8 April 2013, 32( 增刊 ): Mycosystema ISSN CN /Q 2013 IMCAS, all rights reserved.

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1 8 April 2013, 32( 增刊 ): Mycosystema ISSN CN /Q 2013 IMCAS, all rights reserved. Filamentous mycoflora in a Chinese spirit Jiuqu ZHANG Xiang-Min 1 WANG You-Zhi 2 GUO Fang 2 HE Wen-Hua 2 ZHOU Yu-Guang 2* 1 Department of Plant Sciences, University of Saskatchewan, Saskatoon, Canada 2 Institute of Microbiology, Chinese Academy of Sciences, Beijing , China Abstract: Filamentous mycoflora in a Chinese spirit Jiuqu was investigated by using six different culture media and incubating at 25, 32, 42 and 50, respectively. A total of 886 fungal strains were isolated. Forty-five taxa of 20 genera were identified, including 10 Zygomycetes, 7 ascomycetes, and 28 anamorphic fungi. The main components of the functional fermenting fungi were the thermophilic and thermotolerant species, such as Paecilomyces variotii, Absidia corymbifera, Thermomyces lanuginosus, Rhizomucor pusillus, Chrysosporium sp. and Monascus spp. The characters of some important species and their potential roles in the process of fermentation and/or their relevant properties were discussed. Key words: fermentation, Paecilomyces variotii, Absidia corymbifera, Thermomyces lanuginosus, Rhizomucor pusillus, Chrysosporium sp., Monascus spp. 一中国白酒酒曲中的丝状真菌菌群 张向民 1 王有智 2 郭芳 2 何文华 2 周宇光 2* 1 萨斯卡切温大学植物学系萨斯卡通加拿大 2 中国科学院微生物研究所北京 摘要 : 采用 6 种不同的培养基, 分别于 以及 50 培养分离从而对某一中国白酒酒曲中的丝状真菌菌群进行研究 从酒曲中共分离得到 886 株丝状真菌, 分属于接合菌, 子囊菌和无性型真菌的 20 属,45 种 其中最为丰富的是无性型真菌 (28 种 ), 其次是接合菌 (10 种 ) 和子囊菌 (7 种 ) 对发酵起主要作用的为那些嗜热和耐热的种属, 包括 : 宛氏拟青霉, 伞枝梨头霉, 梳棉状嗜热丝孢菌, 微小根毛霉, 金孢霉属一种和红曲属的几个种 文中还对一些在发酵中起重要作用的丝状真菌的特性进行了探讨 关键词 : 发酵, 宛氏拟青霉, 伞枝梨头霉, 梳棉状嗜热丝孢菌, 微小根毛霉, 金孢霉属, 红曲属 * Corresponding author. Zhouyg@im.ac.cn Received: , accepted:

2 ZHANG Xiang-Min et al. / Filamentous mycoflora in a Chinese spirit Jiuqu 225 INTRODUCTION Chinese spirits (alcoholic beverages) have enjoyed a history of over 5,000 years, and have developed specific and peculiar flavors of their own. One characteristic feature in the manufacture of Chinese alcoholic beverages (rice wines, Chinese spirits, etc.) is the use of Jiuqus (fermentation starters). Jiuqu is molded cereals which are source of enzymes necessary for the breakdown of carbohydrates and proteins in grains and also a portion of fermentation substrates (Xu & Bao 2000). Since Shang Dynasty (16th century B.C. 11th century B.C.), most of the alcoholic beverages have been brewed by using Jiuqu in China. China is the only country in the world that makes distilled spirits from fermented rice and other grains. The raw material for most spirit production is sorghum. After being cooked, sorghum is mixed with Jiuqu, and then is put into fermentation pits for about 1 month. Then the fermented grain is distilled into spirit. The distilled grain is returned to the fermentation pits after being mixed with more Jiuqu because it still contains some residual starches. This process is repeated for several times. The spirits collected in every batch is stored separately. The Jiuqu has a significant impact on the aroma and flavor of spirits. Jiuqu is roughly equivalent to the English word malt and yeast, but is somewhat wider in scope, a limited number of studies have indicated that it also includes filamentous fungi (generally known as mold) as well as bacteria, and connotes the starter or inoculum used to initiate various kinds of fermentations (Huang 2000). Jiuqu is traditionally prepared by natural inoculation and growth of molds, bacteria, and yeasts on the grains. Actually, molds, yeasts and bacteria in the environment and on equipment serve as inoculums for preparing Jiuqu. Moistened wheat flour or rice flour are suitable substrates for their growth. The principle of Jiuqu making is summarized in Fig. 1. Fig. 1 Flow scheme of Jiuqu making process. During the growth of these microorganisms, the temperature of Jiuqu goes up to 50 from normal environmental temperature of ca. 25, a lot of hydrolyzing enzymes are excreted and immobilized on the grains which are ready to use as sources of amylase for conversion of starch to sugar, followed by conversion of sugar to ethanol mainly by yeasts. The aim of the study was to understand the microflora of the Jiuqu, to explore what kind of molds, bacteria, and yeasts are (how many species and what species are they?) and their relative quantities in the Jiuqu. This paper focused on the filamentous fungi isolated from the Jiuqu the species isolated at different temperatures, on different media, and the frequency of each species isolated. 1 MATERIALS AND METHODS 1.1 Media for isolation Six different media were used for isolation: PDA (potato dextrose agar); NPDA (PDA+10% glucose); CZA (Czapek agar); NCZA (CZ+20% sucrose); MEA (malt extract agar); and YEPD (10.0g peptone, 5.0g yeast extract, 10.0g glucose, 20.0g agar per 1L of distilled water; ph 8.5). About 2mg/L each of streptomycin, tetracycline and neomycin was added in all the agars. 1.2 Dilution plating Ten grams Jiuqu sample was added to 100mL

3 226 ISSN CN /Q Mycosystema April 8, 2013 Vol.32 (Suppl.) sterile water with glass beads in a 250mL flask, the sample was distributed by shaking with hands. Serial dilutions (10-2, 10-3 ) were carried out by pipette 10mL of the solution to the other 90mL sterile water in a 250mL flask. 0.1mL of each dilution was spread on the agar surface. The plates were incubated at 25, 32, 42 and 50. Three replications were set for each temperature on different media. Colonies appeared were examined after 1 3 days and transferred to adequate agar slants (PDA, MEA), followed by checking the complete isolation. Plates were kept for at least 2 weeks, during which all cultures were examined at frequent intervals (at least three times weekly) and appropriate subcultures were made. 1.3 Taxonomic studies The taxonomic status of the fungi isolated were determined on the basis of macro- and micro-morphological features according to Pitt & Hocking (1997), Samson et al. (2004), Awao & Mitsugi (1973), Awao & Otsuka (1973). 2 RESULTS Mycological survey indicated the presence of 45 taxa distributed among 20 genera of filamentous fungi in the investigated Chinese Jiuqu. The involved genera belong to Zygomycetes, Ascomycetes and anamorphic fungi. Fungal genera, species and strains isolated at various temperatures with different media from Jiuqu are shown in Tables 1, 2 and 3. Paecilomyces, Absidia, Thermomyces, Table 1 Percentage of different filamentous fungal genera isolated from Jiuqu Groups Genera Total Percentage a Zygomycetes Absidia spp Mucor spp Rhizomucor pusillus (Lindt) Schipper Rhizopus spp Ascomycetes Byssochlamys fulva Olliver & G. Sm Emericella quadrilineata (Thom & Raper) C.R. Benj Eurotium spp Monascus spp Thermoascus aurantiacus Miehe Anamorphic fungi Alternaria alternata (Fr.) Keissl Aspergillus spp Basipetospora sp Chrysonilia spp Chrysosporium spp Cladosporium spp Moniliella spp Paecilomyces spp Penicillium spp Scopulariopsis candida (Guég.) Vuill Thermomyces lanuginosus Tsikl Total 886

4 ZHANG Xiang-Min et al. / Filamentous mycoflora in a Chinese spirit Jiuqu 227 Table 2 Fungi species and strains isolated at various temperatures from Jiuqu Groups Species Total Percent a g Zygomycetes Absidia corymbifera (Cohn) Sacc. & Trotter Absidia coerulea Bainier Absidia sp Mucor hiemalis Wehmer Mucor piriformis A. Fisch Mucor racemosus Fresen Mucor sp Rhizomucor pusillus (Lindt) Schipper Rhizopus oligosporus Saito Rhizopus oryzae Went & Prins. Geerl Ascomycetes Byssochlamys fulva Olliver & G. Sm Emericella quadrilineata (Thom & Raper) C.R. Benj Eurotium chevalieri L. Mangin Eurotium herbariorum (F.H. Wigg.) Link Eurotium rubrum W. Bremer Monascus ruber Tiegh Thermoascus aurantiacus Miehe Anamorphic Fungi Alternaria alternata (Fr.) Keissl Aspergillus aureolatus Munt.-Cvetk. & Bata Aspergillus flavus Link Aspergillus fumigatus Fresen Aspergillus japonicus Saito Aspergillus niger v. Tiegh Aspergillus oryzae (Ahlb.) Cohn Aspergillus parasiticus Speare Aspergillus sydowii (Bainier & Sartory) Thom & Church Aspergillus viridinutans Ducker & Thrower Aspergillus wentii Wehmer Aspergillus spp Basipetospora sp Chrysonilia sitophila (Mont.) Arx Chrysonilia sp Chrysosporium farinicola (Burnside) Skou

5 228 ISSN CN /Q Mycosystema April 8, 2013 Vol.32 (Suppl.) Table 2 continued Chrysosporium fastidium Pitt Chrysosporium sp Cladosporium cladosporioides (Fresen.) G.A. de Vries Cladosporium sphaerospermum Penz Moniliella acetoabutens Stolk & Dakin Moniliella sp Paecilomyces variotii Bainier Paecilomyces sp Penicillium islandicum Sopp Penicillium sp Scopulariopsis candida (Guég.) Vuill Thermomyces lanuginosus Tsikl Total Note: a Percentage: percentage of the species isolated. Table 3 Strain numbers of different species isolated from various media from the Jiuqu Groups Species Media CZA NCZA PDA NPDA MEA YEPD Total Zygomycetes Absidia corymbifera (Cohn) Sacc. & Trotter Absidia coerulea Bainier Absidia sp Mucor hiemalis Wehmer Mucor piriformis A. Fisch Mucor racemosus Fresen Mucor sp Rhizomucor pusillus (Lindt) Schipper Rhizopus oligosporus Saito 3 3 Rhizopus oryzae Went & Prins. Geerl Ascomycetes Byssochlamys fulva Olliver & G. Sm Emericella quadrilineata (Thom & Raper) C.R. Benj. 1 1 Eurotium chevalieri L. Mangin Eurotium herbariorum (F.H. Wigg.) Link 3 3 Eurotium rubrum W. Bremer. 3 3 Monascus ruber Tiegh Thermoascus aurantiacus Miehe Anamorphic fungi Alternaria alternata (Fr.) Keissl

6 ZHANG Xiang-Min et al. / Filamentous mycoflora in a Chinese spirit Jiuqu 229 Table 3 continued Aspergillus aureolatus Munt. Cvetk. & Bata Aspergillus flavus Link Aspergillus fumigatus Fresen 1 1 Aspergillus japonicus Saito 1 1 Aspergillus niger v. Tiegh Aspergillus oryzae (Ahlb.) Cohn Aspergillus parasiticus Speare Aspergillus sydowii (Bainier & Sartory) Thom & Church Aspergillus viridinutans Ducker & Thrower Aspergillus wentii Wehmer Aspergillus spp Basipetospora sp. 1 1 Chrysonilia sitophila (Mont.) Arx 1 1 Chrysonilia sp. 1 1 Chrysosporium farinicola (Burnside) Skou Chrysosporium fastidium pitt 4 4 Chrysosporium sp Cladosporium cladosporioides (Fresen.) G.A. de Vries Cladosporium sphaerospermum Penz. 1 1 Moniliella acetoabutens Stolk & Dakin 1 1 Moniliella sp. 1 1 Penicillium islandicum Sopp. 1 1 Penicillium sp Paecilomyces variotii Bainier Paecilomyces sp Scopulariopsis candida (Guég.) Vuill Thermomyces lanuginosus Tsikl Total Rhizomucor, Aspergillus, Chrysosporium, Mucor and Monascus were the most frequently detected genera, with 20.3%, 18.7%, 11.2%, 9.9%, 9.8%, 8.2%, 6.7%, 5.7% of the total strains isolated respectively (Table 1). 71.7% of the isolates were isolated from 25 and 50 (38.2% and 33.5% each). The most popular species grow at 25 were Absidia corymbifera (Cohn) Sacc. & Trotter and Paecilomyces variotii Bainier; while the most frequently isolated species at 50 were Rhizomucor pusillus (Lindt) Schipper, Chrysosporium sp. and Thermomyces lanuginosus Tsikl. The media YEPD,

7 230 ISSN CN /Q Mycosystema April 8, 2013 Vol.32 (Suppl.) NPDA, MEA and NCZA have higher yield than PDA and CZA. 3 DISCUSSION Most of the fungal species are functionally important in the whole fermentation process; some of them are functional in a certain period of fermentation; whereas, a few species like Cladosporium spp. and Chrysonilia spp. that come from the environment might be with little function. The properties of different taxa belonging to the functional flora are discussed as follows. 3.1 Major functional fungi involved and their relevant properties Zygomycetes: Although Zygomycetes are one of the smallest groups of fungi, many are important spoilage organisms in food and indoor environments or even pathogenic to human. However, several species are also important producers of enzymes or organic acids, and have been used in fermentation processes. For example, most of the fermented soybean foods such as fu-ru and tempe (A collective name for fungal fermented beans, cereals or some other food processing by-products. Most probably originates from the island of Java, Indonesia) in Asian involve zygomycetous species like Actinomucor spp., Mucor spp., or Rhizopus spp. Ten species in four Zygomycete genera with a total of 331 strains were isolated from the Jiuqu. The species accounted for 21.7% of the total isolated species, while the strain numbers accounted for 37.2% of the total isolations. Absidia spp. and Rhizomucor pusillus (Lindt) Schipper Three different Absidia species are identified from the Jiuqu by the authors with much higher isolation frequency (Table 2), both of which are worldwide distributed (Samson et al. 2004; Pitt & Hocking 1997). Absidia corymbifera (Cohn) Saccardo & Trotter is the second frequently isolated species from the Jiuqu, with a high frequency of up to 17.7%. Due to its ability to grow under broad temperatures ranging from 14 to 50 (Table 2), and to germinate and grow at low a w down to 0.88, this thermophilic fungus was isolated at all temperatures tested and on all media used. Hocking (1990) recorded that its growth in nitrogen (<1% O 2 ) was similar to that in air. These characters could make it one of the major functional species in the whole fermentation process, and evenly dispersed in the Jiuqu block on surface and in the center. Rhizomucor pusillus, as reviewed by Domsch et al. (1980), commonly existed in nuts, cereals, composted wheat straw, self-heated hay, oil seeds and meat products, can rapidly utilize numerous C sources, and produce various kinds of enzymes, such as: 1,4-β-Xylosidase, endo-1,4-β-glucanase, phosphatase, a strong milk-clotting enzyme, acid proteinase and an alcohol dehydrogenase. With a growth temperature ranging from 20 to 55, this fungus was also found in the composts, and, was recognized as a typical early coloniser of composts, which exploiting simple sugars, amino acids etc. that present initially in the plant material (Deacon 2003). Whereas in Jiuqu, the species Rh. pusillus was only isolated at high temperature of 42 and 50. Two explanations can be made to this phenomenon. First, the Rh. pusillus strains in Jiuqu were all of thermophilic ones that only grow at high temperature. Second, the optimum growth temperature of Rh. pusillis reported are 37 42, at temperatures below 37, the growth of the species was inhibited by the growth of other fungi. Rhizopus spp. and Mucor spp. Rhizopus spp. and Mucor were of important major functional fungi for tempe, sufu (made from soybeans and is an easily digested and nutritious

8 ZHANG Xiang-Min et al. / Filamentous mycoflora in a Chinese spirit Jiuqu 231 protein food), and rice wine making (Nout 1995; Nout & Aidoo 2002). Under certain conditions, these mycelial fungi produce various kinds of enzymes: protease (to degrade soybean protein into peptides, free amino acids and other non-protein nitrogenous substances), α-amylase, α-galactosidase and lipase (hydrolyze soybean oil into fatty acids). These enzymes may start a series of biochemical reactions resulting in changes in flavour, texture, colour and other quality attributes that are desired by the consumer (Nout & Aidoo 2002). It is implicated that these fungi in Jiuqu play a similar role in the Chinese spirit making. The isolation frequency of the genus Mucor from Jiuqu was moderate (6.7%). Four species of the genus Mucor: M. hiemalis Wehmer, M piriformis A. Fisch., M. racemosus Fresen., and a M. sp. were exploited during the study. M. sp. is a thermophile species, further studies are needed to explicit its taxonomy status and physiological properties. R. oligosporus Saito and R. oryzae Went & Prins. Geerl. are the representing species of the genus Rhizopus in Jiuqu. R. oryzae predominates over R. oligosporus with the frequency of 1.6% and 0.3% respectively. Proteinase, polygalacturonase, 1,3-β-glucanase, extracellula lipases, alcohol dehydrogenase and phenol oxidase have been reported produced by the fungus R. oryzae under different conditions Ascomycetes: There are five Ascomycetes genera in Jiuqu: Monascus, Thermoascus, Byssochlamys, Eurotium and Emericella. Most of which are functional fungi that play different roles in fermentation for the spirit making. Eurotium spp. All the Eurotium species are xerophilic (Pitt & Hocking 1997). Three Eurotium species were isolated from the Jiuqu: E. chevalieri L. Mangin, E. herbariorum (F.H. Wigg.) Link and E. rubrum W. Bremer. E. chevalieri has been reported to be the most heat resistant xerophilic fungus: 18% 25% of the ascospores survived heating at 70 for 10min in a medium of 0.98 a w and ph3.8, and up to 0.5% of the ascospores suvived a similar treatment at 80 (Pitt & Christian 1970). Also the species is suggested rank as one of the most common spoilage fungi on earth, especially in warmer regions. It has been isolated from a great variety of foods, especially cereals, including wheat and flour, rice, rice flour and rice bran, maize and cereal flakes (Pitt & Hocking 1997). Just like E. chevalieri, E. rubrum was readily isolated from most food commodities too and as food spoilage fungi, along with E. repens de Bary, it has been found use in food manufacture, as a starter culture in the manufacture of katsuobushi from bonito (Pitt & Hocking 1997). According to Pitt & Hocking (1997), E. herbariorum is a vigorous xerophile and is a far less common species than E. rubrum, the quantity of the two are the same in the tested Jiuqu. It is obvious that the Eurotium spp. plays an active role after the saccharification step when the water activity of the substrate becoming lower, although the specific role of the Eurotium spp. in Jiuqu is still unknown. Only one Emericella quadrilineata (Thom & Raper) C.R. Benj. strain was found in Jiuqu. This might be contamination from the environment, which is a widespread soil fungus (Raper & Fennell 1965). Byssochlamys fulva Olliver & G. Sm This fungus is a well known spoilage fungus in canned or bottled fruit due to its heat resistant characteristic and the ability to grow under very low oxygen tensions (Pitt & Hocking 1997). Its isolation at high temperature (42 ) in Jiuqu demonstrating its vitality, though little is known about how it affect the quality of the product.

9 232 ISSN CN /Q Mycosystema April 8, 2013 Vol.32 (Suppl.) Monascus rubber Tiegh. Monascus is traditionally used to produce Angkak (which has a specific aroma and purple red colour and has been used as a natural colorant in red spirit, red sufu and red rice) by fermentation of cooked rice (Nout & Aidoo 2002). However, their occurrence in Jiuqu was recognized as an indicator of somewhat bad quality of the Jiuqu (Huang 2000). Monascus rubber was the only species of the genus isolated from the Jiuqu in the current study. It is a thermotolerant xerophilous fungus, most of the strains are isolated at higher temperatures from more concentrated media (Table 2 and Table 3). Detected by its high isolation frequency, it may be one of the most important genus in the Jiuqu when it grows at the right time and in a proper proportion with other major fungi. Besides production of the pigment and the typical flavour components, the species also produce a series of enzymes: dextrinase, saccharidase, maltase, and can hydrolysis starch to glucose (Nout & Aidoo 2002). Accumulated studies were focused to the genus since the hypocholesterole function of Monacolin K was recognized (Endo 1979; Liu et al. 2006), further studies are needed to explore its functions in Jiuqu and Chinese spirit making. Thermoascus aurantiacus Miehe This fungus proved to be a tough thermophile with a growth temperature ranging from 30 to 62 and optimum growth temperature around 45. The colonies are bright orange to orange brown (Mouchacca 1997). It was also known for the production of thermo stable xylanases (Mustafa et al. 1994; Tong et al. 1980). The Thermoascus aurantiacus strain isolated from the Jiuqu however, has a preference for media MEA and YEPD, and its optimum growth temperature is 50 (Table 2 and Table 3). Colonies on MEA grow rapidly, attaining 90mm diameter in 4 days at 50 with dull orange and a granular appearance. The reverse of the colonies become red brown in age. A fruit fragrance was produced by the strain, which makes it distinct from other strains reported (Awao & Otsuka 1973; Chen & Chen 1996). Whereas more detailed relevant properties of the fungus in the Jiuqu are still unknown Anamorphic Fungi: Aspergillus spp.: The genus Aspergillus is the most diversify genus of all the genera identified (Table 2). Eleven species altogether accounted for 23.9% of the total fungi species isolated. Only two species have slightly higher isolation frequency, A. flavus Link: Fr. with a frequency of 5.0% and A. parasiticus Speare of 4.3%. Recent study by Chang et al. (2006) showed that A. flavus populations are genetically diverse. A. oryzae (Ahlb.) Cohn isolates may descend from certain atoxigenic L-type A. flavus isolates. Furthermore, the presence of potential toxinogenic species on food products does not always mean that these products contain mycotoxins; various environmental factors also play a part. The conditions, which allow toxin production, are more restricted than those for growth (Filtenborg et al. 2004). Known metabolic products include riboflavin, the volatile compounds 3-methyl-butanol, 3-octanone, 1-octen-3-ol, 1-octanol, cis-2-octen-1-ol and ethylene, free D-amino acids, kojic acid, oxalic acid, β-nitropropionic acid, deferriferrichrysine, asperflavin and anhydroasperflavin, the ergoline alkaloids ergocryptine, agroclavine and elymoclavine et al. (Domsch et al. 1980). The identified metabolites of A. parasiticus include the volatile flavour compounds 1-octen-3-ol, 2-octen-1-ol, 3-octanol, 3-methyl-butanol, kojic acid, the pigment norsolorinic acid, the aflatoxins B 1, B 2, G 1, G 2, the B 1 -related parasiticol and also

10 ZHANG Xiang-Min et al. / Filamentous mycoflora in a Chinese spirit Jiuqu 233 parasiticolide A (Domsch et al. 1980). However, it is also reported that there is a marked reduction in aflatoxin production when A. parasiticus is cultivated simultaneously with either Eurotium chevalieri or A. candidus Link. Furthermore, heavy growth of Paecilomyces variotii Bainier, A. corymbifera, Thermomyces lanuginosus Tsikl. and Rhizomucor pusillis et al. on low water activity media may completely mask the growth of toxigenic Aspergillus such as A. flavus and A. parasiticus. Chrysosporium spp. The Chrysosporium species have been isolated by many researchers from a variety of different habitats around the world. Research revealed considerable biotechnological potential of this genus for recycling keratinous waste in soil and secretion of enzymes and antimicrobials (Kushwaha 2000). Forty-seven species of Chrysosporium along with their isolation information and some salient features were listed by Kushwaha (2000). Most Chrysosporium species are found in soil, on the hair or skin of animals, and are strictly anamorphic. However, circumscribed by Skou (1992) as the Farinicola series, species that occur in foods, are xerophiles, and most likely unrelated to the dermatophyte species (Pitt & Hocking 1997). This is the very case of Chrysosporium species in the Jiuqu: the occurrence of the Chrysosporium species indicated the concentrated nature of Jiuqu at a certain fermentation period. And this is the first time for the genus to be reported to occur in fermented food, though C. farinicola (Burnside) Skou along with its Bettsia Skou teleomorph and C. fastidium pitt were recorded as the most commonly encountered species in foods. Paecilomyces spp. Two Paecilomyces species were isolated from Jiuqu, Paecilomyces variotii and Paecilomyces sp. P. variotii was the most common species in Jiuqu, which also was recorded as a ubiquitous contaminating fungus in foods and raw materials (Pitt & Hocking 1997). The fungus is a xerophile, which grows between about 5 and 50 (Pitt & Hocking 1997; Samson et al. 2004). One hundred and sixty three strains were isolated from the Jiuqu, which accounted for 18.4% of the total isolated strains. This great isolation frequency may due to the fungus property of producing large amounts of conidia, and the conidia are easy to grow on all kinds of media at various temperatures used for isolation. Thermomyces lanuginosus The frequency of the fungus T. lanuginosus isolated from Jiuqu was as high as 11.2%, second only to P. variotii and A. corymbifera. The species was reported be found in hays and soils frequently (Mouchacca 1997), this suggests that its predominance in the Jiuqu was due to their preponderance in the hay used to cover the inoculated wheat flour. Besides this, the fungus grows between 30 and 52 55, which is also extremely common in all types of self-heating materials, in birds nests, and sun-heated soils. It cannot degrade cellulose and seems to live as a commensal with cellulose-decomposing species, sharing some of the sugars released from the plant cell walls by the cellulolytic activities of the cellulose-decomposing species (Deacon 2003). Recently, high productive and thermo stable xylanases, which have significant current and potential uses in industries such as paper and pulp, food, and biofuel were reported to be produced by certain T. lanuginosus strains (Khwanchai & Vichien 2006). 3.2 Succession of fungal communities during fermentation of Jiuqu Fungi grow only under their favorable growing

11 234 ISSN CN /Q Mycosystema April 8, 2013 Vol.32 (Suppl.) conditions. These conditions vary from species to species. Adaptability determines species domination. The specific reason why a particular species dominates in a product is often not known, but it certainly correlated with the species characteristics and the properties of the product. Also the predominance of a species can be due to heavy contamination from ecological niches where the mould has developed (Filtenborg et al. 2004). The whole Jiuqu could be compared to a factory operated by the succession of functional mycoflora. During the incubation process, once the conditions are fit for certain strains, they start to grow. There are thermophilic fungi as well as mesophilic fungi in Jiuqu. Xerophilic fungi may also exist and growth if water activity is below 0.85 (Pitt 1975). The fermentation process of Jiuqu may be initiated by mesophilic organisms, such as: Absidia spp., Rhizopus oryzae, Aspergillus spp. and Paecilomyces variotii, which generate heat by their metabolism and raise the temperature to a point where some of their own activities are suppressed (except Absidia corymbifera and Paecilomyces variotii). Then the strictly thermophilic fungi: Rhizomucor pusillus, Thermomyces lanuginosus, Chrysosporium spp., Thermoascus aurantiacus, Mucor spp., Byssochlamys fulva, and several thermophilic bacteria (e.g. Bacillus spp.) continue the process. 4 CONCLUSIONS Filamentous mycoflora play an important role in fermentation production of Chinese spirits. They are used for their enzymes to degrade polymeric substances, and to combine special aroma and flavor substances of the spirits. The study revealed the community structure of Zygomycetes, Ascomycetes and anamorphic fungi in Jiuqu. There are species that are commonly reported in food fermentation, as well as several species that are newly reported in Jiuqu, i.e. Absidia corymbifera, Rhizomucor pusillus, Thermomyces lanuginosus, Chrysosporium spp. and Thermoascus aurantiacus. Based on current culture-based research result, molecular detection and quantification method could be applied to determine their population dynamics during fermentation and the major roles played by each species in the whole processes. [REFERENCES] Awao T, Mitsugi K, Notes on thermophilic fungi in Japan (1). Transactions of the Mycological Society of Japan, 14: Awao T, Otsuka S, Notes on thermophilic fungi in Japan (2). Transactions of the Mycological Society of Japan, 14: Chang PK, Ehrlich KC, Hua ST, Cladal relatedness among Aspergillus oryzae isolates and Aspergillus flavus S and L morphotype isolates. International Journal of Food Microbiology, 108(2): Chen KY, Chen ZC, A new species of Thermoascus with a Paecilomyes anamorph and other thermophilic Thermoascus species from Taiwan. Mycotaxon, 60: Deacon J, The microbial world: thermophilic microorganisms. microbes/thermo.htm Domsch KH, Gams W, Anderson TH, Compendium of soil fungi, Vols. 1 & 2. Academic Press, London Endo A, Monacolin K, A new hypocholesterole mic agent produced by a Monascus species. The Journal of Antibiotics, 32: Filtenborg O, Frisvad JC, Samson RA, Specific association of fungi to foods and influence of physical environmental factors. In: Samson RA, Hoekstra ES, Frisvad JC (eds.) Introduction to food and airborne fungi. 7th edition. Centraalbureau voor Schimmlecultures, Utrecht

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