**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 dismutase (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.*

#### *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 successful 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 oxidative 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 addition, 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 peroxynitrite). 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 antioxidant 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 nonalcoholic 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 proteasome 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 proteins (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

*Functional Foods and Antioxidant Effects: Emphasizing the Role of Probiotics DOI: http://dx.doi.org/10.5772/intechopen.104322*

**Figure 3.** *Nrf2-keap1-ARE pathway mediated by probiotics.*

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].
