Microalgae and cyanobacteria as microbial substrate and their influence on the potential postbiotic capability of a bacterial probiotic

Abstract

Postbiotics are metabolic by-products from microorganisms that provide health benefits to the host. Their secretion can be influenced by various conditions affecting bacterial metabolism. This study presents a novel approach for producing potential postbiotics, specifically extracellular products (ECPs), from the probiotic strain Shewanella putrefaciens SpPdp11, grown under different culture conditions. These conditions include aquafeed media, with partial or total microalgae/cyanobacteria replacement as the microbial substrate, as well as variations in temperature and growth phase. The use of microalgae/cyanobacteria as substrates may represent a valuable strategy for generating novel postbiotics with unique properties. The ECPs assessed were evaluated for their in vitro cytotoxic, hydrolytic and antimicrobial activities. Three conditions (ECPs derived from aquafeed media with partial (FM2324 and FM1548) or total (M2324) microalgae/cyanobacteria replacement) were non-cytotoxic to various fish cell lines and hydrolysed key nutritional compounds (casein, lipids, amylase and gelatin). Proteomic analysis of these ECP conditions revealed common structural and regulatory DNA-associated proteins, while differentially expressed proteins were associated with amino acid metabolism and antioxidant system (FM2324 and FM1548) and chemotaxis system (M2324). The results highlight the potential of the selected postbiotics as feed additives for future in vivo studies, aligning with sustainable development for aquaculture.

FIGURE 1

Culture conditions for SpPdp11 for ECPs extraction and nomenclature assigned. FM: Partial replacement of aquafeed with a 25% blend of microalgae (Chlorella fusca, Tisochrysis lutea and Microchloropsis gaditana) and cyanobacteria (Arthrospira platensis) (1:1:1:1) (160 g/L) and agar (1.5%); M: Blend of the microalgae and cyanobacteria (50 g/L) and agar (1.5%) (medium M). Internal controls (ICs) were named according to their respective conditions with the addition of ‘Internal Control; IC’.

FIGURE 2

Cytotoxic effect produced by extracellular products (ECPs) from SpPdp11 and its internal controls on brain cell line of European sea bass (DLB‐1). Graphics are distributed according to the different culture media; (A) T media; (B) F media; (C) FM media and (D) M media. The cell viability was determined after 24 h of incubation. The concentration of ECPs tested on all cells were 0.75, 1 and 1.5 μg protein/mL. Values represent the mean ± SD of three replicates. Hash mark (#) and asterisks (*) indicate decreased and increased cell viability, respectively, compared to the different ECPs conditions and the control (p < 0.05).

FIGURE 3

Cytotoxic effect produced by extracellular products (ECPs) from SpPdp11 and its internal controls on brain cell line of mummichogs (FuB‐1). Graphics are distributed according to the different culture media; (A) T media; (B) F media; (C) FM media and (D) M media. The cell viability was determined after 24 h of incubation. The concentrations of ECPs tested on all cells were 0.75, 1 and 1.5 μg protein/mL. Values represent the mean ± SD of three replicates. Hash mark (#) and asterisks (*) indicate decreased and increased cell viability, respectively, compared to the different ECP conditions and the control (p < 0.05).

FIGURE 4

Cytotoxic effect produced by extracellular products (ECPs from SpPdp11 and its internal controls on clearfin livebearer hepatoma cell line (PLHC‐1)). Graphics are distributed according to the different culture media; (A) T media; (B) F media; (C) FM media and (D) M media. The cell viability was determined after 24 h of incubation. The concentration of ECPs tested on all cells were 0.75, 1 and 1.5 μg protein/mL. Values represent the mean ± SD of three replicates. Hash mark (#) and asterisks (*) indicate decreased and increased cell viability, respectively, compared to the different ECP conditions and the control (p < 0.05).

FIGURE 5

Cytotoxic effect produced by extracellular products (ECPs) from SpPdp11 and its internal controls on fibroblast cell line of gilthead seabream (SAF‐1). Graphics are distributed according to the different culture media; (A) T media; (B) F media; (C) FM media and (D) M media. The cell viability was determined after 24 h of incubation. The concentration of ECPs tested on all cells were 0.75, 1 and 1.5 μg protein/mL. Values represent the mean ± SD of three replicates. Hash mark (#) and asterisks (*) indicate decreased and increased cell viability, respectively, compared to the different ECP conditions and the control (p < 0.05).

FIGURE 6

Volcano plots showing the distribution of identified proteins for SpPdp11 ECP conditions: (A) FM1548, (B) FM2324 and (C) M2324. The x‐axis shows log2/fold change), and the y‐axis shows −log10 (adjusted p‐values). The two dotted black vertical lines represent a −log2 of 0.5–2 for the fold change, while the dotted horizontal line denotes −log 10 of our significance threshold (p ≤ 0.05). Blue dots represent upregulated proteins (up); orange dots represent downregulated proteins (down); and grey dots represent non‐differentially expressed proteins (ns).

FIGURE 7

Venn diagram illustrating the distribution (unique or common) of all identified DEPs across different ECP conditions (FM2324 in blue, FM1548 in green and M2324 in yellow) compared to the control.