Development of protective culture production from bacillus subtilis P5-6 as food bio-preservative

Abstract

Halophilic Bacilllus subtilis P5-6 contains bacteriocin encoding genes (subtilin and subtilosin). It has been reported as a potential strain developed to be a protective culture that could reduce viable count of Staphylococcus aureus and prolong the freshness when applied in cheese. The aim of this research was to evaluate bacteriocin generation property of P5-6 and to optimize the bacteriocin production methods for application as food bio-preservative in protective culture form. Through gene assay, only subtilosin gene expression was detected along with housekeeping gene BA-rpoB when co-cultured P5-6 with Staphylococcus aureus. The P5-6 showed low or no inhibitory effect on S. aureus when co-cultured in liquid medium but expressed higher effect on solid medium surface. Spent vegetable brine, adjusted to pH7 then supplemented with 0.04% (w/v) glucose as carbon source and 0.01% (w/v) NH4(SO4)2 as nitrogen source was observed as optimal medium for the P5-6. Cultivation under room temperature at 150 rpm for 16 hours was optimal for this strain to maximally grow as well as inhibit S.aureus at 9.14 log CFU/ mL and 3.3 mm inhibition zone (IZ), respectively. Protective culture was produced from the formula using modified starch as a solid matrix and P5-6 culture at ratio 2:1, then dried with two-step process to have water activity at 0.58. It contained the highest viable count of P5-6 at 8.06 log CFU/ g. Four milligram of the protective culture showed inhibitory effect on S.aureus (4 log CFU/ cm2 density) recorded at 4.55 mm IZ. Beyond 24 hours, when applied protective culture on fresh pork surfaces with non and spiked S.aureus, it was found that protective culture contained viable P5-6 at 6 log CFU (4 log CFU/ cm2) could inhibit the growth of initially contaminated S.aureus in pork and spiked S.aureus. Bacteriocidal effect of protective culture on S.aureus were not clearly observed throughout the storage for 5 days in this food model.