Indigenous Bacteria and Fungi Drive Traditional Kimoto Sake Fermentations

Abstract

Sake (Japanese rice wine) production is a complex, multistage process in which fermentation is performed by a succession of mixed fungi and bacteria. This study employed high-throughput rRNA marker gene sequencing, quantitative PCR, and terminal restriction fragment length polymorphism to characterize the bacterial and fungal communities of spontaneous sake production from koji to product as well as brewery equipment surfaces. Results demonstrate a dynamic microbial succession, with koji and early moto fermentations dominated by Bacillus, Staphylococcus, and Aspergillus flavus var. oryzae, succeeded by Lactobacillus spp. and Saccharomyces cerevisiae later in the fermentations. The microbiota driving these fermentations were also prevalent in the production environment, illustrating the reservoirs and routes for microbial contact in this traditional food fermentation. Interrogating the microbial consortia of production environments in parallel with food products is a valuable approach for understanding the complete ecology of food production systems and can be applied to any food system, leading to enlightened perspectives for process control and food safety. Humans have employed food fermentation since time immemorial to improve the safety, stability, flavor, nutrition, and value of their agricultural products. Traditionally, these processes have been driven by indigenous fungi and bacteria originating in raw materials, in autochthonous starter cultures, or in the processing environment itself (1), organisms that are responsible for these beneficial transformative processes as well as for product spoilage (2, 3). While most modern fermented foods are inoculated with defined starter cultures, traditional, uninoculated products remain celebrated for their historical and cultural significances (4), and indigenous microbial activity is often considered to increase the flavor complexity of these foods (5). The advent of high-throughput sequencing technologies has enhanced our ability to investigate the role of microbial communities in food systems with greater scale and sensitivity than ever possible (4), connecting the transmission of microbial communities in food production and food processing environments to their impact on food products.Sake is the traditional, national alcoholic beverage of Japan. Sake is produced from rice through the saccharification of starch by Aspergillus flavus var. oryzae and subsequent alcoholic fermentation by Saccharomyces cerevisiae. Sake brewing involves a serial propagation process, beginning with koji, a solid culture consisting of rice and A. flavus var. oryzae (6) (Fig. 1). Polished, steamed rice is mixed with the dried spores of A. flavus var. oryzae and incubated for approximately 2 days. Koji is then pitched with more steamed rice, water, and yeast into the moto (seed mash) tank, an open mashing vessel, wherein fermentation occurs for 10 to 25 days. Next, the moto is moved to a larger vessel and mixed with increasing amounts of water, rice, and koji in three additions to form ...

The genomics of microbial domestication in the fermented food environment

“…Successive molecular characterization of the evolving microbiome over the course of lambic fermentations have identified qualitatively distinct, semi-overlapping phases dominated at first by a variety of enterobacteria and non- Saccharomyces yeasts followed by increasing levels of Saccharomyces including S. cerevisiae and S. bayanus [74,75], then LAB, and finally by Brettanomyces bruxellensis [75,76]. Similar microbial communities and progressions have been documented in other types of spontaneously fermented beer [28,77] and in traditionally produced kimoto-style sake [78]. Again, while no overt inoculation of microbes occurs during the production of these beverages, there are nonetheless microbes resident to the brew house environment which take part in the fermentation process and show signatures of adaptation [28,77] [79].…”

Metagenomics insights into food fermentations

“…In other studies, the microbiota was related to raw material origin (Bokulich et al ., 2014a; Rizzello et al ., 2015) or quality (Dolci et al ., 2014; O'Sullivan et al ., 2015; Alessandria et al ., 2016), as well as to development of flavour‐impact compounds (De Pasquale et al ., 2014a, 2016; De Filippis et al ., 2016a). Moreover, food‐related environments were found to harbour a resident microbiota, beneficially involved in dairy (Bokulich and Mills, 2013a; Stellato et al ., 2015; Calasso et al ., 2016), alcoholic (Bokulich et al ., 2012a, 2014b) and sourdough (Minervini et al ., 2015) fermentations, although the presence of potential spoilers was also emphasized in some cases (Bokulich et al ., 2015a; Stellato et al ., 2015). …”

A new perspective on microbial landscapes within food production

“…Thus, high-throughput sequencing technologies are providing the greatest advance to this sector of research, where the increased sample throughput and detection sensitivity support analysis of larger and more complex studies. Amplicon sequencing approaches have been applied to study the microbial ecology of pearl millet fermentations [6], various Asian vegetable fermentations [7–9], seafood fermentations [10], Chinese liquor [11], cheese [12–14], sake [15] and other rice beers [16], olives [17], sourdough [18], kombucha [19], kefir and similar fermentations [20,21], wine [22,23], and spontaneously fermented American coolship ale fermentations [24,25]. What types of questions can be answered from describing the microbial succession in these fermented foods?…”

“…coolship beers) [24], and/or multiple stages with mixed consortia (e.g. rice wine fermentations) [15,16]. Describing the normal and abnormal microbial states at different stages of these fermentations is important to characterizing consistency, identifying biomarkers for product quality or spoilage, diagnosing problem fermentations, and learning to manipulate fermentation conditions for improved process control.…”

“…The aging-room surfaces of different cheesemaking plants harbor distinct, indigenous microbial communities that dominate the surfaces of the cheeses aged therein, forming the basis for a ‘house’ microbiome involved in regional cheesemaking [51]. We see a similar situation in a kimoto (uninoculated) sake brewery, where the bacteria and yeast conducting these fermentations are vectored on equipment surfaces [15]. In beer breweries, season, substrate, and human processes all shape the microbial communities of equipment surfaces [52 •• ].…”