Analysis of the Antioxidant Composition of Low Molecular Weight Metabolites from the Agarolytic Bacterium Alteromonas macleodii QZ9-9: Possibilities for High-Added Value Utilization of Macroalgae

Abstract

Agar accounts for ~60% of the dry weight of some red macroalgae, and the breakdown of this kind of polysaccharide releases high-value compounds; therefore, the resource utilization of agar is of great significance to improve the added value of these macroalgae. Herein, Alteromonas macleodii QZ9-9 isolated from tropical Gracilaria hainanensis in Hainan Island was characterized as an agarolytic bacterium, which displayed a high agar-degrading activity. The highest diameters of the degradation zones of the A. macleodii QZ9-9 and its extracellular-agarase (12.16 U/mL) were 41.46 mm and 22.89 mm, respectively, and the first-order degradation rate constants of those were 0.02 h^-1 and 0.77 U^-1, respectively. Importantly, the fermentation products of A. macleodii QZ9-9 exhibited antioxidant activity, and the peak of DPPH scavenging activity of 50 h fermentation products of this strain was up to 50.79% in the reaction for 1 h; the DPPH scavenging activity of low molecule metabolites (≤3 kDa) in particular was up to ~85.85%. A total of 766 metabolites were detected in the low molecule metabolites by metabolomics. The peptide-like metabolites, such as prolyl-histidine, isoleucyl-histidine, isoleucyl-proline and arginyl-proline, and the antioxidant maculosin were found in the top 20 metabolites with relatively high abundance. Additionally, the antioxidant activity of maculosin was further verified in this work. We concluded that the low molecule metabolites of A. macleodii QZ9-9 with relatively high antioxidant activity are interesting candidates for preparing desirable non-toxic antioxidants, thereby facilitating the high value-added utilization of macroalgae in the fields of cosmetic, food preservation, and pharmaceutical industries.

Keywords: agarase; agarolytic bacterium; antioxidant activity; antioxidant peptides; macroalgae; maculosin.

Figure 1

The morphology of agarolytic bacterial A. macleodii QZ9-9. (A) The colony of strain QZ9-9 on mineral salts agar plate after static incubation for 48 h, and a clear transparent hydrolysis zone surrounds this colony. (B) Hydrolysis zone colored with Lugol’s iodine solution after static incubation for 96 h on mineral salts agar plate. (C) Close-up of the electron microscopy of strain QZ9-9. (D) Phylogenetic tree of A. macleodii QZ9-9 (marked by red color) prepared using Mega 11.0 software. Note: the code in each parenthesis represents the sequence accession number in GenBank, and the number at each branch point is the percentage supported by bootstrap.

Figure 2

The curves of growth and agarase production of A. macleodii QZ9-9 in agar-enriched medium (w/v: containing 0.2% agar, 3% NaCl, 0.5% (NH₄)₂SO₄, 0.2% K₂HPO₄, 0.1% MgSO₄·7H₂O and 0.01% FeSO₄·7H₂O, pH 7.5) with agar as the sole carbon source (A) and agar-deficient medium (w/v: containing 0.5% tryptone, 0.1% yeast extract, 100 mL seawater, pH 7.5) that other carbon instead of agar as the carbon source (B).

Figure 3

The agar-degrading capacity of A. macleodii QZ9-9. (A) Characterization of agar-degrading activity of A. macleodii QZ9-9 on the mineral salts agar plates (stained with Lugol’s iodine solution) at different degradation periods. (B) Kinetics of agar degradation by A. macleodii QZ9-9. Note: The data were fitted according to the first-order rate equation Y_t = Y₀ + a(1 − e^−kx), where Y_t and Y₀ are the degradation fraction at a given culture time t and 0 min, respectively; a is the fitting coefficients; and k is the observed rate constant.

Figure 4

The agar-degrading capacities of agarase secreted by A. macleodii QZ9-9. (A) Characterization of agar-degrading activity of crude agarase with different enzyme activities on the mineral salts agar plates (stained with Lugol’s iodine solution). (B) Kinetics of agar degradation by agarase. Note: The data were fitted according to the first-order rate equation Y_EA = Y₀ + a(1 − e^−kx), where Y_EA and Y₀ are the degradation fraction at a given enzyme activity EA and 0 U/mL, respectively; a is the fitting coefficients; and k is the observed rate constant.

Figure 5

Kinetics of DPPH scavenging activity of fermentation products from two culture periods during the reaction time of 1 h, (A) the period of 0 h to 50 h, and (B) the period of 50 h to 100 h. (C) DPPH scavenging activity of fermentation products during the culture of strain QZ9-9 after 1 h reaction. Note: The reaction time refers to the time consumed by the combination reaction between fermentation products and DPPH solution.

Figure 6

(A) The DPPH scavenging activity of fermentation products with different molecular weights. (B) Comparison of the relative abundance of metabolites in the low molecular weight (≤3 kDa) products. The different superscript letters indicate a significant difference at p < 0.05.

Figure 7

Confirmation of DPPH scavenging activity of maculosin by visual observation (A) and quantification (B). Vitamin C is a positive control.