**3. Discussion**

The MBI has isolated approximately 1000 pigment-producing marine bacteria. We selected 

10 strains, which were identified as rare or novel species by 16S rRNA, including strain HOact23T (*Rubritalea squalenifaciens* sp. nov., phylum Verrucomicrobia), strain iso-3 (*Planococcus maritimus*, the class Bacilli, phylum Firmicutes), strain 04OKA-13- 27 (novel species of the family Flavobacteriaceae), and strain YM6-073 (novel specie of the family Flavobacteriaceae) from these isolated bacteria. 

We found two-type new C30 carotenoids diapolyconedioc acid xylosylesters (compound **<sup>1</sup>**–**<sup>3</sup>**) from HOact23T and methyl 5-glucosyl-5,6-dihydro-apo-4,4<sup>ȝ</sup>lycopenoate (compound **4**) from iso-3 through the isolation and structural analyses of carotenoids produced by these strains. Acyclic C30 carotenoids were previously shown to be contained in land bacteria including *Staphylococcus aureus*, belonging to the class Bacilli, and the methanotrophs *Methylobacterium rhodium* (formerly *Pseudomonas rhodos*), belonging to the class ΅*-*Proteobacteria, and *Methylomonas* sp., belonging to the class 

·-Proteobacteria [17,34]. Thus, acyclic C30 carotenoids are likely to widely exist in domain bacteria (prokaryotes), *i*.*<sup>e</sup>*., they are present not only in some low-GC Grampositive bacteria, but also in some phyla in Gram-negative bacteria. The strong  singlet-oxygen-quenching activities of our C30 carotenoids also indicated that such C30 carotenoids are promising as functional carotenoids, although these *in vivo* functional analyses have not yet been conducted. 

We isolated two rare monocyclic C40 carotenoids with one 3-hydroxy-Ά-ring ((3*R*)- saproxanthin (compound **5**) from 04OKA-13-27 and (3*R*,2<sup>ȝ</sup>*S*)-myxol (compound **6**) from YM6-073), which belong to the family Flavobacteriaceae, phylum Bacteroidetes. (3*R*)-Saproxanthin has only previously 

been detected from *Saprospira grandis*, which belongs to the family Saprospiracea, phylum Bacteroidetes [35]. Hence, marine bacterial strain 04OKA-13-27 was the second species to produce saproxanthin. (3*<sup>R</sup>*,2<sup>ȝ</sup>*S*)-Myxol has only previously been detected in marine bacterial strain 

P99-3 (MBIC03313), belonging to the family Flavobacteriaceae [15], and in the cyanobacterium *Anabaena variabilis* ATCC 29413, phylum Cyanobacteria [36]. Therefore, marine bacterial strain YM6-073 was the third species to produce myxol. Myxoxanthophyll (myxol 2ȝ-fucoside), which is widely distributed in phylum Cyanobacteria, contains myxol as its aglycone. These findings indicated that such monocyclic C40 carotenoids with one 3-hydroxy-Ά-ring exist in phylum Bacteroidetes as well as phylum Cyanobacteria. 

The carotenoids produced by the six other strains isolated were all zeaxanthin, which is a common carotenoid in domain bacteria. Our study may be effective for identifying rare and new carotenoids based on its ratio (4/10). In addition, all the rare and new carotenoids (**1**–**<sup>6</sup>**) isolated possessed potent antioxidant activities. 

## **4. Conclusions**

Marine bacteria are likely to produce carotenoids to protect themselves from activated oxygen produced by sunlight (mainly 1O2); therefore, their potent antioxidant activities were expected 

and reasonable. Therefore, the techniques performed in our study effectively identified new 

antioxidant carotenoids. 

## **Author Contributions**

Kazutoshi Shindo performed the experiments and wrote the text; Norihiko Misawa supervised the project and corrected the manuscript. 
