Multifunctional properties of bacillus cell autolysate as antimicrobial and coloring agents in foods

Abstract

This research aimed to evaluate feasibility of pigments from halophilic Bacillus as coloring agent having multi-bioactivity in foods. Those Bacillus were isolated from Thai traditional salty fermented fish (plara) and identified by 16S rDNA sequencing analysis. Five strains including Bacillus sp. 81-2; 13-3; 77-3, Bacillus amyloliquefaciens 34-12, and Bacillus vietnamensis 39-23, with pink-orange, yellow, and yellow-orange pigmentation were selected as representative of each pigment color shade. All of strains were classified as slight halophilic based on salt tolerance determination. Pigment was extracted and subjected to test for antimicrobial (200 mg/ml) and antioxidant activities (50 mg/ml) through disc diffusion and DPPH assay, respectively. Crude extracts of all strains had inhibitory effect on Escherichia coli, Salmonella Typhimurium, and Bacillus cereus, while only extracts from Bacillus sp. 81-2 and B. amyloliquefaciens 34-12 exhibited inhibitory action against Staphylococcus aureus. DPPH assay showed % scavenging activities of extracts from Bacillus sp. 81-2; 13-3;77-3; B. vietnamensis 39-23; and B. amyloliquefaciens 34-12 as 11.54±1.17; 18.72±1.78; 15.75±0.52; 9.63±5.84; 13.91±0.09, respectively. Pigment extracts were further characterized using UV spectrophotometry, thin layer chromatography (TLC), and fourier transform infrared spectroscopy (FTIR). From spectrophotometric and TLC assay, pigment of all isolates showed similar properties to carotenoid. The FTIR analysis suggested that pink-orange pigment of Bacillus sp. 81-2 comprised lycopene, whereas yellow and yellow-orange pigment of Bacillus sp. 13-3; 77-3, B. amyloliquefaciens 34-12 , and B. vietnamensis 39-23 were xanthophylls. Based on color shade and bioactivities, Bacillus sp. 81-2 was selected for further evaluation including safety evaluation and pigment production. The optimal condition for pigment production was nutrient broth supplemented with 3 % NaCl under room temperature for 72 hours, yielded pigment 0.22 gram per gram of wet cell. Addition of carbon and nitrogen sources gave no impact on both bacterial growth and pigment production. Spectrometric profile of pigment extract showed higher peak of 481 nm than usual profile and its minimum inhibitory concentration (MIC) and/or minimum bactericidal concentration (MBC) against S. aureus were 100 mg/ml and 200 mg/ml, respectively. Addition of 200 mg/ml pigment generated well-incorporated pink-orange pigment in salad dressing matrix with potential expression of bioactivities. This study demonstrated a potential Bacillus pigment containing carotenoid group to be applied into food as colorant having multifunctional properties that could enhance both food quality and safety.