Application of Antimicrobial Bacillus subtilis Strain as a Starter Culture to Improve Qualities and Safety of Fermented Soybean (SIENG) Produced in Cambodia

Fermented foods play a very important role in Cambodian health and nutrition, as well as other developing countries where food preservation methods may be limited. SIENG, a Khmer fermented soybean product, naturally contains both beneficial and pathogenic microorganisms. Traditional fermentation that relies on natural microbial flora and environmental conditions results in variable product quality and can lead to spoilage. A starter culture such as Bacillus subtilis can ensure the safety and stability of the products. The objective of this study is to control the growth of Gram positive pathogens contaminated into traditional fermented soybean (SEING) by using antimicrobial Bacillus subtilis isolated from the same kind of food. Out of 120 SIENG samples, 49 B. cereus strains were isolated, and 12 of B. cereus were positively synthesized compared with the lyophilized control enterotoxin. Two of these strains (BTM8-7 and BTM8-8) produced high levels of enterotoxin. We identified five Bacillus strains with the ability to fight against indicator pathogenic microorganisms. Among the five strains, B. CeM6-2 had the highest activity level against Lactobacillus plantrum ATCC 8014 and the largest diameter. B. CeM6-2 tolerated up to 20 h at 30 °C and 22 h at 37 °C. In testing the strains with PK and PK-PMSF enzymes, bacteriocin produced by the strain B. CeM6PK untreated and B. CeM6-2PK-PMSF had a significantly stronger ability to suppress all the pathogenic indicators from 0 h to 47 h compared to the B. CeM6-2PK. Moreover, CeM6-2 outperformed the Miyagino strains, as it actively produced bacteriocin that fought against all four indicator strains of Gram positive and lactic acid groups, especially against Listeria monocytogenes, Streptococcus pyrogene, Leuconostoc mesenterids and L. plantarum, from 0 h-58 h and 0 h-40 h at 35 °C. CeM6-2 (1%) strain had the highest ability to fight against B. cereus at 24 h and at 34 h to 44 h incubation as well. CeM6-2 (10:0 mL) and CeM6-2 (9:1 mL) have the strongest ability to fight against B. cereus at room temperature (48 h and 72 h). The longer incubation and time at room temperature produce the highest level of bacteriocin. Thus, bacteriocins produced by B. CeM6-2 can be used as a preservative in food processing industries to avoid food spoilage even in higher temperatures and time.