Milk Science Vol. 55, No. 3 2007 Original Report Identiˆcation of Analogous Geotrichum candidum Strains Isolated from Surface Mould Ripened Soft Cheese and Raw Milk Tsunehiko Kawai, Kanae Araki, and Hiroko Kida (Biological Laboratory, Nara University of Education, Takabatake, Nara, 6306528, Consultant Live Stock of Osaka Prefecture) (Biological Laboratory, Nara University of Education, Takabatake, Nara, 6306528) Abstract In the biological laboratory of Nara University of Education 17 strains of analogous Geotrichum candidum have been preserved; 11 strains isolated from 11 sorts of the surface mould ripened on soft cheeses which were manufactured from raw milk by traditional process at Camembert, Normandy and at various places in France; 1 strain isolated from Japanese farm soft cheese; 5 strains isolated from raw milk which was milked at 27 dairies of Camembert cheese making farms in Okayama, Osaka, Nara, Gifu, Nagano and Hokkaido. For the purpose of deˆning these 17 strains we have made observations on the morphological and the physiological characteristics and tests in biochemical properties. We have recognized that all these 17 strains had the morphological characteristics of Geotrichum candidum strains and they satisˆed DE Hoog's9) morphological keys to the taxa (3), (19), (20) and (21) and physiological keys to the taxa (13), (14) of Geotrichum candidum. In addition, tests of biochemical properties resulted in the identiˆcation with those of Geotrichum candidum. Thus, we identiˆed all the 17 strains as Geotrichum candidum strains. With regard to the identiˆcation through biochemical characteristics of the 17 analogous Geotrichum candidum strains, we made miniaturized assimilation tests of the yeasts organism, each containing a dehydrated carbohydrate substrate, using ID 32C API19) (Nippon BioMeriuxá Co. Tokyo). As the result of assimilation tests all the strains were identiˆed with Geotrichum candidum and they were classiˆed as Geotrichum candidum. Key words; Geotrichum candidum, anamorph, teleomorph, arthric conidia, Camembert cheese, raw milk, morphological key tion as a starter for cheese makings2). Besides, Introduction through the remarkable development of the func- tional food research, Camembert cheese has Cheese has always been excellent nutritious become one of the research subject3). Since 1985 food, and many sorts of cheese have been manufac- we have practiced making and studying Camem- tured from raw milk by the traditional methods1). bert cheese by using analogous Geotrichum candi- Many sort of microorganisms exist in bovine raw dum strain as a starter4～8). It is expected that milk and they play more or less an important role in Camembert cheese will be more evaluated and esti- ripening cheese, such as surface mould ripening mated as functional food3). soft type cheese. The genus Geotrichum was described by Link Recentry, the yeast ‰ora, especially Geotrichum (1809) as white hyphomycetes. Taxonomically Ge- candidum, has become a notable microoganism for otrichum candidum is classiˆed as Fungi Imperfecti, researchers, because it has been discovered that Endomyces geotrichum, anamorphs of Galactomyces the Geotrichum candidum strains in raw milk func- geotrichum9,10). Corresponding auther (fax 0742224693) The morphological characteristics of Geotrichum Received 7 August 2006 candidum are as follows: colonies are white, 第巻 farinose or hairy usually dry, hyphae hyalins are research on the identiˆcation of analogous Ge- hygrophilic, fertile hyphae are not or slightly otrichum candidum strains. diŠerentiated, branching at sharp angles and often sickle-shaped or dichotomous. Conidia arthric, oc- Materials and Methods casionally sympodial or percurent, conidia next to each other and without intermediate sterile cells. Analogous Geotrichum candidum and its Keeping The studies of morphological characteristics of We made 33 samples out of 11 sorts of the sur- Geotrichum candidum were opened by Carmichael face mould ripened soft cheese which were (1975)11), succeded by Fiddy and Trinci12),and manufactured by traditional methods using raw have been continued by meny investigators, such milk on farms and farm factories at Camembert, as DE Hoog, Smith, Gueho9)and Kurtzman10) up to Normandy and various place in France and we iso- the present. lated 11 strains of analogous Geotrichum candidum. On the other hand, the studies on Geotrichum Besides, 1 strain isolated from Japanese farm soft candidum in raw milk or natural cheese were only cheese. sterted by Guegen and Jacquet13) in 1982 on the From the fact that Geotrichum candidum was ex- morphological and the biochemical characteristics. tracted from Camembert cheese manufactured us- ItisthefactthateveninFrenchuntiltwenty ing bovine raw milk in France, we inferred that Ge- years ago Geotrichum candidum was a defect- otrichum candidum was probably contained in bo- producing agent excluded from the farms or farm vine raw milk Japan likewise. So from twenty- factories, which are traditional manufacturing suc- seven dairy ferms or farm houses manufacturing cessors of Camembert cheese and other soft type Camembert cheese in Okayama, Osaka, Nara, cheeses, because it formed ``toad skin or Gifu, Nagano and Hokkaido we obtained raw milk, sliminess14,16)'' and stored ``excessive fat'', on the andprepared152milksamples,andisolated5 surface of soft type cheese. analogous Geotrichum candidum strains. From This microoganism, has a place as a defect- these samples we isolated 17 analogous Geotrichum producing agent giving an inhibitory evil eŠect on candidum strains, designated as Ga, Gb, Gd, Gg, the growth and sporulation of Penicillium caseico- Gh, Gisi, Gk, GMu, Gn, Go, Gy, GYd, GIz, GAs, lum, but on the other hand, it has an inhibitory ˆne GDur, GM'do, and GRe.(Table 1) eŠect on the growth and sporulation of Mucor and The standerd strains prepared for identiˆcation Listeria monocytogenes15,17). and classiˆcation were as follows; for identiˆca- Dr. Mourgues, Bergere and Vassal16) were the tion, Geotrichum candidum (JCM No. 6263) pur- ˆrst to use this trouble-maker Geotrichum candi- chased from RIKEN Bioresource Center, and for dum as a starter of cheese making in France16). classˆcation (teleomorph; posessed copulating Since then in France studies have been carried on gametangia): Galactomyces geotrichum (JCM No. by French investigators17) in various aspects; clas- 1945),andDipodascus albidus (JCM No. 5297) siˆcation, morphology, physiology, metabolism, purchased from RIKEN Bioresource Center. (JCM pathogenicity, applied and technical milk products stands for Japan Collection of Microoganisms) and molecular biology. However in Japan, the These strains were stored at 3°C on potato-dex- report of Geotrichum candidum in raw milk had not trose agar (Nissui-Seiyaku Ltd Tokyo) slant. been found until the reports made by Kawai et al. Media and culture conditions from 19914～6) to 19987,8). For the cutivation of samples we prepared liquid We have done research in the morphological and 5 GYP and 0.5 GYP (used for observation of the biochemical characteristics for the purpose of anamorph) medium (glucose 5 g and 0.5 g, pep- deˆning classiˆcation of analogous Geotrichum can- tone 〈Difco Laboraories, Michigan USA〉 1g, didum which has been preserved at the Biological yeast extract〈Difco Laboraories, Michigan USA〉 Laboratory of Nara University of Education. We 1 g, distilled water 100 ml), and liquid MEYA have also tried it to be useful strains as cheese mak- medium (malt extract〈Difco Laboraories, Michi- ing starter. We will report several results of our gan USA〉4 g, yeast extract〈Difco Laboraories, 第号 Table 1 The list of analogous Geotrichum candidum strains made observation on the germination or micro isolated from the soft type cheese (ripened by Penicillium growth of conidia of the sample strains inoculated camemberti on surface) and raw milk. in 20 ml of MEYA within 50 ml capasity sterilized strains origin year ‰ask, and cultured on a reciprocal shaker at 40°C Ga Camembert, farm factory at Camembert, in 1997 Normandy and 37°Cfor7days9). Gb Brie de Meaux, farm factory in Ile de 1997 The identiˆcation of Geotrichum candidum France Gd Camembert, farm house at Mr. Durand, at 1989 Morphological and physiological key to the taxa Camembert The identiˆcation of Geotrichum candidum was Gg Raw milk, farm factory of Nogei High 1993 School, Osaka made according to the morphological keys to the Gh Chaource, farm factory in Champagne 1997 taxa (1～34 keys) and the physiological keys to the Gisi Camembert, factory in Normandy 1996 taxa (1～14 keys) by DE Hoog et al.9), which clas- Gk Raw milk, farm house at Katsuragi-Kogen 1996 in Nara sify the genus Geotrichum and its teleomorph. GMu MunsterGerom á e,à farm factory in Alsace 1999 The identiˆcation through the biochemical Gn Neufchatel,â farm factory in Normandy 1997 characteristics Go Brie de Coulommiers, farm factory in Ile de 1997 France The identiˆcation through the biochemical Gy Camembert, farm house at Kibi-Kogen in 1990 characteristics was made using the ID 32C API19) Okayama which consists of 32 cupules, with miniaturized as- GYd Raw milk, dairy farm at Sayama Osaka 1999 similation tests of the yeasts organism, each con- GIz Raw milk, dairy farm at Akkeshi Hokkaido 1999 GAs Raw milk, dairy farm at Asuka in Nara 1999 taining a dehydrated carbohydrate substrate. As- GDur Camembert, farm house at Mr. Durand, at 2000 similation tests of 31 substrates, as follows; D Camembert Galactose GAL, Cycroheximide ACT, DSac- GMdO Vacherin Mont d`O, farm factory