Heat stress as an innovative approach to enhance the antioxidant production in Pseudooceanicola and Bacillus isolates

Abstract

It is well known that the quality and quantity of bioactive metabolites in plants and microorganisms are affected by environmental factors. We applied heat stress as a promising approach to stimulate the production of antioxidants in four heat-tolerant bacterial strains (HT1 to HT4) isolated from Aushazia Lake, Qassim Region, Saudi Arabia. The phylogenetic analysis of the 16S rRNA sequences indicated that HT1, HT3 and HT4 belong to genus Bacillus. While HT2 is closely related to Pseudooceanicola marinus with 96.78% similarity. Heat stress differentially induced oxidative damage i.e., high lipid peroxidation, lipoxygenase and xanthine oxidase levels in HT strains. Subsequently, heat stress induced the levels of flavonoids and polyphenols in all strains and glutathione (GSH) in HT2. Heat stress also improved the antioxidant enzyme activities, namely, CAT, SOD and POX in all strains and thioredoxin activity in HT3 and HT4. While GSH cycle (GSH level and GPX, GR, Grx and GST activities) was only detectable and enhanced by heat stress in HT2. The hierarchical cluster analysis of the antioxidants also supported the strain-specific responses. In conclusion, heat stress is a promising approach to enhance antioxidant production in bacteria with potential applications in food quality improvement and health promotion.

Figure 1

Neighbor-joining phylogenetic tree showing the relationship between the heat-tolerant isolate HT2 from the present study and the closely related species in genus Pseudooceanicola. Bacillus subtilis ATCC 6051 was used as the out‐group.

Figure 2

Neighbor-Joining phylogenetic tree showing the relationships between the heat-tolerant isolates (HT1, HT3 and HT4) from the present study and the closely related species of genus Bacillus. Microbacterium oleivorans T13 KC764962 was used as the out‐group.

Figure 3

The oxidative damage markers of the four heat-tolerant bacterial isolates (HT1, HT2, HT3 and HT4) in terms of (A) Lipid peroxidation (MDA), (B) Lipoxygenase activity and (C) Xanthine oxidase activity under control and heat stress conditions. Data are represented by the mean of at least 3 replicates ± standard error. Different small letters (a, b, c…) above bars indicate significant differences between means at p < 0.05.

Figure 4

The overall antioxidant capacity of the four heat-tolerant bacterial isolates (HT1, HT2, HT3 and HT4) in terms of (A) FRAP, (B) DPPH % and (C) superoxide scavenging (SOS) under control and heat stress conditions. Data are represented by the mean of at least 3 replicates ± standard error. Different small letters (a, b, c…) above bars indicate significant differences between means at p < 0.05.

Figure 5

The antioxidant metabolites of the four heat-tolerant bacterial isolates (HT1, HT2, HT3 and HT4) in terms of (A) Flavonoids, (B) Polyphenols, (C) Reduced GSH, (D) oxidized GSH (GSSG) and (E) total GSH per g of bacterial cell weight under control and heat stress conditions. Data are represented by the mean of at least 3 replicates ± standard error. Different small letters (a, b, c…) above bars indicate significant differences between means at p < 0.05.

Figure 6

The antioxidant enzyme activities of the four heat-tolerant bacterial isolates (HT1, HT2, HT3 and HT4) including (A) CAT, (B) SOD, (C) GPX and (D) POX under control and heat stress conditions. Data are represented by the mean of at least 3 replicates ± standard error. Different small letters (a, b, c…) above bars indicate significant differences between means at p < 0.05.

Figure 7

The antioxidant enzyme activities of the four heat-tolerant bacterial isolates (HT1, HT2, HT3 and HT4) including (A) GR, (B) Grx (C) TRD, and (D) GST activity under control and heat stress conditions. Data are represented by the mean of at least 3 replicates ± standard error. Different small letters (a, b, c…) above bars indicate significant differences between means at p < 0.05.

Figure 8

Strain-specific responses of the four heat-tolerant bacterial isolates (HT1, HT2, HT3 and HT4) to heat stress at both physiological and biochemical levels. The measured parameters represented by damage markers, overall antioxidant capacity, antioxidant metabolites and antioxidant enzymes are grouped into four main clusters based on their responses to heat stress.