Novel method for predicting the risk of spoilage by lactic acid bacteria during the storage of Japanese sake

Lactobacillus acetotolerans and Fructilactobacillus fructivorans are responsible for the spoilage of Japanese sake, and sake brewers want to control these bacteria to prevent product-quality deterioration. Due to these bacteria's slow growth, a long time must pass before a product's spoilage can be identified by a culture-dependent method; a method enabling the rapid detection of spoilage is thus desired for maintaining sake's quality and reducing processing times. Herein, we developed a novel, simple method for determining the ori copy and ter copy numbers of both bacterial species by a single multiplex digital polymerase chain reaction (PCR). Our investigation about the ori and ter copy numbers of L. acetotolerans RIB9124 strain revealed that ( i ) the ter-region copy number was highly correlated with the colony-forming units, and ( ii ) the ori:ter ratio during log phase (mean: 1.36) was higher than that during non-log phase (mean: 1.16). The relationship between the ori:ter ratio and the bacterial doubling time was parabola-shaped, not linear. We constructed a model for estimating the two species' doubling times based on the ori:ter ratio and then created a generalized linear model with a probit link function using the ori:ter ratio, the doubling time predicted by the ori:ter ratio, and the ter copy number as parameters. The model was able to predict whether sake inoculated with either species would spoil within 4 months (spoilage-prediction error rate validated with 45 samples: 15.6 %). In light of these results, we suggest some criteria for judging the risk of Japanese sake spoiling within 4 months.