm [80]. That shows that the biofilm formation is one of the important mechanisms of B. breve UCC2003 activated against bile stress in the GIT transit. In another study on bile stress, when probiotic L. salivarius Ren exposed to MRS containing 0.75 g/L bile for 14 h, various changes such as maltose and glycerol were utilized in carbohydrate metabolism to produce additional energy, overproduction of the enzymes involved in cell surface charge modification, secretion of cell envelope bound haemolysin-like protein (hinder bile penetration), overexpression of ATP-binding cassette (ABC) transporters (for expulsion of toxic intracellular bile), and overexpression of proteolytic system (to give additional amino acids to repair damaged proteins) were recorded [81]. Figure 4 shows various bile response mechanisms discovered and defined in Lactobacilli. Bile stress responses in various beneficial bacteria are given in Table 2.

Fig. 4
figure 4

Bile response mechanisms identified and characterized in Lactobacilli [69]

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Table 2 Bile stress response in various beneficial bacteria
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Cold Stress

Probiotic may be exposed to low temperatures during the storage of bacterial formulations before they are utilized in food production and during refrigerated storage of food products. Moreover, freezing and freeze drying are general techniques to preserve and concentrated probiotics lead to cold stress to the cells.

Low storage temperatures of probiotics can cause stiffening of the cell membranes, reducing vital enzyme functionality and lower down RNA transcription and protein translation rate, which may lead to growth arrest in a cell. Moreover, the ice crystals generated during freezing can permanently injure the bacterial cell envelope by punching and piercing. Furthermore, solutes begin to accumulate inside the cell during refrigerated storage due to conversion of liquid water into solid ice. Hence, cells experience desiccation and osmotic pressure gradient under cold environment. Since most probiotics are sold in freeze-dried form, the ability of probiotic to remain alive in cold environments is extremely important. It was reported that P. freudenreichii elevate the concentration of branched chain fatty acids in cytoplasmic membrane by synthesizing from branched amino acids, eventually maintaining required fluidity under the cold stress [55]. In probiotic Lactobacilli, cold stress stimulates many antifreeze and cold shock proteins (CSP) that bind to RNA, which prevent secondary structure generation and bolster transcription, translation, and ribosomal activity to keep the cell active under stress [82, 83]. Freezing resistant enzymes secreted by lactic bacteria are also capable of supporting both RNA and protein synthesis at extremely low temperatures [83].

The damage and piercing caused by the ice crystal formation during freezing were prevented by expression of antifreeze proteins by the probiotic Lactobacilli [84]. Osmotic pressure gradient formed during low temperature storage was found to balance by bacteria through secretion and accumulation of compatible solutes such as glycerol, trehalose, and amino acids like glycine, glutamate, lysine, arginine, betaine, and proline [85]. In a study, L. delbrueckii subsp. bulgaricus LBB.B5 were exposed to milk at 4 °C for 5 days and 37 °C for 16 h. Increased levels of many stress tolerance proteins like AddB, UvrC, RecA, and DnaJ were observed at lower temperature of exposure [86]. The cold-stress response of probiotic L. plantarum K25 was measured by comparing differentially expressed (DE) protein profiles after incubation at 10 °C for 72 h and 37 °C for 14 h. Various proteins upregulated after exposure to 10 °C compared to 37 °C were DNA repair, transcription, translation, quorum sensing, and ABC transporters [87]. Cold stress response in various beneficial bacteria is given in Table 3.

Table 3 Cold stress response in various beneficial bacteria
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Heat Stress

Heat is a common technological stress because probiotics frequently have to deal with it at different phases of food production. Probiotics may encounter high temperatures like 60 °C during various stages of food preparation. Even immensely high temperature during spray drying can result in brief heat shocks of up to 200 °C. When biomolecules like DNA, RNA, and protein are subjected to elevated temperatures, they denature and loses their native property, which hampers metabolic activity [88]. Additionally, heat stress increases cell membrane flexibility, affecting the vital activity of cell, and may irreversibly damage cells and causes cell death [89]. Bacterial cells can tolerate milder heat challenges up to 65 °C, but it may reduce the stability of non-covalent bonds, causes cell envelope disruption, affecting function of ribosome, and leads to proteins denaturation [34].

Promoting the synthesis of particular proteins is one of the adaptive methods found in L. kefiranofaciens M1 to prevent cell damage [90]. These proteins were HSP, phosphoenolpyruvate-protein phosphotransferase, chaperone, chaperonins, and cofactors. They are crucial for facilitating proper packing and eventual transport of nascent polypeptides [91]. The GroEL/GroES chaperonin is one of the chaperone proteins that probiotic Lactobacilli use to deal with heat stress [92]. It was observed that B. longum synthesize HSP in heat stress and also adapt to higher temperature [93]. Interestingly, several HSP from lactic bacteria have capacity to attach and stabilize cell membranes. Such HSP is also known as lipochaperone [94, 95]. Saturated and straight-chain fatty acids that contribute to optimal liquidity necessary for normal membrane function were found in LAB that grew under heat stress [96].

The effect of heat adaptation (pre-exposure to sub-sublethal stress) on production of EPS by B. bifidum was checked in a study. The cells were exposed to 42 °C for 5 min and then grown in MRS containing 0.5 g/L of L-cysteine at 37 °C for 24 h anaerobically. L-cystein acts as oxygen remover and addition of this creates anaerobic conditions in the media, thus, enhancing the growth of anaerobic bacteria, B. bifidum. The EPS production was significantly higher in pre-exposed cells than the non-heat-exposed cells [97]. This finding shows the crucial role of EPS in protecting B. bifidum during heat stress. The SDS PAGE analysis of intracellular proteins of L. casei, heat stressed at 45 °C, 50 °C, 55 °C, and 60 °C for 60 min, was carried out. In all heat-stressed cells, variations in protein content at 40–55 kDa, 60 kDa, and 70 kDa were linked to overexpression of DnaJ, GroEL, and DnaK [98].

Adherence to HeLa cells and fatty acid composition of L. casei introduce to two heat treatments such as 37 °C for 10 min and 45 °C for 10 min were measured. HeLa cells adherence and ratio of unsaturated fatty acids (USFA) to SFA in 45 °C for 10 min treatment were 31.33% and 0.36, respectively, whereas in 37 °C for 10 min treatment were 28.66% and 0.40, respectively [96]. In case of 45 °C for 10 min treatment, the significant (p < 0.05) rise in adherence ability indicates the L. casei cell attaches to other cells to get away from the stressful condition, whereas significant reduction in the ratio of USFA to SFA means there is increase in concentration of SFA which prevents melting of cell membrane at high temperature and maintains the proper fluidity required for the normal functioning of the cell.

In one of the studies, cells of probiotic Enterococcus faecium HL7 were kept at 52 °C and 47 °C for 15 min for heat adaptation. Then % survival was calculated for the control and heat-adapted cells at 60 °C after every 10 min up to 40 min. Complete death of all the cells occurred in control, while heat-adapted cell survival was found to reduce at the end of 60 min. Cells adapted to 52 °C exhibited greater survival than 47 °C. Cells heat adapted to 52 °C had comparatively higher SFA and lower USFA than the remaining treatments. Cells with a reduced level of USF or with increased level of SFA have a reduced cytoplasmic membrane flexibility which is correlated to greater heat resistance [99]. Viability of heat-treated (52 °C/15 min) and non-heat-treated E. faecium HL7 cells after subjected to different stress, such as 0.01% hydrogen peroxide (oxidative stress), 20% ethanol, 3 pH (acid stress), and 12 pH, was evaluated. It was discovered that the heat-adapted cells managed to survive at significantly (p < 0.05) higher number than the non-adapted cells [99]. When cells are exposed to a particular stress, it activated various general stress response mechanisms that help the cell to survive and fight against other stressful conditions. This mechanism is also known as cross protection. In the above study, when E. faecium HL7 cells were previously treated with sublethal heat stress, it improved the survival rate under various stresses. It indicated the cross protection in the heat-adapted cells. Heat stress response in various beneficial bacteria is given in Table 4.

Table 4 Heat stress response in various beneficial bacteria
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Osmotic Stress

Osmotic stress is experienced by probiotic bacteria in the growth medium, in the course of food production and in the GIT. Probiotic bacteria undergo osmotic stress when solute contents in food preparation were changed like salt in cheese, high sugary foods, pickle, etc. [100]. Water moves out of the cell as osmotic pressure rise, triggering cell contraction, loss of cell turgor pressure, and altering cytoplasmic solute content. All of which have a negative impact on bacterial survival [101]. Compatible solutes either from the surrounding medium or secreted by the cell were extremely helpful to combat the osmotic stress. Most of the compatible solutes do not have any charge at pH 7; hence, without interfering with the metabolism, uncharged compatible solutes can be collected in large concentrations. Compatible solutes were observed to preserve proteins in their correct conformation during osmotic pressure.

Osmotic stress could stimulate the secretion of trehalose in P. freudenreichii and L. casei [42]. Lactobacilli were reported to modulate the cytoplasmic level of amino acids such as proline and glutamate during osmotic adaptation [34]. The osmotically induced OpuABC (or Bus ABC) transporter accumulates glycine betaine in P. freudenreichii during osmotic stress [42]. The high levels of sugar in the carrier matrix of LAB allow formation of metabolites such as mannitol (non-fermentable carbohydrate). The non-fermentable carbohydrates were found to boost cell survival during spray drying by raising osmotic pressure and causing cells to osmotically adapt [102]. The LAB cells activated several defence mechanisms when pre-exposed to sub-lethal osmotic stress, could have increased survival after spray drying [42]. The LAB produced EPS in the surrounding medium, thereby prevented cell damage due to dehydration in osmotic stress. The hydroxyl group present on the polysaccharides of EPS was responsible for the water binding ability [103]. The Leu. mesenteroides 406 produced 25.83 g/L EPS in MRS broth containing 5% NaCl compared to 16.02 g/L EPS in control after 48 h at 28 °C [104].

Osmotic stress triggers production or import of K+ or compatible solute in probiotics, which protect cell against lethal damage. L. acidophilus and L. casei secrete protective molecules like DnaK and HtrS operon proteins that safeguard the bacteria against damage caused by high salt concentration in medium [105]. The impact of osmoadaptation of B. bifidum CCFM16 cells on multiplication rate during osmotic stress was determined in a study. Medium added with 0.3% NaCl gives osmotic stress of 100 mOsm/kg. In MRS containing 1 g/L L-cysteine, B. bifidum CCFM16 was exposed to an osmotic environment that increased steadily over the course of 1000 generations. Then the hyperosmotic-tolerance mutant and parent strains were exposed to 1300 mOsm/kg osmotic stress. The generation time of extremely high osmotic pressure–resistant mutant B. bifidum CCFM16m is 1/3 of its parent strain B. bifidum CCFM16 [106]. The finding of this study suggests that the B. bifidum CCFM16 develops various defense mechanisms to cope against harsh osmotic stress when gradually grown in increasing level of osmotic pressure. Such mutant strain has potential to survive in higher number during freeze dying and refrigerated storage in food matrix.

Lactobacillus rhamnosus GG was previously exposed to 4% NaCl and 4.5 pH in MRS for 24 h for stress adaptation. The cell count reduction of stress-adapted L. rhamnosus GG (0.2 log reduction) was comparatively lesser than non-stress-adapted L. rhamnosus GG (0.5 log reduction) in yoghurt matrix during refrigerated storage [107]. This indicates the adaptive response of L. rhamnosus GG to sublethal osmotic and pH stress had improved the survival in the carrier matrix during low-temperature storage.

Osmotic stress response in various beneficial bacteria is given in Table 5.

Table 5 Osmotic stress response in various beneficial bacteria
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Oxidative Stress

Aerobic conditions experienced by probiotics during food processing as well as GIT transit lead to oxidative stress to the sensitive bacterial strain. The oxygen acts as toxic compound by reacting with iron of heme-dependent cytochrome oxidase in electron transport chain to create ROS [108]. Metabolic conversion of oxygen generates ROS like superoxide (O2−), hydroxyl radicals (HO•), and hydrogen peroxide (H2O2). These ROS are highly unstable entities and are responsible for the oxidative chain reaction which damages several critical biomolecules like proteins, DNA, RNA, and lipids, which affects cells viability. ROS can freely pass through the semipermeable membrane and greater amount of ROS ceases LAB cell multiplication. Since many LAB and bifidobacteria lack catalase and superoxide dismutase (SOD) enzyme activity, they were unable to neutralize hydrogen peroxide and ROS, which made them vulnerable to oxygen [109].

Certain LAB like L. plantarum does not have ROS-neutralizing enzymes such as SOD. They defend themselves by other strategies, i.e., concentration of manganese inside the cell and utilizing Mn-dependent mechanisms of superoxide neutralization [110]. Aerotolerant anaerobe LAB Leu. mesenteroides, which lacks SOD, neutralized ROS by encouraging EPS production and cellular aggregation. EPS expel dissolved oxygen from medium to relieve oxidative stress [111]; EPS also neutralizes the ROS by binding with them and cellular aggregation protects the inner cells from the ROS. The fatty acid constituents in bifidobacterial cell membrane were found to alter in oxidative stress [112], suggesting that such modifications might strengthen tolerance to ROS.

The redox mechanisms of Lactobacillus spp. may contribute to the downregulation of ROS-forming enzymes. In addition, nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf-2) and nuclear factor kappa B (NF-κB) were two common transcription factors, through which Lactobacillus spp. modulated oxidative stress [113]. The active cells of L. plantarum CAUH2 were suspended in MRS supplemented with 3 mM, 4 mM, 5 mM, 6 mM, and 7 mM H2O2 to study the oxidative stress response. The concentrations 6 mM and 7 mM H2O2 inhibited L. plantarum CAUH2 growth, but cells were able to survive up to 5 mM H2O2 stress. It was also observed that L. plantarum CAUH2 changed its carbon source utilizing profile and modified glycolytic pathway to produce more ATP under 5 mM H2O2 stress. At transcription level, the antioxidant enzymes like NADH peroxidase, thioredoxin reductase, and glutathione peroxidase were 6.11, 36.76, and 6.23 times upregulated under 5 mM H2O2 stress [114]. The surviving cell numbers of L. rhamnosus hsryfm 1301 increased from 3.7 log CFU in non-adapted cells to 7.8 log CFU in pre-adapted (5 mM H2O2 for 1 h) cells in the presence of 0.5 mM H2O2 [115]. Since oxidative stress improved the survival rate of adapted cells, pre-adaptation to oxidative stress has potential to ameliorate the aerotolerance in probiotic bacteria during food processing.

Oxidative stress response in various beneficial bacteria is given in Table 6.

Table 6 Oxidative stress response in various beneficial bacteria
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Conclusion

Various environmental stresses are encountered by probiotics during food preparation and passage through the GIT. All the response mechanisms used by probiotics to various stresses are species and strain dependent. While under stress, probiotics respond by triggering a number of regulatory functions, such as control gene expression, modification in cell membrane composition, and alteration in metabolic pathways. Exposure of probiotic bacteria to sublethal stress activates the multiple stress tolerance mechanisms in the stress-adapted cell. This review will be helpful to the people involved in development of a new/novel probiotic food, i.e., selection of the carrier matrix with most effective probiotic strain in active physiological state giving maximum therapeutic benefit during GIT transit. Furthermore, this information lays the path for the development of biological and technology strategies to enhance probiotic robustness. It is required to conduct more genomic studies for better understanding of bacterial cell’s behavior under stress. Additionally, there is also requirement to perform animal studies for the stressed cells to check for any pathogenicity, efficacy, and effectiveness. Trials can be conducted to prepare customized foods by utilizing stress-adapted probiotics.