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4. The mechanisms of action of probiotics in antioxidation
There are different types of probiotics, so a variety of resistance mechanisms in different probiotic strains to cope with oxidative stress can be expected (Figure 2).
4.1 Metal chelating ability
Some probiotics exert their antioxidant potential by preventing metal ions from oxidizing. They use chelators to trap metal ions. These chelators include bathophen- anthroline disulfonic acid (BPS), desferrioxamine, and ethylene diamine tetraacetic acid (EDTA) [45].
Different strains of probiotic bacteria were studied for this antioxidant mecha- nism; for example, among the various strains capable of chelating iron (II) or copper (II), the strains of Lactobacillus casei KCTC 3260 and Streptococcus thermophilus 821 have a much higher ability to chelate iron (II) and copper (II) [46]. Also, the high potency of Lactobacillus helveticus CD6 intracellular cell-free extraction in chelating iron (II) ions can be mentioned [47].
These chelating agents are not fully understood in probiotic bacteria. However, studies have shown their role in inhibiting phosphate ester displacement enzymatic reactions, as well as the production of radicals (such as peroxyl radical and alkoxyl radical) due to the decomposition of hydroperoxide compounds [48].
4.2 Antioxidant enzymes system
Mitochondria are sources of superoxide production. Superoxide is one of the most prevalent ROS. To reduce the risk of this compound, an enzyme called superoxide dis- mutase (SOD), as one of the essential enzymes in antioxidant enzyme systems, is needed.
This enzyme breaks down the high-risk compound superoxide and converts it into less dangerous compounds, such as hydrogen peroxide and water. Therefore, it can be said that SOD in animals and also in prokaryotes plays an important role in regulating ROS [49].
Types of SOD enzymes have been identified in mammals and bacteria, which are used against oxidative stress. Fe-SOD and Mn-SOD have been observed in bacteria [50]; for example, the Mn-SOD in Lactobacillus fermentum E-3 and E-18 was reported by Kalisar et al. [35]. While cytoplasmic and extracellular Zn-SOD and mitochondrial Mn-SOD have been observed in mammals [50].
Figure 2.
The action modes of probiotic bacteria in antioxidation.
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Functional Foods and Antioxidant Effects: Emphasizing the Role of Probiotics DOI: http://dx.doi.org/10.5772/intechopen.104322
SOD enzymes need suitable transporters, that can be used in the local delivery of these enzymes [51] because despite its antioxidant activity [52–54], the limited bioavailability of SOD (due to its short half-life in the circulating) has questioned its therapeutic application. The use of probiotic bacteria for this purpose led to suc- cessful results; for example, the use of probiotic bacteria as a transporter for topical delivery of SOD was effective in counteracting the oxidative stress induced by ROS in people with intestinal diseases. Also, according to a study, engineered strains of Lactobacillus casei BL23 (capable of producing SOD) improved the inflammatory status and increased enzymatic activity of mice with Crohn's disease, which shows the beneficial effect of using probiotic bacteria as a carrier [51].
Another enzyme that can be found in probiotic bacteria (except LAB) is called catalase (CAT), Which acts as an antioxidant under oxidative stress. CAT is involved in a reaction known as Fenton. In this reaction, CAT inhibits the production of hydroxyl radicals by the decomposition of hydrogen peroxide, in this way, exerts its antioxidant role [55]. To determine the antioxidant effect of the enzyme CAT, studies have been performed on bacteria that contain this enzyme. CAT-producing probiotic bacteria include Lactococcus lactis and the engineered strains of Lactobacillus casei BL23 [56].
Another action of probiotics inside the host is to enhance antioxidant activity by increasing the levels and activity of several enzymes. For example, according to studies, the probiotic Lactobacillus fermentum increases the levels of SOD, GPx, CAT and Cu, and Zn-SOD enzymes [57], yeast probiotics increase the activity of GPx enzyme [58], and Bacillus amyloliquefaciens SC06 probiotic increases the expression of genes such as CAT and glutathione S-transferase (GST) in the studied animals [7].
In addition, people with type 2 diabetes show increased antioxidant activity by taking the probiotics Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12, which is due to increased activity of antioxidant enzymes such as SOD and GPx in red blood cells [59].
4.3 Antioxidant metabolites
Probiotics can exert their antioxidant power in other ways, for example, they produce various metabolites with antioxidant properties. These metabolites include glutathione (GSH), butyrate, and folate.
The properties of folate include the acceptance of mono carbon units, its use in various metabolic pathways, and its necessity in DNA synthesis and regeneration, DNA methylation, and cell division [60].
Studies showed an increase in folate levels in the body of rats and humans after treatment with bifidobacteria [61, 62] and an increase in the level of this metabolite and vitamin B12 in people treated with the probiotic Lactobacillus acidophilus La1 [63]. Evidence also showed that patients with type 2 diabetes experience oxida- tive stress, and in the absence of metabolites, such as folate and vitamin B12, this condition is exacerbated. Therefore, host treatment with these types of probiotics can increase the levels of these antioxidant metabolites in patients [64]. In addi- tion, according to the study, intact cells of Lactobacillus helveticus CD6 producing folate and intracellular cell-free extract of this probiotic have similar antioxidant power [47]. Vitamins, such as vitamin B1, can make cells more resistant to oxidative stress. According to research, consumption of some probiotics can lead to increased absorption of this vitamin in individuals, which helps protect cells against oxidative stress [65–67].
GSH is another example of a non-enzymatic antioxidant, which is involved in the removal of radicals (such as hydrogen peroxides, hydroxyl radicals, and peroxyni- trite). GSH works in conjunction with the selenium-dependent enzyme glutathione peroxidase [68]. Probiotics may contain GSH, and have antioxidant properties under oxidative stress. According to studies, Lactobacillus fermentum E-3, E-18, and ME-3 are among the probiotics with large amounts of GSH [35, 69].
During the fermentation of a series of indigestible substances, the microbiota makes a short-chain fatty acid (SCFA) called butyrate [70]. Butyrate has an anti- oxidant role under oxidative stress by inducing antioxidants. Some probiotics can produce butyrate; for example, based on evidence, MIYAIRI 588 strain of Clostridium butyricum with the production of butyrate has been able to improve rats with non- alcoholic fatty liver and exposed to oxidative stress [71].
4.4 Antioxidant signaling pathway mediated by probiotic bacteria 4.4.1 Nrf2-Keap1-ARE
Under oxidative stress, the expression of genes involved in the detoxification of ROS can be mediated through a pathway called Nrf2-Keap1-ARE (Figure 3) [72, 73]. In this pathway, the binding of Nrf2 to the antioxidant response element (ARE) sequences in the nucleus leads to the expression of factors related to the detoxification of ROS [74–76]. The activation or inhibition of Nrf2 depends on the amount of ROS. When the amount of ROS is low, the cytoplasmic inhibitor Keap1 binds to Nrf2, causing its protea- some degradation by polyubiquitination [77]; however, under oxidative stress, the functional structure of Keap1 changes due to the influence of the amino acid cysteine, which leads to the activation of Nrf2 and its entry into the nucleus and binding to the ARE sequences [74–76, 78].
Probiotics can exert their antioxidant effects by regulating the Nrf2-Keap1-ARE pathway. According to research, probiotics such as Lactobacillus Plantarum FC225, Lactobacillus Plantarum CA16, and Lactobacillus Plantarum SC4 can increase the level of Nrf2 in the liver cells of hypertensive mice [79, 80]. The effect of Clostridium butyricium MIYAIRI 588 [71] and Bacillus amyloliquefaciens SC06 on increasing the level and regulation of Nrf2 expression in the studied animals has been shown [7].
4.4.2 NFκB
In inflammatory conditions, the expression of inflammatory cytokines is mediated by the transcription factor NFκB. This factor is activated by ROS. It can be said that NFκB is the first transcription factor that responds to oxidative stress [19]. Evidence suggests that probiotics may inhibit NFκB by their antioxidant power, and thus play a role in preventing inflammation. Inhibition of NFκB and stimulation of heat shock pro- teins (Hsps) in colon epithelial cells by probiotic mixture VSL # 3 and also the effect of Bacillus sp. strain LBP32 in the prevention of inflammation in RAW 264.7 macrophages are examples of the antioxidant effect of probiotics by inhibiting NFκB [81, 82].
4.4.3 MAPK
Among the four subfamilies of mitogen-activated protein kinases (MAPKs), c-jun N-terminal kinase (JNKs) and p38-MAPK are key enzymes involved in response to various stresses (UV irradiation and osmotic shock), and extracellular regulated
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Functional Foods and Antioxidant Effects: Emphasizing the Role of Probiotics DOI: http://dx.doi.org/10.5772/intechopen.104322
protein kinases (ERKs) have an important role in anabolic metabolisms [83, 84].
These are the best-known mitogen-activated protein kinases [85].
Based on studies, some probiotics, such as Lactobacillus GG, can activate MAPK in the young adult mouse colon (YAMC) cells. Soluble agents in the conditioned media from the probiotic Lactobacillus GG (Lactobacillus GG-CM) in these cells stimulate Hsp25 and Hsp72. MAPK signaling pathways are involved in the expression and stimulation of Hsps in treated cells, so inhibition of p38 and JNK in the YAMC and then treatment with the probiotic Lactobacillus GG-CM stops the expression of Hsp72 [86]. In addition, the soluble proteins p40 and p75 produced by the probiotic Lactobacillus rhamnosus GG via the MAPK pathway can correct the dysfunction of epithelial cell barriers caused by a potent oxidant [85].
4.4.4 PKC
The control of the phosphorylation function of hydroxyl groups of serine and threonine residues in proteins is associated with Protein kinase C (PKC). PKC is also cellular messenger molecule that plays a role in various pathways, including regula- tion of cell growth and death and response to stresses. This molecule is very sensitive to redox modification, as well [87–89]. On the other hand, some probiotics can affect PKC activity. Corresponding to the previous report (Seth et al., 2008), inhibition of Ro-32-0432 (PKC inhibitor) by soluble proteins p40 and p75 produced by L. rhamno- sus GG improves H2O2-induced epithelial barrier disorder [85].
4.5 Regulation of the ROS producing enzymes
Production of ROS through enzymatic reactions and various chemical processes in the host body is essential [90]; because they play an important role in defense
Figure 3.
Nrf2-keap1-ARE pathway mediated by probiotics.
and messaging functions [91]. The human NADPH oxidase (NOX) complex, as the main source of reactive oxygen species [92–94], has seven homologs (NOX1–5, dual oxidase 1 (DUOX1), and DUOX2) [91]. Membrane-bound NOX2 catalytic subunits and p22phox in combination with cytosolic agents (e.g., p40phox, p47phox, p67phox, and small GTPase RAC1, called by neutrophils) cause a respiratory burst (Figure 4) [95, 96]. However, oxidative stress occurs if there is an imbalance in the production of ROS or a decrease in the level of oxygen-scavenging proteins, which leads to tissue damage and cell death [93].
Probiotics are able to affect the production of ROS by the NOX complex. Based on researches, the probiotic Bacillus amyloliquefaciens SC06 reduces the activity of NOX and expression of p47phox (H2O2-induced IPEC-1) and the probiotics Lactobacillus fermentum CECT5716, Lactobacillus coryniformis CECT5711 (K8) and Lactobacillus gasseri CECT5714 (LC9) reduce NOX activity and decrease the mRNA expression of NOX-1 and NOX-4 enzymes (in hypertensive rats), as a result, they reduce the production level of ROS [7, 97].
On the other hand, ROS can be produced during prostaglandin biosynthesis. For example, cyclo-oxygenase (COX) participates in process of prostaglandin biosyn- thesis [98]. In some diseases, such as atherosclerosis, COX-2 expression is increased [99]. Therefore, it can be said that the production of vascular prostanoids is caused by overexpression of the COX-2 enzyme [100]. Studies have shown that some pro- biotics can reduce the expression of COX-2 in the host; for example, the commercial probiotic Lacidofil, when used in mice infected with Helicobacter pylori [101].
Furthermore, it has been established that the expression of COX-2 is reduced by Lactobacillus acidophilus in Catla thymus macrophages [102].
Improper functioning of cytochrome P450 (CYP) enzymes is associated with over- production of ROS and oxidative stress conditions [103]; because they are involved in the metabolism of xenobiotic substances [104]. Some probiotics reduce the expression of these enzymes. Matuskova et al. reported Lactobacillus casei is involved in reducing the expression of CYP1A1 enzyme in the intestines of male rats [105].
Figure 4.
NADPH oxidase (NOX) complex regulated by probiotics.
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Functional Foods and Antioxidant Effects: Emphasizing the Role of Probiotics DOI: http://dx.doi.org/10.5772/intechopen.104322
4.6 Modulation of the intestinal microbiota
Studies have shown that some probiotics can be used to treat intestinal diseases [106, 107] because they have the ability to improve the oxidative stress created by changing the composition of the microbiota. In fact, probiotics show their antioxidant properties by settling in the gastrointestinal tract [108, 109] to regulate the altered microbiota composition and prevent the proliferation of harmful bacteria. Based on researches, Lactobacillus and Bifidobacterium are among the probiotics that prevent the growth of pathogenic bacteria by lowering the pH of the intestine; as a result, a balance is established in the composition of the microbiota [110, 111]. In addition, some probiotics produce toxic compounds (such as organic acids, bactericides, and biosurfactants) against pathogenic microorganisms [112]. For example, Lactobacillus rhamnosus GG, suppresses different bacteria by producing antimicrobial compounds [113]. As previously mentioned, the use of probiotics leads to improving oxidative stress by modulating the composition of gut microbiota and reducing the abundance of harmful bacteria.