Isolation and identification of hyper-ammonia-producing bacteria from commercial fermented skates (Raja kenojei)

Abstract

Ammonia-producing bacteria were isolated and identified from five commercial fermented skates (A1, A2, A3, A4, and A5). In addition, the pH, ammonia nitrogen, total volatile nitrogen (TVBN), trimethylamine nitrogen (TMAN), and amino nitrogen contents of skate samples were also determined. A total of 88 strains of ammonia-producing bacteria was isolated and seven hyper-ammonia-producing bacteria isolates (A2-2, A2-3, A2-12, A2-18, A2-20, A3-6 and A3-14) were selected based on ammonia nitrogen producing ability. Those isolates were identified as Proteus hauseri (three strains), Providencia rustigianii (three strains), and Kurthia gibsonii. The pH and ammonia nitrogen content in skate samples were ranged from 8.63 to 9.03, and 4.86 to 7.31 g/kg, respectively. No significant difference of pH values was observed in skate samples A2, A3, A4 and A5. While, skate samples A3, A4 and A5 showed similar level of TVBN and TMAN content. Skate sample A2 showed the highest amino nitrogen content among all samples, which indicated the highest degree of protein degradation of skate muscle during fermentation. Bivariate cluster analysis showed that skate samples A3, A4, and A5 clustered together at a relatively high level, implying a similar microbial environment during fermentation. The cluster analysis allowed different commercial fermented skates to be clearly differentiated based on the characteristics determined in this study. This study can provide important information for investigating the mechanisms underlying ammonia flavor formation in skate muscle during fermentation.

Keywords: Ammonia producing bacteria; Cluster analysis; Fermented skate; Identification; Isolation.

Fig. 1

Phylogenetic analysis of 16S rRNA gene sequences from hyper-ammonia-producing (HAP) bacterial strains (A2-2, A2-3, A2-12, A2-18, A2-20, A3-6 and A3-14). Bacterial species indicated are sequences obtained from Genbank

Fig. 2

pH (a) and ammonia nitrogen content (b) in different commercial fermented skate samples. Error bars represent standard deviation from three independent experiments; ^a–cP < 0.05 indicates statistical significance

Fig. 3

Total volatile base nitrogen (TVBN) (a) and trimethylamine nitrogen (TMAN) (b) content in different commercial fermented skate samples. Error bars represent standard deviation from three independent experiments; ^a–dP < 0.05 indicates statistical significance

Fig. 4

Amino nitrogen content in different commercial fermented skate samples. Error bars represent standard deviation from three independent experiments; ^a–dP < 0.05 indicates statistical significance

Fig. 5

Cluster analysis of different commercial fermented skate samples based on chemical characteristics