Development of a Novel Restrictive Medium for Monascus Enrichment From Hongqu Based on the Synergistic Stress of Lactic Acid and Ethanol

Abstract

Hongqu is a famous fermented food produced by <i>Monascus</i> and has been used as food coloring, wine starters and food additives for thousands of years in China. Excellent <i>Monascus</i> strain is an important prerequisite for producing high-quality Hongqu. However, the isolation of <i>Monascus</i> pure culture from Hongqu samples is time-consuming and laborious because it is easily interfered by other microorganisms (especially filamentous fungi). Therefore, the development of restrictive medium for <i>Monascus</i> enrichment from Hongqu is of great significance for the preparation and screening of excellent <i>Monascus</i> strains. Results of this study showed that <i>Monascus</i> has good tolerance to lactic acid and ethanol. Under the conditions of tolerance limits [7.5% lactic acid (v/v) and 12.0% ethanol (v/v)], <i>Monascus</i> could not grow but it still retained the vitality of spore germination, and the spore activity gradually decreased with the increasing concentrations of lactic acid and ethanol. More interestingly, the addition of lactic acid and ethanol significantly changed the microbial community structure in rice milk inoculated with Hongqu. After response surface optimization, <i>Monascus</i> could be successfully enriched without the interference of other microorganisms when 3.98% (v/v) lactic acid and 6.24% (v/v) ethanol were added to rice milk simultaneously. The optimal enrichment duration of <i>Monascus</i> by the restrictive medium based on the synergistic stress of lactic acid and ethanol is 8∼24 h. The synergistic stress of lactic acid and ethanol had no obvious effects on the accumulation of major metabolites in the progeny of <i>Monascus</i>, and was suitable for the enrichment of <i>Monascus</i> from different types of Hongqu. Finally, the possible mechanisms on the tolerance of <i>Monascus</i> to the synergistic stress of lactic acid and ethanol were preliminarily studied. Under the synergistic stress of lactic acid and ethanol, the cell membrane of <i>Monascus</i> defends against lactic acid and ethanol into cells to some extent, and the superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities of <i>Monascus</i> were higher than those of other fungi, which significantly reduced the degree of lipid peroxidation of cell membrane, while secreting more amylase to make reducing sugars to provide the cells with enough energy to resist environmental stress. This work has great application value for the construction of <i>Monascus</i> strain library and the better development of its germplasm resources.