Bacterial dynamics during yearlong spontaneous fermentation for production of ngari, a dry fermented fish product of Northeast India

Abstract

Ngari is the most popular traditionally processed non-salted fish product, prepared from sun-dried small cyprinid fish Puntius sophore (Ham.) in Manipur state of Northeast India. The microbial involvement in ngari production remained uncertain due to its low moisture content and yearlong incubation in anaerobically sealed earthen pots without any significant change in total microbial count. The culture-independent PCR-DGGE analysis used during this study confirmed a drastic bacterial community structural change in comparison to its raw material. To understand the bacterial dynamics during this dry fermentation, time series samples collected over a period of nine months through destructive sampling from two indigenous ngari production centres were analysed by using both culture-dependent and culture-independent molecular methods. A total of 210 bacteria isolated from the samples were identified by amplified ribosomal DNA restriction analysis (ARDRA) based grouping and 16S rRNA gene sequence similarity analysis. The dominant bacteria were Staphylococcus cohnii subsp. cohnii (38.0%), Tetragenococcus halophilus subsp. flandriensis (16.8%), a novel phylotype related to Lactobacillus pobuzihii (7.2%), Enterococcus faecium (7.2%), Bacillus indicus (6.3%) and Staphylococcus carnosus (3.8%). Distinct bacterial dynamics with the emergence of T. halophilus at third month (106CFU/g), L. pobuzihii at sixth month (106CFU/g), S. carnosus at three to six months (104CFU/g) and B. indicus at six to nine months (105CFU/g) in both the production centres was observed during ngari fermentation. However, the other two dominant bacteria S. cohnii and E. faecium were isolated throughout the fermentation with the population of 106CFU/g and 104CFU/g respectively. Culture-independent PCR-DGGE analysis further showed the presence of additional species, in which Kocuria halotolerans and Macrococcus caseolyticus disappeared during fermentation while Clostridium irregulare and Azorhizobium caulinodans were detected throughout the fermentation. Principal component analysis showed a drastic bacterial community structural change at the sixth month of fermentation. These identified dominant bacterial cultures of T. halophilus, L. pobuzihii, S. carnosus and B. indicus could be effectively utilised for designing starter culture and optimizing fermentation technology for industrialisation of ngari production.