Study of the yeasts during the ripening process of Armada cheeses from raw ’s milk Maria E. Tornadijo, José M. Fresno, Roberto Martin Sarmiento, Javier Carballo

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Maria E. Tornadijo, José M. Fresno, Roberto Martin Sarmiento, Javier Carballo. Study of the yeasts during the ripening process of Armada cheeses from raw goat’s milk. Le Lait, INRA Editions, 1998, 78 (6), pp.647-659. ￿hal-00929623￿

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Original article

Study of the yeasts during the ripening process of Armada cheeses from raw goat's milk

Maria E. Tornadijo", José M. Fresno", Roberto Martin Sarmiento", Javier Carballo?"

a Departamento de Higiene y Tecnologfa de los Alimentos, Universidad de Leon, 24071 Leon, Spain b Area de Tecnologfa de los Alimentos, Facultad de Ciencias de Orense, Universidad de Vigo, 32004 Orense, Spain

(Received 12 May 1997; accepted 25 May 1998)

Abstract - The counts, species and characteristics of yeasts were studied throughout the manu- facturing and ripening of four batches of Armada chee se, a craft variety made in the north of Spain from raw goat's milk. Counts in Oxytetracycline Glucose Yeast-Extract Agar (1.82 x 104 - 1.15 x lOS cfu·g-1 in milk) increased one log. unit in curd and another log. unit during the first week ofripening, remaining stable in two- and four-week-old cheeses and decreasing from this point to the end of the ripening process. The yeast percentage of the isolates carried out remained practically constant at around 75 % throughout the ripening process, dropping to 27 % at the last sampling point (16-week-old cheese). Of the 181 isolated yeast strains, 60 were identified as Geotrichum candidum, 58 as Candida lambica, II as Candida krusei, 4 as Candida lipolytica, 2 as Candida ingens, 1 as Candida catenu- lata, 1 as Candida pintolopesii, 1 as Candida colliculosa, 19 as Kluyveromyces lactis, II as Kluyveromyces marxianus, 10 as Saccharomyces unisporus and 3 as Trichosporon beigelii. G. can- didum predominated in 1- and 2-week-old cheeses and C. lambica was the most abundant species in milk and in 4- and 8-week-old cheese while C. krusei dominated in 16-week-old cheese. Judging from their metabolic characteristics, the yeasts in Armada cheese seem to participate in the initial acidifi- cation of the curd and in the consumption of lactic acid which takes place throughout the ripening pro- cess, though, with the exception of G. candidum, they do not seem to play a relevant role in the lipolysis, either way its resistance to NaCI is not very high. However, they seem to play an important role in the degradation of the biogenic amines; of the 181 strains identified, 136 assimilated the four biogenic amines tested (cadaverine, histamine, putrescine and tyramine) and ail the strains assimilated at least one ofthese amines. © Inra/Elsevier, Paris.

Armada cheesel goat cheesel yeast speciesl yeast count 1 techno1ogical property

Résumé - Étude des levures pendant la fabrication et l'affinage des fromages de chèvre au lait cru de la variété Armada. L'étude a porté sur le dénombrement, l'identification et quelques carac-

* Correspondence and reprints. 648 M.E. Tomadijo et al. téristiques technologiques des levures au cours de l'élaboration et de l'affinage de quatre fabrications de fromage Armada, une variété artisanale élaborée au nord de l'Espagne à partir de lait cru de chèvre. Le nombre de microorganismes en milieu OGYEA (1,82 x 10 - 1,15 X 105 ufc·g-I de lait) augmentait d'une unité logarithmique dans le caillé et d'une autre unité logarithmique pendant la pre- mière semaine de maturation, restait stable jusqu'à 4 semaines puis diminuait jusqu'à la fin du pro- cessus d'affinage. Le pourcentage de levures identifiées à chacun des stades d'échantillonnage repré- sentait environ 75 % des isolements, excepté pour les fromages de 16 semaines (27 %). Des 181 souches de levures isolées, 60 étaient identifiées à Geotrichum candidum, 58 à Candida lambica, Il à Candida krusei, 4 à Candida lipolytica, 2 à Candida ingens, 1 à Candida catenulata, 1 à Can- dida pintolopesii, 1 à Candida colliculosa, 19 à Kluyveromyces lactis, Il à Kluyveromyces marxia- nus, 10 à Saccharomyces unisporus et 3 à Trichosporon beigelii. G. candidum était l'espèce majoritaire dans les fromages âgés de 1 et 2 semaines, C. lambica était l'espèce la plus abondante dans le lait et dans les fromages âgés de 4 et 8 semaines, tandis que C. krusei prédominait dans les fromages de 16 semaines. À en juger d'après leurs caractéristiques métaboliques, dans le fromage Armada les levures dont la résistance au sel n'est pas très élevée contribueraient à l'acidification initiale du caillé et à la consommation d'acide lactique tout au long du processus de maturation, mais, excepté G. candidum, elles interviendraient peu dans l'hydrolyse des lipides. En revanche, elles pourraient jouer un rôle important dans la dégradation des amines biogènes. Des 181 souches de levures identifiées, 136 assimilaient les quatre amines biogènes testées (cadaverine, histamine, putrescine et tiramine), et toutes les souches au moins l'une de ces amines. © Inra/Elsevier, Paris. fromage Armada / fromage de chèvre / espèce de levure / nombre de levure / propriété technologique

1. INTRODUCTION nological properties suggest an important intervention in the ripening process. Armada cheese is a hard variety made in In previous works we have enumerated the north of Spain from raw goat's milk throughout the ripening process the micro- using artisanal procedures without addition bial groups of technological interest iden- of starter cultures, which means that the tifying the lactic acid bacteria and entero- ripening process is performed only by the cocci [39] and we have also studied the natural flora of the milk. This, together with rnicroorganisms of the Micrococcaceae fam- the absence of any rigorous control of tem- ily [40]. The yeast and mould counts perature and relative humidity during the throughout the process (log cfu-g! about ripening process, makes the final product 5-6) [39] suggest that the microorganisms of very variable. This lack of uniformity limits this group could play an important role in its acceptance and distribution in the mark- the ripening of this cheese. This is why we ets. The use of its own specifie starters and have deemed it necessary to identify the the control of the ripening conditions would mycoflora at species level and to study the allow the making of a uniform product of main characteristics of the isolates through- constant quality capable of opening its way out the ripening process. to markets both at home and abroad. The yeasts can have many functions In order to elaborate an adequate starter throughout the ripening of the cheese. The culture for this cheese variety it is neces- yeasts which assimilate lactose contribute sary to carry out an extensive microbiolog- together with the lactic acid bacteria towards ical study of the artisanal cheese, identifying the drop in pH values which occur in the the microbial species which are present and earlier stages of the ripening. The yeasts selecting those whose abundance and tech- which also have fermentative ability, metab- Yeasts of Armada goat's milk cheese 649

olize lactose to ethanol and COz and from 2.2. Microbiological analysis ethanol esters of fatty acids are originated, which are responsible for the development Fifty g of each sample (after discarding the rind of the cheeses) were homogenized with of flavour in certain types of cheese. 200 mL of a sterile solution of 2 % sodium citrate Through the production of COz, the yeasts at 40--45 "C for 1 min in a Stomacher 400 Lab can also participate together with hetero- Blender (Seward Medical, London, England), thus making a 1/5 dilution. Consecutive decimal fermentative lactobacilli and leuconostoc dilutions were prepared by mixing 10 mL of the on opening the curd. Sorne yeasts have pro- previous dilution with 90 mL of 0.1 % sterile teolytic and lipolytic activities and can also peptone water. One mL of each dilution was play an important role in the development of inoculated in duplicate in 20 mL of Oxytetracy- cline Glucose Yeast-Extract Agar (Oxoid, Uni- the flavour in the cheese. Finally, other inter- path Ltd, Basingstoke, Hampshire, England) [28] esting activities of sorne yeasts are the influ- and mixed before solidification. Plates were incu- ence on the elimination of biogenic amines bated for 5 d at 22 oc. After incubation, plates with 30 to 300 colonies were counted. and the ability to metabolize lactic acid, thus allowing for the stablishing of other micro- bial groups sensitive to acid pHs as micro- 2.3. Isolation and identification cocci. ofstrains Therefore, the yeasts actively partake in From the count plates, 10 random colonies the transformations which occur during were taken from each sampling point with the ripening as they have a great enzymatic aid of a Harrison di sc [20]. The 274 isolates potential and contribute to the development obtained were purified and maintained in Sabouraud dextrose agar (Oxoid) tubes under of flavour and texture [29]. They have also refrigeration, covered with sterile parafin in order been the subject of many studies for being to reduce their metabolic activity and to avoid the most important contaminants in certain the agar dehydration. Of the 274 isolates carried types of rnilk products in which low pH val- out throughout the manufacturing and ripening process, 188 were considered yeasts and were ues offer a selective medium for their growth identified according to the methods and criteria [34]. proposed by Lodder [24] and Kreger- Van Rij [23]. The nomenclature of the species was made This article aims at identifying of yeast according to Barnett et al. [1]. Of each isolate species present throughout the ripening pro- the following characteristics were studied. cess of Armada goat' s cheese and the study of their possible role in the maturation. 2.3.1. Morphological characteristics

Morphology of the cells and type of vegetative reproduction, presence of pseudomycelium 2. MATE RIALS AND METHODS and/or true mycelium, production of ballis- tospores, and arthroconidia formation. 2.1. Cheese samples 2.3.2. Cultural characteristics Four batches of Armada cheese were elabo- rated by experienced cheesemakers according to Growth on solid medium (2 % Glucose-yeast- the tradition al methods as described by Tomadijo peptone agar after 2-3 d of incubation at 25 "C), et al. [39]. From each batch, milk, curd, and 1-, The aspect of the colonies (smooth, rough, level, dome shape, mucous), the colour and the pig- 2-,4-,8- and 16-week-old chee se samples were ment production were observed. Growth on liquid taken. Each cheese sample was constituted of medium (2 % Glucose-yeast-peptone broth after one whole cheese. Samples were transported to 2-3 d of incubation at 25 "C). The film formation, the laboratory under refrigeration (below 5 "C) aspect of the film, ring formation and sediment and analyzed on arrivaI. formation were observed. 650 M.E. Tornadijo et al.

2.3.3. Sexual characteristics 3. RESUL TS

Asci and ascospores formation and interfer- Table 1 shows the evolution of the counts tility of the yeasts. The presporulation media in OGYE agar during the manufacturing yeast malt broth and yeast malt agar were used, and ripening, and the percentage of yeasts at incubating at 25 "C for 1 or 2 d. From the cultures the different sampling times. Counts in growth in the presporulation media, cultures in OGYE agar increased one log unit in curd different sporulation media were made (the fol- and another log unit during the first week lowing media were tested before confirming that of ripening, remaining stable in cheese of they did not produce ascospores: Gorodkowa two and four weeks of ripening. From this agar, McClary's acetate agar, Kleyn's acetate point onwards, counts decreased until the agar. Vegetable juice agar. Corn me al agar, end of ripening. The percentage of yeasts Potato glucose agar, and Starkey's ethanol agar), in the isolates carried out remained fairly incubating at 25-28 "C and at 20 "C and observ- ing the production of ascospores at 3, 7, 14, and constant throughout the process, only falling 21 d of incubation. In order to investigate the at the last sampling time (l6-week-old interfertility of the yeasts and to prove that it did cheese). not de al with haploid types of heterothallic Of the 188 isolated yeast strains, 60 were species, the strains studied which did not pro- identified as Geotrichum candidum (Galac- duce any ascospores in the previously mentioned tomyces geotrichum), 58 as Candida lam- media were used. To do so, strains were taken four by four (strains of a same previously iden- bica (Pichia fermentans), Il as C. krusei tified species) and were inoculated in the afore- (Issatchenkia orientalis), 4 as C. lipolytica mentioned sporulation media in order to observe (Yarrowia lipolytica), 2 as C. ingens (Dipo- if there was ascospores formation. dascus ingens), 1 as C. catenulata, 1 as C. pintolopesii (Arxiozyma telluris), 1 as C. colliculosa (Torulaspora delbrueckii), 2.3.4. Biochemical characteristics 19 as Kluyveromyces lactis, Il as K. marx- ianus, 10 as Saccharomyces unisporus and 3 as Trichosporon beigelii. Seven strains Assimilation of glucose, lactose, maltose, raf- were not identified at species level in a reli- finose, cellobiose, galactose, sucrose, trehalose, ribitol, L-sorbose, D-xylose, L-arabinose and able way. Table II shows the distribution of inositol; fermentation of glucose, lactose, galac- the isolated species in the sampling points. tose, sucrose, maltose and melibiose; nitrate G. candidum (32 % of the isolated species) assimilation; urease. In order for a better iden- dominated in 1- and 2-week-old cheese. tification, in sorne strains the growth in a medium Among the typical yeasts, a clear predomi- without vitamins, and in presence of 100 ppm nance of anascosporogenous ones was of cycloheximide were studied. observed; C. lambica (31 % of the isolated species) was the predominant species in milk and in 4- and 8-week-old cheese, being C. krusei dominant in l ô-week-old cheese. 2.4. Study of sorne properties Of the sporogenous yeasts, K. lactis, of technological interest K. marxianus and S. unisporus predomi- nated. According to the methods described by Besançon et al. [3], on each isolate the following Table III shows the assimilation and the technological properties were studied: growth in fermentation of tested sugars by the isolated 5, 10, 15 and 20 % of salt; assimilation of lactose species. Table rv shows the morphological, at 0.5 %; assimilation of lactic acid at 0.5 %; cultural and sexual characteristics of the assimilation of tributyrin; assimilation of cadav- strains. The properties of technological erine, histamine, putrescine and tyramine. interest of these strains are shown in table V. Yeasts of Armada goat's milk cheese 651

4. DISCUSSION

Counts of moulds and yeasts throughout the ripening process of Armada cheese were similar to those observed in Valdeteja goat milk cheese [19]. However, they were higher than those observed in other Spanish goat

00 milk cheese varieties as [12], Ibores [26], and Cendrat dei Montsec [27]. According to our data, the oxytetracy- cline is not effective against bacteria at 100 %. Of the 274 strains isolated from OOYE agar during the manufacturing and ripening of Armada cheese, 188 were iden- tified as yeasts, 30 as moulds, and 56 were bacteria. The moulds and yeasts represented C'l <') only 80 % of the total strains isolated. ci o +1 <') Geotrichum candidum was one of the ~o -.0 main yeast species during the manufacturing and ripening of Armada cheese, being dom-

00 inant in the earliest stages of the ripening. ci- The majority of the strains of G. candidum +1 isolated from Armada cheese and from.the 0\ \0 milk used in its making morphologically -.0 belong to the type 1 described by Guéguen

o and Jacquet [17], only 9 strains could be Vl considered somewhat closer to type 2 of "E ci ::l +1 Guéguen and Jacquet as they showed a whit- U ~ ish and dusty aspect. G. candidum is present Vl on the surface of many cheeses [16] where the high levels of fatty acids found are mainly due to the action of their enzymes. o <') This species can exert a protection against the development of undesirable moulds of the Mucor genus [6,9, 18]. In our study we isolated Candida lam- bica at high proportions in milk, curd and cheese at different stages of ripening which indicates that it can easily adapt to the Aw, pH and salt concentration conditions found .@ in this cheese and it is highly competitive . ~00 BaroiIIer and Schmidt [2] isolated w >-- anascosporogenous yeasts at high levels o from agricultural exploitations (silages, o .S water, soil, and so on) with C. lambica being C/J particularly important. However, these 'i:: ::l o authors did not isolate this species in milk U not even during the ripening of the cheeses. 652 M.E. Tornadijo et al.

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They regarded it as contaminant flora, which can play an important role in the aroma in cheese is seen as being displaced by other development of the cheese increasing the species better adapted to this food, such as volatile fatty acid content [ID, 33], its pres- K. lactis and its asexual form Candida· ence in different types of cheeses has been sphaerica. In the evolution of C. lambica widely demonstrated [10, 25, 30]; Nahabieh throughout the ripening process of Armada and Schmidt [29] isolated this species in chee se only a slight drop in one- and two- French goat's cheeses in its sexual and asex- week-old chee se was observed where ual forms in proportions of 13 % of isola- G. candidum predominated, and in sixteen- tions, being the dominant species in Crot- week-old chee se where it is hardly present tin de Chavignol cheese throughout the and is surpassed by C. krusei. ripening process. It is unusual to isolate C. lambica as the K. lactis was isolated in important per- main yeast species from chee se, although it centages in one-week-old cheese, represent- is usually found in not quite negligible per- ing approximately 28 % of the isolated centages in sorne studies [7, 37]~ C. lam- yeasts at this sampling point. This species bica was also isolated in French goat's approximately made up 10 % of ail isolated cheeses such as Crottin de Chavignol and yeasts in Armada cheese. K. marxianus rep- Pouligny Saint-Pierre [29], although at not resented 6 % of all isolated yeasts in Armada important levels. Moreover C. lambica is cheese, with its presence being outstanding the imperfect state of Pichia fermentans, in one-week-old cheese. The importance of which was found in butter milk, kefyr and K. lactis and K. marxianus in prod- cheese [1]. In addition, in the review by ucts, because they are able to metabolize Fleet [Il] the perfect state P. fermentans is lactose, is weil known. The reported data reported as a frequently found species in confirm that point. Sorne authors [10] have dairy products. attributed to K. lactis the ability to act on roqueforti by stimulating the Other species of the genus Candida iso- production of metilketones. Georgantas [15] lated in Armada chee se were C. lipolytica, also attributes it a stimulating effect on the C. krusei, C. ingens, C. catenulata, C. pin- production of acid through lactococci. tolopesii and C. colliculosa. C. krusei was also isolated by Sala Trepat and Burgos [35] Saccharomyces unisporus was the only in Cabrales cheese, by Deiana et al. [8] in isolated species of the Saccharomyces genus goat's cheese from different places in Sar- and constituted 5 % of ail the identified· dinia and by Cooke and Brazis [5] from raw yeasts from Armada cheese. This species milk, pasteurized milk and different milk was also identified by Nüûez et al. [32] from products, cheese being included. Nüüez et al. Cabrales cheese, making up 3 % of the iso- [32] also described this species {Candida lates obtained in this cheese. krusei was previously denominated Pichia Trichosporon beigelii is a species which membranaefaciens) as being dominant in always cornes from contaminations. In our the inner of Cabrales cheese during ripening. study it represented 1.5 % of isolates car- C. catenulata was frequently observed in ried out. Ntiûez et al. [32] isolated this Pont-L' Evêque cheeses, representing 24 % species in milk destined for the making of of the isolates in this cheese varietie [4]; Cabrales cheese. Nahabieh and Schmidt [29] this species was also isolated by Nooitgedagt isolated it in very low proportions, similar to and Hargot [31] from Brie and Camembert ours, in goat's cheese. In sorne cheeses, cheeses. C. pintolopesii was also identified however, it makes up the main species; in by Nahabieh and Schmidt [29] from goat's Kopanisti cheese, for example, Karninar- cheese at a percentage of 15 % of the total ides and Anifantakis [22] identified as isolates, nevertheless these authors point T. beigelii 61 of the 86 yeast strains iso- out its presence to be unusual. C. lipolytica lated. Yeasts of Armada goat's milk cheese 657

With regard to the biochemical and tech- C. lipolytica, 2 from C. ingens and 3 from nological characteristics of the yeasts isol- T. beigelii) were able of hydrolyzing the ated from Armada cheese, only the K. lactis tributyrin. and K. marxianus strains were able to assim- Of the 181 strains identified, 13 (60 from ilate and ferment lactose which in agree- G. candidum, 58 from C. lambica, Il from ment with Cooke and Brazis [5] leads us to C. krusei, 4 from C. lipolytica, 2 from conclude that the most part of the species C. ingens and 1 from C. pintolopesii) assim- we isolated are casual contaminants and not ilated the four biogenic amines tested. Ail 'selfsame flora' of chee se and milk. The the identified strains assimilated at least one K. lactis and K. marxianus strains possibly of these amines. contribute, together with the lactic acid bac- teria, to the degradation of lactose which Ali the G. candidum strains isolated takes place in the early stages of the ripen- showed a good growth at 5 % NaCl. G. can- ing process of this cheese [13]. The 90 % didum has been known for a long time to of the strains identified (the G. candidum, be particularly sensitive to salt with a total K. lactis, K. marxianus, C. lambica, C. kru- inhibition at concentrations of 5-6 % [36]. sei, C. lipolytica, C. catenulata and C. pin- In this sense these strains have an excep- tolopesii strains) showed the ability to assim- tional behaviour. ilate lactic acid. These species could actively The isolated yeasts do not seem to be participate in the consumption of lactic acid very resistant to salt and only a few of them and be responsible for the fall in D and L are capable of growing at NaCI concentra- lactic acid contents observed in Armada tions higher than 5 %. Only 12 strains of cheese from the first week of ripening [13]. K. lactis and 4 of K. marxianus were able to grow at a 15 % NaCI concentration. The G. candidum seems to play a role in the resistance to high salt concentrations is lipolysis in Armada cheese. According to regarded as being a very important charac- Sidebottom et al. [38] G. candidum pro- teristic in choosing starter cultures for cheese duces 2 lipases: lipase A (not specifie) and making [3]. lipase B (specifie for esters of cis-6-9 fatty acids) with a preference for C18:1(oleic acid) [21], although it also frees to a lower extent ACKNOWLEDGEMENTS saturated fatty acids. In view to the profile of free fatty acids observed in Armada cheese This study was financially supported by the throughout ripening [14], G. candidum Junta de Castilla y Leon (the Regional Govern- seems to play an important role in the degra- ment). Author M E Tornadijo was awarded a dation of the lipids in the first stages of grant from the Consejerfa de Agricultura y ripening. In this cheese, the ratio of the main Ganaderfa of the Junta de Castilla y Leon. free fatty acids CI6/CI8:1 is very low (0.5) and favourable to C18:1in the first weeks of ripening when G. candidum predominates, REFERENCES which agrees with the lipase B activity of [1] Barnett J.A., Payne R.W., Yarrow D., Yeasts: G. candidum. This ratio increases up to 0.85 characteristics and identification, 2nd. ed., Bar- as G. candidum decreases from the second nell J.A., Payne R.W., Yarrow D. (Eds.), Cam- month of ripening. bridge University Press, Cambridge, UK, 1990. [2J Baroiller C, Schmidt J.L., Contribution à l'étude The rest of yeasts from Armada chee se de l'origine des levures du fromage de Camem- do not seem to take part in the lipid hydrol- bert, Lait 70 (1990) 67-84. ysis which takes place throughout the ripen- [3] Besançon X., Smet C., Chabalier c., Rive- male M., ReverlJel J.P., Ratomahenina R., Galzy P., ing process of this chee se. Of ail the typical Study of surface yeast flora of cheese, yeast strains isolated, only 9 (4 from Int.J. Food Microbiol. 17 (1992) 9-18. 658 M.E. Tornadijo et al.

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