Isolation of Bacteria from Freeze-Dried Samples and the Functional Characterization of Species-Specific Lactic Acid Bacteria with a Comparison of Wild and Captive Proboscis Monkeys

Abstract

Previously, we isolated a novel lactic acid bacteria species (Lactobacillus nasalidis) from the fresh forestomach contents of a captive proboscis monkey (Nasalis larvatus) in a Japanese zoo. In this study, we isolated two strains of L. nasalidis from the freeze-dried forestomach contents of a wild proboscis monkey inhabiting a riverine forest in Malaysia. The samples had been stored for more than six years. Phenotypic analysis showed that strains isolated from the wild individual had more diverse sugar utilization and lower salt tolerance than strains previously isolated from the captive counterpart. These phenotypic differences are most likely induced by feeding conditions; wild individuals consume a wide variety of natural food, unlike their zoo-raised counterparts that consume formula feed with sodium sufficiency. Since 16s rRNA sequences of L. nasalidis were detected in the previously created 16S rRNA libraries of wild, provisioned, and captive proboscis monkeys in Malaysia and Japan, L. nasalidis may be an essential bacterium of the foregut microbial community of the proboscis monkey. The currently established method for the isolation of gut bacteria from freeze-dried samples under storage will be applicable to many already-stored precious samples.