Journal of Fungi Review Making Traditional Japanese Distilled Liquor, Shochu and Awamori, and the Contribution of White and Black Koji Fungi Kei Hayashi 1,*, Yasuhiro Kajiwara 1, Taiki Futagami 2,3 , Masatoshi Goto 3,4 and Hideharu Takashita 1 1 Sanwa Research Institute, Sanwa Shurui Co., Ltd., Usa 879-0495, Japan; [email protected] (Y.K.); [email protected] (H.T.) 2 Education and Research Center for Fermentation Studies, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan; [email protected] 3 United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-0065, Japan; [email protected] 4 Department of Applied Biochemistry and Food Science, Faculty of Agriculture, Saga University, Saga 840-8502, Japan * Correspondence: [email protected]; Tel.: +81-978-33-3844 Abstract: The traditional Japanese single distilled liquor, which uses koji and yeast with designated ingredients, is called “honkaku shochu.” It is made using local agricultural products and has several types, including barley shochu, sweet potato shochu, rice shochu, and buckwheat shochu. In the case of honkaku shochu, black koji fungus (Aspergillus luchuensis) or white koji fungus (Aspergillus luchuensis mut. kawachii) is used to (1) saccharify the starch contained in the ingredients, (2) produce citric acid to prevent microbial spoilage, and (3) give the liquor its unique flavor. In order to make delicious shochu, when cultivating koji fungus during the shochu production process, we use a Citation: Hayashi, K.; Kajiwara, Y.; unique temperature control method to ensure that these three important elements, which greatly Futagami, T.; Goto, M.; Takashita, H. affect the taste of the produced liquor, are balanced without any excess or deficiency. This review Making Traditional Japanese Distilled describes in detail the production method of honkaku shochu, a distilled spirit unique to Japan Liquor, Shochu and Awamori, and and whose market is expected to expand worldwide, with special attention paid to the koji fungi the Contribution of White and Black cultivation step. Furthermore, we describe the history of the koji fungi used today in the production Koji Fungi. J. Fungi 2021, 7, 517. of shochu, and we provide a thorough explanation of the characteristics of each koji fungi. We also https://doi.org/10.3390/jof7070517 report the latest research progress on this topic. Academic Editors: Katsuhiko Kitamoto and Keywords: koji; shochu; honkaku shochu; awamori; Aspergillus luchuensis; Aspergillus luchuensis Yujiro Higuchi mut. Kawachii; Aspergillus awamori; multiple parallel fermentation; amylolytic enzymes system; citric acid synthesis; wide variety of flavors; genome editing technology Received: 29 May 2021 Accepted: 25 June 2021 Published: 28 June 2021 1. Introduction Publisher’s Note: MDPI stays neutral Traditional Japanese distilled liquors include shochu and awamori [1]. Shochu is di- with regard to jurisdictional claims in vided into two types, single distilled shochu and continuously distilled shochu, depending published maps and institutional affil- on the distillation method used under the Japanese Liquor Tax Law. Among single dis- iations. tilled liquors, those using koji and yeast with designated ingredients are specifically called “honkaku shochu”. Koji is a fermented grain, such as rice and barley, with filamentous fungi (called koji fungi). Koji produces saccharification enzymes, which convert the starch contained in the ingredients into mono- or disaccharides which can be further utilized by Copyright: © 2021 by the authors. yeast to produce ethanol [2]. Licensee MDPI, Basel, Switzerland. Originally, honkaku shochu was mainly enjoyed in the Kyushu region in the southern This article is an open access article part of Japan, but it began to spread throughout Japan around 1980; in recent years, it has distributed under the terms and also been enjoyed overseas. Honkaku shochu is made using local agricultural products as conditions of the Creative Commons fermented ingredients. There are various types of honkaku shochu, such as barley shochu, Attribution (CC BY) license (https:// sweet potato shochu, rice shochu, and Japanese buckwheat (soba) shochu. In addition to creativecommons.org/licenses/by/ alcohol, it contains an abundance of other characteristic flavor components. In contrast, 4.0/). J. Fungi 2021, 7, 517. https://doi.org/10.3390/jof7070517 https://www.mdpi.com/journal/jof J. Fungi 2021, 7, x FOR PEER REVIEW 2 of 10 J. Fungi 2021, 7, 517 2 of 10 shochu. In addition to alcohol, it contains an abundance of other characteristic flavor components.awamori is a In distilled contrast, liquor awamori that is has a distilled been enjoyed liquor that mainly has inbeen the enjoyed Okinawa mainly Prefecture in the Okinawasince the RyukyuPrefecture Kingdom since the era. Ryukyu The characteristic Kingdom era. of its The making characteristic method isof that its ricemakingkoji methodproduced is usingthat rice black kojikoji producedfungus is using used asblack an ingredient,koji fungus and is used fermentation as an ingredient, is carried and out fermentationin a single step is [carried3]. out in a single step [3]. In this review, wewe discussdiscuss thethe preparationpreparation ofof singlesingle distilleddistilled shochu,shochu, in which koji fungi are an important factor, and describedescribe the types and characteristics of koji fungi used in the making of shochu. 2. Making Making Method of Single Distilled Shochu The conventional firstfirst stepstep in in the the making making of of single single distilled distilled shochu, shochu, grain grain ingredients, ingredi- ents,such assuch rice as and rice barley, and barley, are inoculated are inoculated with koji withfungi, koji andfungi, it isand fermented it is fermented in a solid in statea solid to statemake tokoji make(Figure koji1 ).(Figure The main 1). The purpose main of purpose using koji of usingin the koji making in the of making liquor is of to liquor provide is anto provideenzyme sourcean enzyme that decomposessource that starch,decomposes protein, starch, and lipids, protein, as welland aslipids, to supply as well vitamins as to supplythat enhance vitamins the growththat enhance of yeast the in growth mash [ 4of]. ye Inast addition, in mash other [4]. characteristicsIn addition, other of koji charac-fungi teristicsare crucial of forkojithe fungi making are crucial of honkaku for the shochu making and of awamori. honkaku Currently, shochu and yellow awamori.koji fungus Cur- rently,is used yellow for making koji fungus sake, whereas is used many for making shochu sake, factories whereas use black many and shochu white factorieskoji, because use koji blackthey canand produce white koji large, because amounts they of can citric produce acid. Bylarge using amountswith of citric a high acid. content By using of citric koji acid, the pH of the mash can be kept low, and the growth of microorganisms other than the with a high content of citric acid, the pH of the mash can be kept low, and the growth of yeast involved in alcoholic fermentation can be suppressed [5]. As a result, a stable and microorganisms other than the yeast involved in alcoholic fermentation can be sup- good fermentation becomes possible even in the hot, humid, and harsh climate of Japanese pressed [5]. As a result, a stable and good fermentation becomes possible even in the hot, summers or in an open environment. humid, and harsh climate of Japanese summers or in an open environment. Figure 1. Making method for single distilled barley shochu. The second step is alcoholic fermentation.fermentation. In honkaku shochu, fermentation is is gen- erally divided into two stages [6]. [6]. Briefly, Briefly, shochu yeast and water are added to the koji produced inin thethe first first step step to to create create a starter a starter culture culture (seed (seed mash). mash). Once Once the yeast the hasyeast grown has grownsufficiently sufficiently in the starter in the starter culture, culture, the main the ingredients main ingredients are added are added to make to themake main the mash,main mash,and alcoholic and alcoholic fermentation fermentation takes place. takes During place. thisDuring period, this multipleperiod, multiple parallel fermentation parallel fer- mentationis performed, is performed, in which saccharification in which saccharific of the ingredientation of the by ingredient each enzyme by each derived enzyme from de- the rivedkoji fungus from the and koji fermentation fungus and by fermentation yeast occur simultaneously.by yeast occur simultaneously. In multiple sequentialsequential fermentation, fermentation, which which separates separates saccharification saccharification and and fermentation fermenta- tion(e.g., (e.g., beer beer and whiskey),and whiskey), the alcohol the alcohol content content at the at end the of end fermentation of fermentation is approximately is approxi- mately5–8%. In5–8%. multiple In multiple parallel parallel fermentation, fermentation, saccharification saccharification and fermentationand fermentation occur occur in a well-balanced manner, and the alcohol content at the end of fermentation can be as high as 15–20%. J. Fungi 2021, 7, 517 3 of 10 The third step is distillation. While whiskey is distilled twice or more, honkaku shochu is distilled once by a single distillation method (pot still) [7]. Because the alcohol content in the mash stage is high, a high concentration of alcohol can be obtained by distillation. A characteristic of this