Prevalence and host specificity of Bacillus subtilis-targeting bacteriophages in traditional Thai Thua Nao

Abstract Thua Nao, a traditional northern Thai fermented soybean product, relies on Bacillus subtilis for protein hydrolysis, flavour development, and texture formation. Bacteriophages infecting B. subtilis have been reported in other fermented soybean foods, where they can affect fermentation, but their prevalence in Thua Nao remains largely unknown. In this study, 20 Thua Nao samples (dried, fresh, and paste) from northern Thailand and a Korean fermented soybean paste (Doenjang) were analysed for phage presence, host range, and lytic activity. All samples exhibited high bacterial populations (>6 log CFU g −1 ) and pH values between 5 and 7, providing favourable conditions for phage–host interactions. Phage-containing filtrates were tested against eight Bacillus strains, including seven B. subtilis and one Bacillus licheniformis . Most filtrates lysed at least one B. subtilis strain, particularly ASA, TN3, and S1-13, while thirteen of twenty-one filtrates also lysed B. licheniformis TISTR1109, suggesting broad host ranges or mixed phage populations. Eight representative phages were purified, and their host specificity was reassessed using spotting and double-layer agar techniques. Spotting assays indicated broader host susceptibility, whereas double-layer agar confirmed productive infection and revealed host-dependent differences in plaque size. These findings demonstrate that B . subtilis -targeting bacteriophages are prevalent in Thua Nao and provide a valuable platform for future studies on phage biodiversity and its potential role in shaping microbial communities during soybean fermentation.