Starter culture selection for making Chinese sesame-flavored liquor based on microbial metabolic activity in mixed-culture fermentation

Abstract

Selection of a starter culture with excellent viability and metabolic activity is important for inoculated fermentation of traditional food. To obtain a suitable starter culture for making Chinese sesame-flavored liquor, the yeast and bacterium community structures were investigated during spontaneous and solid-state fermentations of this type of liquor. Five dominant species in spontaneous fermentation were identified: Saccharomyces cerevisiae, Pichia membranaefaciens, Issatchenkia orientalis, Bacillus licheniformis, and Bacillus amyloliquefaciens. The metabolic activity of each species in mixed and inoculated fermentations of liquor was investigated in 14 different cocultures that used different combinations of these species. The relationships between the microbial species and volatile metabolites were analyzed by partial least-squares (PLS) regression analysis. We found that S. cerevisiae was positively correlated to nonanal, and B. licheniformis was positively associated with 2,3-butanediol, isobutyric acid, guaiacol, and 4-vinyl guaiacol, while I. orientalis was positively correlated to butyric acid, isovaleric acid, hexanoic acid, and 2,3-butanediol. These three species are excellent flavor producers for Chinese liquor. Although P. membranaefaciens and B. amyloliquefaciens were not efficient flavor producers, the addition of them alleviated competition among the other three species and altered their growth rates and flavor production. As a result, the coculture of all five dominant species produced the largest amount of flavor compounds. The result indicates that flavor producers and microbial interaction regulators are important for inoculated fermentation of Chinese sesame-flavored liquor.

FIG 1

Ratio of dominant species in liquor fermentation. (A) Ratio of B. licheniformis to the total bacterial population; (B) ratio of B. amyloliquefaciens to the total bacterial population; (C) ratio of S. cerevisiae to the total yeast population; (D) ratio of I. orientalis to the total yeast population; (E) ratio of P. membranaefaciens to the total yeast population. The total bacterial and yeast populations were obtained from the counts of all bacterial and yeast species. Yeast species were not detected after 15 days in liquor fermentation.

FIG 2

Populations of single and double combinations of five dominant species at the end of different mixed-culture fermentations. The initial cell density of each species was adjusted to 1 × 10⁶ CFU/g. (A) B. licheniformis; (B) B. amyloliquefaciens; (C) S. cerevisiae; (D) I. orientalis; (E) P. membranaefaciens. Bl, B. licheniformis; Ba, B. amyloliquefaciens; Sc, S. cerevisiae; Io, I. orientalis; Pm, P. membranaefaciens.

FIG 3

Populations of five dominant species at the end of different mixed-culture fermentations. The initial cell density of each species was adjusted to 1 × 10⁶ CFU/g. (A) B. licheniformis; (B) B. amyloliquefaciens; (C) S. cerevisiae; (D) I. orientalis; (E) P. membranaefaciens. Bl, B. licheniformis; Ba, B. amyloliquefaciens; Sc, S. cerevisiae; Io, I. orientalis; Pm, P. membranaefaciens. Numbers in parentheses and below the x axis refer to trial numbers.

FIG 4

Differential profiling of different mixed-culture fermentations. (A) Hierarchical clustering by using ANOVA test samples of volatile compounds. The tree represents 42 mixed fermentations of the five selected strains after 30 days of fermentation at 30°C, based on the ANOVA-filtered volatile compounds detected by GC-MS. (B) Pair plot showing the differences in levels of volatile compounds detected in different mixed fermentations. The compounds displayed are the flavor compounds most important in discriminating sample types by PLS. Names of compounds in the squares on the diagonal indicate the compound displayed on the axes in the rows or columns of graphs intersecting with that square. The level increases from left to right and from bottom to top in the squares. The pair plot visualizes which compounds allow discrimination of different culture groups. The pair plot showed, for instance, that the purple solid circle (trial 1) was in the top of the squares in the row and in the right of the squares in the phenethyl butyrate column (as shown in the red dotted box). This indicates that a high concentration of phenethyl butyrate was highly discriminative for the combination B. licheniformis-B. amyloliquefaciens-S. cerevisiae-I. orientalis-P. membranaefaciens (trial 1), whereas the black solid diamond (trial 7) was at the bottom of the squares in the row and in the left of the squares in the guaiacol column (as shown in the green dotted box). This indicates that a low concentration of guaiacol was highly discriminative for the mixed culture of S. cerevisiae, I. orientalis, and P. membranaefaciens (trial 7). Bl, B. licheniformis; Ba, B. amyloliquefaciens; Sc, S. cerevisiae; Io, I. orientalis; Pm, P. membranaefaciens.