Isolation and identification of histamine-producing Enterobacteriaceae from Qu fermentation starter for Chinese rice wine brewing

Abstract

Histamine (HIS) producers in fermented wines are generally believed to be lactic acid bacteria (LAB), and other microorganisms have received little or no attention. In this work, HIS-producing bacteria were isolated from Qu fermentation starter for Chinese rice wine brewing by decarboxylase medium, and their identity was confirmed by RP-HPLC and PCR. Surprisingly, the histidine decarboxylase gene (hdc) was present in only 2 out of 26 isolates. All 26 isolates were genotyped using the randomly amplified polymorphic DNA (RAPD)-PCR assay, which revealed the presence of 21 biotypes. Single type isolates were identified via 16S rRNA sequence analysis, in some cases coupled with partial sequencing of the rpoB or dnaJ gene. All isolates belonged to the Enterobacteriaceae, and included Enterobacter asburiae, Enterobacter cloacae, Enterobacter hormaechei, Citrobacter amalonaticus and Cronobacter sakazakii. All these strains were capable of producing >3.5 mg/L of HIS in TS medium without ethanol, but did not grow in TS medium with 8% ethanol. Small-scale Chinese rice wine fermentation revealed that HIS contents exhibited the same trend as the LAB and ethanol no matter what kinds of Qu were used. However, in the early stages of fermentation (from day 2 to day 4), the HIS contents had a stronger correlation with Enterobacteriaceae (0.943) than with LAB (0.369) when the Qu fermented samples are analyzed as a whole. Moreover, the lowest HIS content was measured in Xiao Qu (Q) fermented sample at the end of fermentation, which suggests that the formation of HIS in the early stages of fermentation has a decisive effect on HIS content in the final product. Our results demonstrate that Enterobacteriaceae from Qu are an important cause for HIS formation in Chinese rice wine. Consequently, selecting Qu with a low content of Enterobacteriaceae contaminants and inhibiting the growth of Enterobacteriaceae in the early stages of fermentation are useful approaches for preventing excessive amounts of HIS formation in Chinese rice wine brewing.