Article pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Ethyl Carbamate Formation Regulated by Lactic Acid Bacteria and Nonconventional Yeasts in Solid-State Fermentation of Chinese Moutai-Flavor Liquor Hai Du, Zhewei Song, and Yan Xu* The Key Laboratory of Industrial Biotechnology of the Ministry of Education, State Key Laboratory of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, China S Supporting Information *
ABSTRACT: This study aimed to identify specific microorganisms related to the formation of precursors of EC (ethyl carbamate) in the solid-state fermentation of Chinese Moutai-flavor liquor. The EC content was significantly correlated with the urea content during the fermentation process (R2 = 0.772, P < 0.01). Differences in urea production and degradation were found at both species and functional gene levels by metatranscriptomic sequencing and culture-dependent analysis. Lactobacillus spp. could competitively degrade arginine through the arginine deiminase pathway with yeasts, and most Lactobacillus species were capable of degrading urea. Some dominant nonconventional yeasts, such as Pichia, Schizosaccharomyces, and Zygosaccharomyces species, were shown to produce low amounts of urea relative to Saccharomyces cerevisiae. Moreover, unusual urea degradation pathways (urea carboxylase, allophanate hydrolase, and ATP-independent urease) were identified. Our results indicate that EC precursor levels in the solid-state fermentation can be controlled using lactic acid bacteria and nonconventional yeasts. KEYWORDS: ethyl carbamate, Moutai-flavor liquor, solid-state fermentation, urea metabolism, microbiota
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products.12−14 However, research about the control of EC in Chinese liquor is still lacking. The knowledge gap remains between the complexity and the function of the microbial ecosystem. The formation of several EC precursors, such as urea, citrulline, and carbamoyl phosphate, has aided the development of biochemical approaches to generate EC using yeasts and lactic acid bacteria (LAB).15,16 Urea, a major cause of EC production, is mainly produced by yeasts through arginine metabolism.17,18 The arginine deiminase pathway in the malolactic fermentation of wine has been characterized in certain isolates, including Oenococcus oeni.19−21 The metabolism of arginine and the urea cycle are of oenological concern because they can result in the excretion of citrulline and urea, which can spontaneously react with ethanol to form EC.22 In this study, we employed amplicon and metatranscriptomic sequencing to explore nitrogen regulation in fermentation microbiota that produce and degrade EC precursors, particularly urea. Relationships were established between key microorganisms and the amounts of urea, which could provide important information for controlling the EC content in SSF processes.
INTRODUCTION Ethyl carbamate (EC; C2H5OCONH2) occurs naturally in fermented foods and beverages, and may have carcinogenic and mutagenic effects. EC has been classified as a group of 2A carcinogen (i.e., probably carcinogenic to humans) by the World Health Organization’s International Agency for Research on Cancer (IARC, 2010). EC is widely present in fermented foods and beverages such as bread, wine, cheese, soy sauce, and vinegar.1−4 The average excess cancer risk of an alcoholdrinking adult is twice that of an adult who does not drink.5 Accordingly, the amount and production mechanism of EC in fermented alcoholic beverages have attracted much attention.6 As traditional fermented alcoholic beverages are particularly important sources of EC intake, the health risks posed by this EC are of concern.7 Solid-state fermentation (SSF) is a commonly used technology for the production of traditional alcoholic beverages and can improve the texture and flavor of the product.8 Chinese Moutai-flavor liquor (53% alcohol by volume) production is a typical SSF process originating from ancient fermented beverages and driven by multistrain microbial communities.9 Exploring the presence and function of food microbiota during SSF processes has long been popular. The growth and metabolism of microbes are key factors influencing liquor fermentation.10 Some of the microbes decompose raw materials for the growth of microbiota and promote fermentation,8 while metabolites such as lactic acid can inhibit the growth of pathogenic and corrupting bacteria.11 A recent trend toward mixed-culture inoculations (including nonconventional yeasts [i.e., non-Saccharomyces cerevisiae]) has already proven profitable, producing improvements in the quality of a number of © XXXX American Chemical Society
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MATERIALS AND METHODS
Reagents and Materials. Urea, amino acid standards, EC, and d5EC were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.) with Received: Revised: Accepted: Published: A
October 31, 2017 December 11, 2017 December 13, 2017 December 13, 2017 DOI: 10.1021/acs.jafc.7b05034 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry their purity >99.8%. A stock solution of EC (1000 mg L−1) was prepared in ethanol and was diluted with 40% (v/v) ethanol solution before application. N-Methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA; Sigma-Aldrich) was used as the derivatization agent. All solvents used were of HPLC grade. Ultrapure water was obtained using a Milli-Q system (Millipore, Milford, MA, U.S.A.). Hydrochloric acid (37%, m/m), acetic acid, and sodium acetate were obtained as analytical grade reagents from Sinopharm Chemical Reagent Group Corporation (Shanghai, China). Acetonitrile, dichloromethane, diethyl ether, and ethanol were obtained from CNW (Germany). Sample Collection. Samples were taken from a distillery of the Moutai-flavor type in southwest China. Samples were collected from the same batch in two distinct phases of liquor production, namely heaping and fermentation. Heaping-phase samples were the grain mixture that had been heaped on the ground for 1, 2, or 3 days. Fermentation-phase samples were the grain mixture fermented in the anaerobic environment and taken at 0, 5, 10, 15, 20, 25, 30, and 32 days (the latter being the end of the fermentation). To obtain representative samples, fermented grains were collected at three different points from the top to the bottom of the pile and pool and then mixed thoroughly. About 500 g of sample were transferred to sterile bags, sealed and stored at −80 °C. DNA Extraction, PCR Amplification, and Sequencing. Genomic DNA was extracted using a Soil DNA Kit (Omega biotek; Norcross, GA). For high-throughput 16S rRNA gene paired-end sequencing, the V3−V4 regions of 16S rDNA genes were amplified using the universal primer set 338F (5′-GTACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GTGGACTACHVGGGTWTCTAAT3′).23 PCR products were purified using a Qiagen Gel Extraction Kit (Qiagen, Germany). The barcoded PCR amplicons were sequenced on a MiSeq Benchtop Sequencer for 300 bp paired-end sequencing (Illumina, San Diego, CA). Raw reads were filtered using Mothur software24 with removal of (i) sequences that had quality scores
