Daqu- A traditional fermentation starter in China: microbial ecology and functionality Xiao-Wei Zheng Thesis committee Promotors Prof. Dr M.H. Zwietering Professor of Food Microbiology Wageningen University Prof. Dr E.J. Smid Personal chair at the Food Microbiology Laboratory Wageningen University Co-promotor Dr M.J.R. Nout Associate professor, Food Microbiology Laboratory Wageningen University Other members Prof. Dr H. Smidt, Wageningen University Prof. Dr J. Schnürer, Swedish University of Agricultural Sciences, Uppsala, Sweden Dr J. Dijksterhuis, CBS-KNAW Fungal Biodiversity Centre, Utrecht Prof. Dr B.J. Zwaan, Wageningen University This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). Daqu- A traditional fermentation starter in China: microbial ecology and functionality Xiao-Wei Zheng Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Thursday 21 May 2015 at 11 a.m. in the Aula. Xiao-Wei Zheng Daqu - A traditional fermentation starter in China: microbial ecology and functionality, 188 pages. PhD thesis, Wageningen University, Wageningen, NL (2015) With references, with summaries in English, Dutch and Chinese ISBN 978-94-6257-280-5 天道酬勤 地道酬善 人道酬诚 业道酬精 Table of Contents Abstract Chapter 1 General introduction and thesis outline 1 Chapter 2 Review: Daqu - A traditional Chinese fermentation starter 13 Chapter 3 Characterization of the microbial community in different types of Daqu 35 Chapter 4 Distribution of microbiota in a Chinese fermentation starter (Fen-Daqu) - comparison of inner and outer Daqu 55 Chapter 5 Microbiota dynamics related to environmental conditions during the fermentative production of Fen-Daqu 73 Chapter 6 Characterization of bacteria and yeasts isolated from traditional fermentation starter (Fen-Daqu) through a 1H NMR-based metabolomics approach 93 Chapter 7 General discussions and future perspectives 121 List of references 137 Summary 155 Samenvatting 159 概要 163 Acknowledgements 167 List of publications 171 Curriculum vitae 173 Overview of completed training activities 175 Abstract Fermented products have high nutritional value and constitute an important part of the Chinese dietary profile; they are also gaining popularity throughout the world. Daqu is a traditional natural fermentation starter culture that has a significant impact on the quality and flavour of Chinese liquor and vinegar. A review of the literature was conducted focusing on the classification, composition, and manufacture of Daqu. The review provided a preliminary understanding of the link between the fermentation process and the characteristics of the final Daqu product. Then the occurrence, levels, and diversity of microorganisms were studied in different types of Daqu produced by various fermentation processes. The results showed that Bacillus licheniformis and Saccharomycopsis fibuligera were present in all the tested samples of Daqu. Regional comparisons showed Staphylococcus gallinarum and Staphylococcus saprophyticus in southern Daqu. The fungi Sm. fibuligera and Lichtheimia ramosa were found in low/medium-temperature Daqu and Thermomyces lanuginosus occurred in high- temperature Daqu. In order to study the functionality of Daqu and the contribution of the predominant microorganisms to alcoholic fermentation, the mesophilic and thermophilic bacteria and spores, Enterobacteriaceae, lactic acid bacteria, yeasts, and moulds present in the core and outer portions of Fen-Daqu were isolated. The isolates were identified by culture- dependent sequencing of rRNA genes (16S rRNA for bacteria; 18S rRNA, 26S rRNA, and ITS rRNA for fungi). A succession of fungi, lactic acid, and Bacillus spp. was associated with prevailing acidity, moisture content, and temperature during Daqu fermentation. The predominant species in fermentation were B. licheniformis, Pediococcus pentosaceus, Lactobacillus plantarum, Pichia kudriavzevii, Wickerhamomyces anomalus, Sacchromyces cerevisiae, and Sm. fibuligera. One strain of the each of the above-mentioned predominant species, with the highest starch degrading ability and alcohol tolerance, was selected and used in different combinations to perform alcoholic fermentation. Metabolite composition differed significantly between various fermentation trials. S. cerevisiae provided superior ethanol production. Sm. fibuligera and B. licheniformis provided the amylolytic activity that converted starch and polysaccharides into fermentable sugars. Finally, W. anomalus was found to be an important contributor to formation of the liquor aroma. Understanding the microbial diversity and functional activity, as well as the production dynamics and safety of Daqu will enable commercial producers to improve and/or scale-up traditional processes and enhance product quality and safety, thus facilitating entry into international markets. Chapter 1 General introduction and thesis outline Chapter 1 Chinese fermented foods have received increased attention owing to their specific flavours, nutritional value, and health benefits, and are a major part of the food production system in China. Alcoholic beverages including beer, Chinese liquor, and wine are important indigenous fermented products. In particular, Chinese liquor is an economically vital cultural commodity in China that is enjoyed by consumers at various social occasions. The liquor is typically generated by natural solid-state fermentation (SSF), and the product quality depends primarily on the skills of process operators; in general, the latter have little knowledge of microbiological and biochemical events occurring during the production process. However, non-standardised process parameters often lead to products with inconsistent quality. Several industries have tried to automate their production technology or replace some of the indigenous ingredients, but these attempts have resulted in the loss of unique product flavours due to circumvention of traditional methods. A detailed investigation of the microbiological and biochemical processes involved in the production of traditional fermented foods is therefore necessary for successful commercial-scale production. 1.1 Background Solid-State Fermentation (SSF) SSF is a process in which microbial cultures are grown on a solid matrix in the absence of a liquid (aqueous) phase (Barrios-González, 2012). This method has gained an increase in scientific and industrial attention in the past 20 years as a cost-saving measure for the efficient utilisation of agricultural products and waste (Barrios-González, 2012). Several fermented foods and beverages such as soy sauce, men, meju, and Chinese liquor are traditionally produced by SSF (Kim et al., 2011b; Tanaka et al., 2012; Wang et al., 2008b). SSF can also be applied for the production of enzymes that hydrolyse macromolecules and facilitate fermentation by bacteria and fungi (Singhania et al., 2009). In addition, SSF can provide high yields and can improve the functionalities of a variety of biomolecules including flavours, colourants, preservatives, and sweeteners, which could add value to the fermented products (Couto et al., 2006). SSF-associated indigenous microbiota include different lactic acid bacteria (LAB), Bacillus spp., yeast, and fungi 2 General introduction and thesis outline (Nout, 2009). These microorganisms convert substrates into fermentation end-products and also synthesise a variety of aroma compounds. For example, 2,5-dimethylpyrazine and tetramethylpyrazine are important flavour enhancers that are generated by Bacillus natto and Bacillus subtilis, respectively (Couto et al., 2006). A downside of SSF however, is that microbial growth and community composition are difficult to control; nutrient diffusion, enzyme stability, metabolic activities, and aerobic processes are influenced by various parameters including moisture content, temperature, pH, particle size, aeration, and agitation (Krishna, 2005). A better understanding of the microbiological and biochemical mechanisms underlying SSF would enable better control of the process and assure the quality of the final product. Production of Chinese liquor (Baijiu) Chinese liquor (known as Baijiu in Chinese) is a potent, distilled spirit containing 40- 65% alcohol by volume. The annual production of Chinese liquor has steadily increased in recent years and currently exceeds ten million metric tons annually (Anonymous, 2014). Hundreds of different types of Chinese liquor are produced by various processes in different regions of China, and can be previously classified as strong, light, sauce and miscellaneous, according to their flavour (Shen, 2005), nowadays more flavours such as rice, sesame, feng, chi, fuyu, yao, teyi, laobaigan appear. Representatives of six major Chinese liquor types are shown in Figure 1.1. Traditionally, Baijiu contained average ethanol levels of 50-65% v/v, but most products now contain around 40% v/v through the addition of water and adjustment of flavours (Fan et al., 2006). Techniques used to produce Chinese liquor have a long history and have been passed on through many generations. Chinese liquor is typically produced from grains such as sorghum, wheat, rice, glutinous rice, and maize by a complex SSF process, which comprises (i) material preparation, (ii) grinding