Isolation and Identification of B. coagulans strains
The strains were subjected to polyphasic taxonomic analyses based on their phenotypic characteristics and phylogenetic analysis (Fig. 1). Strain ZC2-1 form white opaque circular colonies with a white dot in the center (Fig. 1a), and its cells are short rod shape of 0.4-0.8 µm wide by 2.5-4.0 long (Fig. 1e). Strain ZA-1 and ZC-9 both from light milky white small round colonies with moist surface and viscous texture (Fig. 1b and Fig. 1d). There cells are of rod shape of 0.5-1.5 µm wide by 5.0–8.3 µm long (Fig. 1f) and 0.4–0.8 µm wide by 1.8–3.2 µm long (Fig. 1h), respectively. The colony of ZB-1 is white colony with irregular edge, rough surface, and viscous texture (Fig. 1c), and its cells are slender rods with the size of 0.4-0.8 µm wide by 1.8–3.2 µm long (Fig. 1g).
16S rRNA sequences of four B. coagulans ZC2-1, ZA-1, ZB-1, ZC-9 (shown in Supplementary Material) were deposited in GenBank with the accession numbers MW195020, MW504830, MW504831, MW504832. B. coagulans ZC2-1 is numbered 22951 in CGMCC.
The 16S rRNA sequence of ZC2-1 exhibited 99.93% identity with a B. coagulans strain (MT604689.1). The 16S rRNA sequence of ZA-1 revealed 100% identity with a B. coagulans strain (MT611810.1). The 16S rRNA sequence of ZB-1 showed 100% identity with a B. coagulans strain (MT611733.1). The 16S rRNA sequence of ZC-9 has 99.93% identity with a B. coagulans strain (MT626077.1). ZC2-1, ZA-1, ZB-1, ZC-9 was located in B. coagulans clade (Fig.2), and their closest relative were B. coagulans strain KCCM203098 and B. coagulans strain E21.
Establishment of coculture system
The four B. coagulans strains ZC2-1, ZA-1, ZB-1, ZC-9 were co-fermented with C. butyricum DL-1, after 24 h culture, the C. butyricum spore concentration of four co-culture system were 5.5×105, 4.5×105, 4.8×105, 5.1×105 CFU/mL, respectively. The highest C. butyricum spore concentration was obtained in coculture system composed of B. coagulans ZC2-1 and C. butyricum DL-1. Therefore, this coculture system was studied further.
The growth curve and pH curve of C. butyricum DL-1 and B. coagulans ZC2-1
The lag period of C. butyricum DL-1 lasted only 4 h in the proliferation medium (Fig S1 in Supporting Information). Then the logarithmic phase began with the cell multiplying rapidly and pH dropping sharply. The stable period lasted from 12 h to 20 h, and fermentation process entered the decay period at 20 h with pH value increasing continuously.
B. coagulans ZC2-1 grew slowly in the proliferation medium in the lag period with pH decreasing slowly (Fig S2 in Supporting Information). From the 8th hour, the bacteria rapidly propagated into the logarithmic phase and reached the plateau at 16 h.
There is a markedly negative correlation between the changing trend of cell concentration and pH value. In the stable and decay period, bacterial growth was inhibited by low pH, low nutrients concentration and high harmful metabolites concentration caused by bacterial growth. The increasing pH value in the decay period may be related to the autolysis of the bacteria cells. At late logarithmic phase, the bacterial cell concentration reached the highest, and the cells exhibited the highest viability and fertility in the same time. Therefore, the culture broth of C. butyricum DL-1 and B. coagulans ZC2-1 harvested after cultured for 16 h was used as inoculum for co-fermentation process.
Optimization of mixed fermentation medium composition
It was found that the concentration of B. coagulans cells in co-fermentation broth is far inferior to the culture result in purebred fermentation. Additionally, B. coagulans spore yield in co-fermentation process is almost negligible. Therefore, the concentration of C. butyricum viable bacteria and its spore yield was used as medium optimization criterion in this study.
Effect of carbon source types on the viable counts and spore yield of C. butyricum DL-1
When bran is used as carbon source, the number of viable bacteria and spores of C. butyricum in the culture broth reached the highest (Fig. 3). Besides provision carbon source, bran contains trace metal ions, various amino acids, vitamins and other grow factors which facilitate bacterial growth (Ritthibut et al. 2020). Bran served as much better fermentation performance than other carbon sources (Fig. 3), so it was used as the only carbon source in further study.
Effect of nitrogen source sorts on the viable counts and spore yield of C. butyricum
When corn steep powder was used as nitrogen source, both the number of viable counts and spores of C. butyricum were the highest (Fig. S3), reaching 5.6×107 CFU/mL and 3.5×107 CFU/mL respectively. In order to further increase the viable counts and spore yield of C. butyricum DL-1, composite nitrogen source with more comprehensive nutrition was studied. The results are shown in Table 1:
The concentration of viable bacteria and spores of C. butyricum DL-1 of Group 2 was the highest (Table 1), reaching 7.1×107 CFU/mL and 5.8×107 CFU/mL respectively. The Group 1̍s spore rate was the highest, but the number of viable counts and spore was lower than that of Group 2. With rich protein, amino acids, vitamins, minerals and trace grow factors, corn steep powder can provide comprehensive nutrient for the growth of microbes (Zeng et al. 2018). Peptone contains vitamins and other growth factors (Setiari et al. 2016). They are both good choices for bacterial nitrogen source.
Effect of the carbon and nitrogen source concentration on the viable counts and spore yield of C. butyricum DL-1.
A L9 (33) orthogonal table (Table 2) was designed to optimize the concentration of carbon source and nitrogen source,bran(A), peptone(B) and corn steep powder(C) concentration were set as factors in this orthogonal test. The results were analyzed as shown in Table 3.
According to the range analysis results (Table 3), the three factors had the similar influence on the viable counts and spore yield of C. butyricum DL-1. The order of importance was corn steep powder concentration>peptone concentration >bran concentration. The optimum contents of carbon source and nitrogen source were determined as 10 g/L bran, 15 g/L peptone, and 15 g/L corn steep powder. The verification results conducted under the optimal condition combination were as follows: the viable counts and spore yield of C. butyricum reached 8.8×107 and 7.6×107 CFU/mL, respectively. The results were better than all those shown in the orthogonal table, further verified the conclusion drawn by the orthogonal experiment.
Effect of inorganic salts on the viable counts and spore yield of C. butyricum DL-1
Inorganic salts play an important role in the growth of microorganisms, since many metal ions in them serve as cofactors of metabolic enzymes. It could be seen from Fig. 4 that K2HPO4 was the top factor in promoting C. butyricum’s growth and sporulation. Sodium acetate trihydrate, MnSO4, and MgSO4 also exhibited marked enhancement effect, so the combination of K2HPO4 and these the three inorganic salts was further studied. The results shown that the optimal inorganic salts combination for coculture of C. butyricum DL-1 and B. coagulans ZC2-1 was K2HPO4 and MnSO4 (Table S1 in Supporting Information), with the viable counts and spores rate reaching 1.04×108 CFU/mL and 91.3%, respectively. The concentration of K2HPO4 and MnSO4 was optimized by a L9(32) orthogonal experiment (Table S2 and Table S3 in Supporting Information). With K2HPO4 (A) and MnSO4 (B) content as factors.
According to the range analysis results (Table S3 in Supporting Information), K2HPO4 is the most important influence factor for the viable bacteria and spore rate of C. butyricum DL-1. The optimum inorganic salt combination was 1 g/L K2HPO4 and 0.5 g/L MnSO4. Under this condition, the viable counts and spore rate of C. butyricum DL-1 reached 1.3×108 CFU/mL and 92.3%, respectively.
Fermentation result in the optimal medium
Different from the growth curves in proliferation medium, B. coagulans ZC2-1 started growth earlier than C. butyricum DL-1. At 0-12 h, B. coagulans ZC2-1 used the nutrients in medium to multiply preferentially (Fig. 5), and consumed the dissolved oxygen in the medium in the same time, providing an anaerobic environment for C. butyricum. Consequently, C. butyricum DL-1 entered the logarithmic phase at 12 h after a long lag phase and began to spore at 20 h. At 36 h, the viable counts and spores yield of C. butyricum DL-1 both reached the peak value of 1.5×108 CFU/mL and 1.4×108 CFU/mL respectively. Therefore, 36 h was the high time to stop the mixed fermentation process.