2.4 Molecular identification

Molecular identification was performed according to Screening Criteria of Lactic Acid Bacteria for Feeding Aquatic Animals (TCSWSL016-2019). Four isolates were incubated overnight at 37 °C in MRS broth, and then genomic DNA was extracted using TIANamp bacteria DNA extraction kit (TIANGEN, Beijing, China). Molecular identification was performed by amplifying the 16S rRNA gene using universal primers (27F 5′-AGAGTTTGATCCTGGCTCAG -3′; 1492R, 5′-GGTTACCTTGTTACGACTT-3′) and previously described polymerase chain reaction (PCR) reaction conditions (Vaz-Moreira et al., 2009). PCR products were separated by 1.2% agarose gel electrophoresis and confirmed by sequencing (Genewiz, Suzhou, China). The obtained 16S rRNA sequences of the strains were compared with the EzBioCloud databases to identify the species (https://www.ezbiocloud.net/). Phylogenetic tree was constructed based on 16S rRNA sequences by MEGA software (version 7.0) with a Kimura two-parameter model for distance options and a Neighbor Joining (NJ) method for clustering with 1000 bootstrap replicates (Kumar et al., 2016).