Specificity of Amino Acid Profiles Produced in Soybean Fermentations by Three Bacillus spp

Abstract

We compared the salt tolerance and proteolytic activity of 120 strains of each of Bacillus subtilis, Bacillus velezensis, and Bacillus licheniformis. Most B. subtilis strains exhibited growth in 12% (w/v) NaCl and showed proteolytic activity in 10% or 11% NaCl. The majority of B. velezensis strains grew in 14% NaCl and showed proteolytic activity in 12% or 13% NaCl. Most B. licheniformis strains grew in 14% NaCl and exhibited proteolytic activity in 5%-7% NaCl. We selected nine representative strains of each species based on their proteolytic activities and analyzed the free amino acid (FAA) profiles produced by culture of the bacteria on soybean. Statistical analyses of the 22 FAAs quantified in the cultures revealed clustering of FAA production profiles at the species level. The FAA production profiles of B. subtilis and B. velezensis were similar, and both differed from that of B. licheniformis. These trends persisted in cultures containing 7% NaCl. These results suggest that FAA production profiles are characteristic of each Bacillus species. Specifically, in soybean cultures compared with uninoculated soybeans, B. subtilis increased the amounts of leucine and phenylalanine; B. velezensis increased the amounts of leucine, phenylalanine, and tyrosine; and B. licheniformis increased the amounts of alanine, glutamic acid, tyrosine, and ornithine, and dramatically decreased the amount of arginine. The proteolytic activity of B. velezensis strains correlated with the quantity of FAAs in their soybean cultures. Considering its salt tolerance and proteolytic activity, B. velezensis showed high potential for contributing to the ripening of high-salt fermented soybean foods. Our results regarding the specific production of amino acids at the species level and correlations between proteolytic activities and produced amino acid quantities will facilitate the determination and selection of target strains for functional Bacillus-fermented foods.

Keywords: Bacillus licheniformis; Bacillus subtilis; Bacillus velezensis; amino acid profile; proteolytic activity; salt tolerance.

Fig. 1. Growth and proteolytic activities of nine representative strains of each of Bacillus subtilis (A) B. velezensis (B) and B. licheniformis (C) on tryptic soy-agar plates containing 2% skim milk.

B. subtilis strain abbreviations: S1, CH3-25; S2, F4241; S3, F1387; S4, SRCM103637; S5, F1237-2; S6, D12-5; S7, CHKJ 1253045; S8, F1323-1; and S9, PJ-7. B. velezensis strain abbreviations: V1, F2204; V2, GWHC B6; V3, MJ5-41; V4, SBK B3; V5, HCD2; V6, F1174-2; V7, Ms005; V8, 7BDL15; and V9, Rs501. B. licheniformis strain abbreviations: L1, SRCM100040; L2, F4112; L3, F3099-1; L4, 7DA21; L5, 7DA6; L6, 14DA18; L7, 8MS01; L8, 8MI08; and L9, SRCM100165.

Fig. 2. Principal component analysis (PCA) plots (A, B) and heatmaps (C, D) of the free amino acid (FAA) profiles of soybean cultures (A, C) and soybean cultures supplemented with 7% NaCl (B, D).

B. subtilis strain abbreviations: 1 and S1, CH3-25; 2 and S2, F4241; 3 and S3, F1387; 4 and S4, SRCM103637; 5 and S5, F1237-2; 6 and S6, D12-5; 7 and S7, CHKJ 1253045; 8 and S8, F1323-1; and 9 and S9, PJ-7. B. velezensis strain abbreviations: 1 and V1, F2204; 2 and V2, GWHC B6; 3 and V3, MJ5-41; 4 and V4, SBK B3; 5 and V5, HCD2; 6 and V6, F1174-2; 7 and V7, Ms005; 8 and V8, 7BDL15; and 9 and V9, Rs501. B. licheniformis strain abbreviations: 1 and L1, SRCM100040; 2 and L2, F4112; 3 and L3, F3099-1; 4 and L4, 7DA21; 5 and L5, 7DA6; 6 and L6, 14DA18; 7 and L7, 8MS01; 8 and L8, 8MI08; and 9 and L9, SRCM100165.

Fig. 3. PCA results for the FAA profiles from soybean cultures of B. subtilis (A) and B. velezensis (B) analyzed at the species level.

B. subtilis strain abbreviations: 1, CH3-25; 2, F4241; 3, F1387; 4, SRCM103637; 5, F1237-2; 6, D12-5; 7, CHKJ 1253045; 8, F1323-1; and 9, PJ-7. B. velezensis strain abbreviations: 1, F2204; 2, GWHC B6; 3, MJ5-41; 4, SBK B3; 5, HCD2; 6, F1174-2; 7, Ms005; 8, 7BDL15; and 9, Rs501. The analysis results for sterilized soybean samples were used as the control. Semi-quantitative determination of proteolytic activity used the proteolytic index (PI) value and Bacillus strains were classified as “high,” “medium,” and “low,” based on their PI values.