Carotenoid Cocktail Produced by An Antarctic Soil Flavobacterium with Biotechnological Potential

Abstract

Carotenoids are highly important in pigmentation, and its content in farmed crustaceans and fish correlates to their market value. These pigments also have a nutritional role in aquaculture where they are routinely added as a marine animal food supplement to ensure fish development and health. However, there is little information about carotenoids obtained from Antarctic bacteria and its use for pigmentation improvement and flesh quality in aquaculture. This study identified carotenoids produced by Antarctic soil bacteria. The pigmented strain (CN7) was isolated on modified Luria-Bertani (LB) media and incubated at 4 °C. This Gram-negative bacillus was identified by 16S rRNA analysis as Flavobacterium segetis. Pigment extract characterization was performed through high-performance liquid chromatography (HPLC) and identification with liquid chromatography-mass spectrometry (LC-MS). HPLC analyses revealed that this bacterium produces several pigments in the carotenoid absorption range (six peaks). LC-MS confirms the presence of one main peak corresponding to lutein or zeaxanthin (an isomer of lutein) and several other carotenoid pigments and intermediaries in a lower quantity. Therefore, we propose CN7 strain as an alternative model to produce beneficial carotenoid pigments with potential nutritional applications in aquaculture.

Keywords: Antarctic Flavobacterium; aquaculture; carotenoids; lutein; zeaxanthin.

Figure 1

(A) Sample area at King George Island, Antarctica (S62°10′42.5″, W58°55′59.5″). (B) Intense orange colonies recovered from topsoil growth on LB modified medium. (C) Microscope analysis of CN7 strain. According to the Gram staining, the CN7 strain is a Gram-negative bacillus.

Figure 2

Dendrogram of phylogenetic reconstruction based on analyzing ribosomal RNA 16S of F. segetis strain CN7 (KJ943556.1) (red circle). Phylogenetic reconstruction was performed using the neighbor-joining (NJ) method within the Mega X program. The evolutionary distances were computed using Jukes–Cantor with the pairwise deletion option. The percentage of bootstrap values is shown next to the branches based on 10.000 bootstrap replications. The sequence of interest is shown with a red circle.

Figure 3

HPLC profile of total pigments extracted from isolated CN7. (A) Commercial astaxanthin (peak 1) and canthaxanthin (peak 2); insert shows both UV-visible spectrograms. (B) Chromatogram (recorded at 472 nm) of acetone-extracted carotenoids, showing two main peaks (1 and 2) with larger absorbance levels; insert shows UV-visible spectrograms of the main 6 carotenoid peaks extracted from CN7 strain.

Figure 4

LC-UV-MS/MS profile at 450 nm of carotenoid pigments produced by strain CN7. Identification analysis was carried out using the mass spectra determined for each peak. The proposed identifications are detailed in Table 1.

Figure 5

Possible metabolic pathways of CN7 for producing carotenoid pigments. Compounds determined by LC-UV/vis-MS/MS are marked in green boxes. Figure adapted from the work carried out by Kopsell and Kopsell (2006) and Chi et al. (2015) [60,61].