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 in France- 2003 Comte charose SAC, NAcetylGlucosamine NAG, Lactic GRe Camembert, farm factory in Normandy 2005 acid LAT, LArabinose ARA, DCellobiose CEL, DRa‹nose RAF, DMaltose MAL, DTrehalose TRE, Potassium 2Ketogluconate 2KG, Methyla Michigan USA〉 0.5 g, distilled water 100 ml). DGluconate MDG, DSorbitol SOR, DXylose Medium was sterilized at 120°C for 20 min. The XYL, DRibose RIB, Glyserol GLY, LRhamnose culture was perfomed on a reciprocal shaker at 110 RHA, Palatinose PLE, Erythritol ERY, D times/min at 30°C for 72 hours. We used potato- Melibiose MEL, Sodium Glucuronate GRT, D dextrose agar for solid medium. The culture medi- Melezitose MLZ, Potassium Gluconate GNT, um was diluted adequately and 3 plates were pre- Levulinic acid LVT, DMannitol MAN, DLactose paredforeachliquidculturesample. (bovine origin)LAC, Inositol INO, DGlucose The observation of morphological and physiologi- GUL, LSorbose SBE, Glucosamine GNL, Esculin cal characterticsis ferric citrate ESC. The observation of morphological characteristics To prepare the inoculum we used one or several was made on the cultivated samples mounted on identical colonies of young culture (24～48 hours plates. We observed the process of germination old) which were taken from the culture medium of and micro growth of conidia 4 hours after inocula- PDA,andsuspendedthemin2ml API C Medium, tion. In order to conˆrm the existence of fertile and prepared homogenous suspension with turbidi- hyphae and gametangial copulation that prove tel- ty equivalent to 2 Mc-Faland. Then, approximately eomorph of the sample strains, we observed on the 0.25 ml of culture medium suspension was trans- micro growth or expansion of hyphae on the 7th ferred into the API C ampoule. day and the 21st day after inoculation on PDA in 0.14 ml of the inoculum was dispensed in each petri dishes or on microscoupe slide18). cupule on plate, and incubated at 30°Cfor24～48 The cellular tissue, thalli and conidia were hours. We compared the turbidity of each cupule scrutinized with the phase diŠerence microscope of with the control (0) so as to know how much the in- 400 magniˆcations9,10). oculum in each cupule grew. If we observed any In regard to physiological characteristics we turbidity in the suspension by visual reading, we 第巻 recorded it as positive. Identiˆcation was obtained strains according to DE Hoog's9) morphological using the identiˆcation table (Nippon Bio-Meriuxá keys to the taxa of Geotrichum candidum as men- Co. Tokyo). tioned above; In the morphological key to the taxa of items Results and discussion (1), colonies attein less than 10 mm diameter in 10 days, cushion-shaped, cerebriform, moist, glassy; Isolation of yeast hyphal cells capsulated, locally amyloid; anamorph We isolated 12 sorts of analogous Geotrichum absent. (Dipodascopis) candidum from surface mould ripened on soft The colonies of 17 analogous Geotrichum candi- cheese which was manufactured by traditional dum strains and JCM No. 6263 (the whole of sam- process using raw milk at farms and farm factories ples) measured 70～90 mm in diameter in 10 days, at Camembert, Normandy and various place in with dry surface, no copulating gametangia and no France, and 5 sort from bovine raw milk which was fertile hyphae. (Fig. 1, Fig. 2) This shows that tel- milked at dairy ferms in Nara, Osaka and Hok- eomorph is absent. We decided that they were not kaido. In addition we isolated Kluyveromyces applicable to the key of items (1),buttothekeyof marxianus, Saccharomyces cerevisiae, Zygosac- items (3). Because ascospore was absent, we charomyces rouxii, Schzosaccharomyces pombe20). selected the key of items (18). (Table 2) In the key of items (18) hyphae consist of coher- Several sorts of microoganisms, such as Kluyve- ent chains of regular, broadly ellispoidal cells with romyces, Debaromyces, Saccharomyces, Zygosac- broad septa. (Geotrichum anamorph of Dipodascus charomyces, were isolated by Lenoir21) from bovine macrosporus) raw milk and surface mould ripened on soft cheese Hyphae have even width throughout, and if con- manufactued by ˆve farm houses in Normandy, stricted at the septa, they easily disarticulate. The France. Gueguen and Jacquet13) also isolated 96 whole samples were applicable to the morphologi- strains of Geotrichum candidum. This fact shows cal key to the taxa of items (18). that the similar sorts of yeasts exist in bovine raw On the basis that hyphae were 4～10 mm wide, as milk both in Japan and France. hyphae grew on their even width throughout, and if Identiˆcation of Geotrichum candidum constricted at the septa, easily disarticulated and Identiˆcation of Geotrichum candidum by mor- formed arthroconidia, we accordingly selected the phological keys to the taxa key of items (19). The expansion growth of colo- The identiˆcation of Geotrichum candidum nies attained over 50 or 60 mm diameter in 10 strains was made through the observations of mor- days, they were applicable to the morphological phological and physiological characteristics of the key to the taxa of items (20). Because the expan-
Table 2 Disribution of yeast ‰ora isolated from raw milk for cheese-makings collected from farm houses in Japan.
Farm12345678Total Species Samples 12 48 12 12 4 50 16 18 152
Kluyveromyces marxianus 10 24 10 8 4 24 16 16 113 Geotricum candidum 0 2 110024 9 Saccharomyces cerevisiae 12 48 12 12 4 30 16 18 152 Zygosaccharomyces rouxii 2683317101261 Schizosaccharomyces pombe 086026104 36 Others 12 24 12 10 4 30 2 2 96
Region of farms; 1Kibi-Kogen Okayama, 2Mihara Osaka, 3Katsuragi-Kogen Nara, 4Asuka Nara, 5Kamo Kyoto, 6 Hirugano-Kogen Gifu,including ˆve farms, 7Hirakata, Sakai, Sayama, Sennan Osaka, 8Urahoro, Shihoro, Shintoku, Hamanaka, Akkeshi, Bekkai, Tsubetsu, Bihoro, Nemuro, Nakashibetsu Hokkaido. From 27 dairy farms and farm houses manufacturing Camembert cheese in Japan we obtained bovine raw milk and prepared 72 sam- ples, which were selected from 152 samples. We isolated 6 analogous Geotrichum candidum strains. Recognized only in October and November during the screening tests of yeasts throughout the year. 第号
branching, they were applicable to the morphologi- cal key to the taxa of items (20). Thus the 17 analogous Geotrichum candidum strains were examined by morphological keys to the taxa (3), (19), (20) and (21) of Geotrichum candidum, we identiˆed them as Geotrichum candi- dum strains. Taxonomically Geotrichum candidum22) is classi- ˆed as Ascomycotia, Archiasocmycetes, Endomyce- tales, Endomycetaceae Endomyces and Endomyces- geotrichum, but according to Sugiyama's paper22) as Geotrichum candidum is anamorph, its teleo- Fig. 1 Photograph of Dipodascus albidus JCM No. morph is Galactomyces geotrichum. 5297. Copulating gametangia(a) and ar- Physiological key to the taxa throconidia(b). 9) Development of copulating gametangia(a) as Also, according to DE Hoog's physiological follow; Hyphal elements, either germinating keys to the taxa of Geotrichum candidum,thewhole close to or through one or later through both ter- minal scars, or slightly in‰ating and disarticulat- samples were inoculated into MEYA medium, and ing further, then ˆnally becoming rounded oŠ, cultivated on a reciprocal shaker at 40°Cand37°C 9) cubical to subglobose(b) . 9) Microscope slide on PDA culture with the phase for 7 days . The conidia were absent. diŠerence microscope of ×400 Thus 17 Geotrichum candidum strains were iden- Scale: mm tiˆed and classˆed as Geotrichum candidum strains by physiological keys to the taxa (13), (14). The identiˆcation through biochemical charac- teristics With regard to the identiˆcation through biochemical characteristics of the 17 analogous Ge- otrichum candidum strains, we made miniaturized assimilation tests of the yeasts organism, each con- taining a dehydrated carbohydrate substrate, using ID 32C API19). The whole of samples assimilated to DGalactose GAL, Cyclohexamide ACT, Lactic acid LAT, DSorbitol SOR, DXylose XYL, Glycerol GLY, DMannitol MAN, DGlucose GLU, LSorbose SBE, and does not assimilated to DSaccharose SAC, NAcetylGlucosamine NAG, Fig. 2 Photograph of Galactomyces geotrichum JCM LArabinose ARA, DCellobiose CEL, D No. 1945. On MEYA, fertile hyphae with asci, immature asci in self-sporulating cultures. During Ra‹nose RAF, DMaltose MAL, DTrehalose the present study self-sporulating strains were TRE, Potassium 2Ketogluconate 2KG, Methyla invariably seen to be monopoidal, the asci being formed laterally or terminally on short, narrow, DGluconate MDG, DRibose RIB, LRhamnose often ‰exuose hyphae. Asci were often abortive, RHA, Palatinose PLE, Erythritol ERY, D and mature spores were rate9).2ofmature spores(a) and several of abortive spores(b). Melibiose MEL, Sodium Glucronate GRT, D Microscope slide on MEYA culture with the Melezitose MLZ, Potassium Gluconate GNT, phase diŠerence of microscope of ×400 Scale: mm Levulinic acid LVT, DLactose (bovine origin) LAG, Inositol INO, Glucosamine GNL and Esculin ferric citrate ESC. As the result of assimilation sion growth of branches of hyphae attained from 4 tests all the strains were identiˆed with Geotrichum ～10 mm wide, main branches of hyphae 9～10 mm candidum and they were classiˆed as Geotrichum wide too, and expanding hyphae had dichotomous candidum. 第巻
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表面かび熟成型軟質チーズおよび生乳より単離した Geotrichum candidum 様酵母の同定
河合恒彦・荒木哉衣・木田博子 (奈良教育大学教育学部生物学教室) (奈良教育大学教育学部生物学教室 大阪府畜産コンサルタント)
Geotrichum candidum 様酵母に注目し，これをスターターに用いるカマンベールチーズ製造研究を通して，チーズの 性質，レオロジー，化学成分の生成，酵母の形態学的特徴を報告してきた。Geotrichum candidum 様酵母は，フランス の伝統的製法の表面白かび熟成型チーズ，本邦産チーズ，奈良県，大阪府，北海道の酪農家産生乳より単離した17株 で，菌株は本学生物学教室に保管されている。酵母の分類学的位置を明確にするため，形態学的性状の観察と生化学的 性状の試験を通して同定をおこなった結果，17株の全てが Geotrichum 属の形態学的検索9)に相当したので，Ge- otrichum candidum に分類することができた。生化学的性状の判定も同様であった。 これらの結果より，Geotrichum candidum であると同定された。