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

Transcription

1 Study f the yeasts during the ripening prcess f Armada cheeses frm raw gat s milk Maria E. Trnadij, Jsé M. Fresn, Rbert Martin Sarmient, Javier Carball T cite this versin: Maria E. Trnadij, Jsé M. Fresn, Rbert Martin Sarmient, Javier Carball. Study f the yeasts during the ripening prcess f Armada cheeses frm raw gat s milk. Le Lait, INRA Editins, 1998, 78 (6), pp <hal > HAL Id: hal Submitted n 1 Jan 1998 HAL is a multi-disciplinary pen access archive fr the depsit and disseminatin f scientific research dcuments, whether they are published r nt. The dcuments may cme frm teaching and research institutins in France r abrad, r frm public r private research centers. L archive uverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusin de dcuments scientifiques de niveau recherche, publiés u nn, émanant des établissements d enseignement et de recherche français u étrangers, des labratires publics u privés.

2 Lait (1998) 78, InralElsevier, Paris 647 Original article Study f the yeasts during the ripening prcess f Armada cheeses frm raw gat's milk Maria E. Trnadij", Jsé M. Fresn", Rbert Martin Sarmient", Javier Carball?" a Departament de Higiene y Tecnlgfa de ls Aliments, Universidad de Len, Len, Spain b Area de Tecnlgfa de ls Aliments, Facultad de Ciencias de Orense, Universidad de Vig, Orense, Spain (Received 12 May 1997; accepted 25 May 1998) Abstract - The cunts, species and characteristics f yeasts were studied thrughut the manufacturing and ripening f fur batches f Armada chee se, a craft variety made in the nrth f Spain frm raw gat's milk. Cunts in Oxytetracycline Glucse Yeast-Extract Agar (1.82 x x los cfu g- 1 in milk) increased ne lg. unit in curd and anther lg. unit during the first week fripening, remaining stable in tw- and fur-week-ld cheeses and decreasing frm this pint t the end f the ripening prcess. The yeast percentage f the islates carried ut remained practically cnstant at arund 75 % thrughut the ripening prcess, drpping t 27 % at the last sampling pint (16-week-ld cheese). Of the 181 islated yeast strains, 60 were identified as Getrichum candidum, 58 as Candida lambica, II as Candida krusei, 4 as Candida liplytica, 2 as Candida ingens, 1 as Candida catenulata, 1 as Candida pintlpesii, 1 as Candida clliculsa, 19 as Kluyvermyces lactis, II as Kluyvermyces marxianus, 10 as Saccharmyces unisprus and 3 as Trichsprn beigelii. G. candidum predminated in 1- and 2-week-ld cheeses and C. lambica was the mst abundant species in milk and in 4- and 8-week-ld cheese while C. krusei dminated in 16-week-ld cheese. Judging frm their metablic characteristics, the yeasts in Armada cheese seem t participate in the initial acidificatin f the curd and in the cnsumptin f lactic acid which takes place thrughut the ripening prcess, thugh, with the exceptin f G. candidum, they d nt seem t play a relevant rle in the liplysis, either way its resistance t NaCI is nt very high. Hwever, they seem t play an imprtant rle in the degradatin f the bigenic amines; f the 181 strains identified, 136 assimilated the fur bigenic amines tested (cadaverine, histamine, putrescine and tyramine) and ail the strains assimilated at least ne fthese amines. Inra/Elsevier, Paris. Armada cheesel gat cheesel yeast speciesl yeast cunt 1 techn1gical prperty Résumé - Étude des levures pendant la fabricatin et l'affinage des frmages de chèvre au lait cru de la variété Armada. L'étude a prté sur le dénmbrement, l'identificatin et quelques carac- * Crrespndence and reprints.

3 648 M.E. Tmadij et al. téristiques technlgiques des levures au curs de l'élabratin et de l'affinage de quatre fabricatins de frmage Armada, une variété artisanale élabrée au nrd de l'espagne à partir de lait cru de chèvre. Le nmbre de micrrganismes en milieu OGYEA (1,82 x 10-1,15 X 10 5 ufc g- I de lait) augmentait d'une unité lgarithmique dans le caillé et d'une autre unité lgarithmique pendant la première semaine de maturatin, restait stable jusqu'à 4 semaines puis diminuait jusqu'à la fin du prcessus d'affinage. Le purcentage de levures identifiées à chacun des stades d'échantillnnage représentait envirn 75 % des islements, excepté pur les frmages de 16 semaines (27 %). Des 181 suches de levures islées, 60 étaient identifiées à Getrichum candidum, 58 à Candida lambica, Il à Candida krusei, 4 à Candida liplytica, 2 à Candida ingens, 1 à Candida catenulata, 1 à Candida pintlpesii, 1 à Candida clliculsa, 19 à Kluyvermyces lactis, Il à Kluyvermyces marxianus, 10 à Saccharmyces unisprus et 3 à Trichsprn beigelii. G. candidum était l'espèce majritaire dans les frmages âgés de 1 et 2 semaines, C. lambica était l'espèce la plus abndante dans le lait et dans les frmages âgés de 4 et 8 semaines, tandis que C. krusei prédminait dans les frmages de 16 semaines. À en juger d'après leurs caractéristiques métabliques, dans le frmage Armada les levures dnt la résistance au sel n'est pas très élevée cntribueraient à l'acidificatin initiale du caillé et à la cnsmmatin d'acide lactique tut au lng du prcessus de maturatin, mais, excepté G. candidum, elles interviendraient peu dans l'hydrlyse des lipides. En revanche, elles purraient juer un rôle imprtant dans la dégradatin des amines bigènes. Des 181 suches de levures identifiées, 136 assimilaient les quatre amines bigènes testées (cadaverine, histamine, putrescine et tiramine), et tutes les suches au mins l'une de ces amines. Inra/Elsevier, Paris. frmage Armada / frmage de chèvre / espèce de levure / nmbre de levure / prpriété technlgique 1. INTRODUCTION Armada cheese is a hard variety made in the nrth f Spain frm raw gat's milk using artisanal prcedures withut additin f starter cultures, which means that the ripening prcess is perfrmed nly by the natural flra f the milk. This, tgether with the absence f any rigrus cntrl f temperature and relative humidity during the ripening prcess, makes the final prduct very variable. This lack f unifrmity limits its acceptance and distributin in the markets. The use f its wn specifie starters and the cntrl f the ripening cnditins wuld allw the making f a unifrm prduct f cnstant quality capable f pening its way t markets bth at hme and abrad. In rder t elabrate an adequate starter culture fr this cheese variety it is necessary t carry ut an extensive micrbilgical study f the artisanal cheese, identifying the micrbial species which are present and selecting thse whse abundance and technlgical prperties suggest an imprtant interventin in the ripening prcess. In previus wrks we have enumerated thrughut the ripening prcess the micrbial grups f technlgical interest identifying the lactic acid bacteria and enterccci [39] and we have als studied the rnicrrganisms f the Micrcccaceae family [40]. The yeast and muld cunts thrughut the prcess (lg cfu-g! abut 5-6) [39] suggest that the micrrganisms f this grup culd play an imprtant rle in the ripening f this cheese. This is why we have deemed it necessary t identify the mycflra at species level and t study the main characteristics f the islates thrughut the ripening prcess. The yeasts can have many functins thrughut the ripening f the cheese. The yeasts which assimilate lactse cntribute tgether with the lactic acid bacteria twards the drp in ph values which ccur in the earlier stages f the ripening. The yeasts which als have fermentative ability, metab-

4 Yeasts f Armada gat's milk cheese 649 lize lactse t ethanl and COz and frm ethanl esters f fatty acids are riginated, which are respnsible fr the develpment f flavur in certain types f cheese. Thrugh the prductin f COz, the yeasts can als participate tgether with heterfermentative lactbacilli and leucnstc n pening the curd. Srne yeasts have prtelytic and liplytic activities and can als play an imprtant rle in the develpment f the flavur in the cheese. Finally, ther interesting activities f srne yeasts are the influence n the eliminatin f bigenic amines and the ability t metablize lactic acid, thus allwing fr the stablishing f ther micrbial grups sensitive t acid phs as micrccci. Therefre, the yeasts actively partake in the transfrmatins which ccur during ripening as they have a great enzymatic ptential and cntribute t the develpment f flavur and texture [29]. They have als been the subject f many studies fr being the mst imprtant cntaminants in certain types f rnilk prducts in which lw ph values ffer a selective medium fr their grwth [34]. This article aims at identifying f yeast species present thrughut the ripening prcess f Armada gat' s cheese and the study f their pssible rle in the maturatin. 2. MATE RIALS AND METHODS 2.1. Cheese samples Fur batches f Armada cheese were elabrated by experienced cheesemakers accrding t the traditin al methds as described by Tmadij et al. [39]. Frm each batch, milk, curd, and 1-, 2-,4-,8- and 16-week-ld chee se samples were taken. Each cheese sample was cnstituted f ne whle cheese. Samples were transprted t the labratry under refrigeratin (belw 5 "C) and analyzed n arrivai Micrbilgical analysis Fifty g f each sample (after discarding the rind f the cheeses) were hmgenized with 200 ml f a sterile slutin f 2 % sdium citrate at "C fr 1 min in a Stmacher 400 Lab Blender (Seward Medical, Lndn, England), thus making a 1/5 dilutin. Cnsecutive decimal dilutins were prepared by mixing 10 ml f the previus dilutin with 90 ml f 0.1 % sterile peptne water. One ml f each dilutin was inculated in duplicate in 20 ml f Oxytetracycline Glucse Yeast-Extract Agar (Oxid, Unipath Ltd, Basingstke, Hampshire, England) [28] and mixed befre slidificatin. Plates were incubated fr 5 d at 22 c. After incubatin, plates with 30 t 300 clnies were cunted Islatin and identificatin fstrains Frm the cunt plates, 10 randm clnies were taken frm each sampling pint with the aid f a Harrisn di sc [20]. The 274 islates btained were purified and maintained in Saburaud dextrse agar (Oxid) tubes under refrigeratin, cvered with sterile parafin in rder t reduce their metablic activity and t avid the agar dehydratin. Of the 274 islates carried ut thrughut the manufacturing and ripening prcess, 188 were cnsidered yeasts and were identified accrding t the methds and criteria prpsed by Ldder [24] and Kreger- Van Rij [23]. The nmenclature f the species was made accrding t Barnett et al. [1]. Of each islate the fllwing characteristics were studied Mrphlgical characteristics Mrphlgy f the cells and type f vegetative reprductin, presence f pseudmycelium and/r true mycelium, prductin f ballistspres, and arthrcnidia frmatin Cultural characteristics Grwth n slid medium (2 % Glucse-yeastpeptne agar after 2-3 d f incubatin at 25 "C), The aspect f the clnies (smth, rugh, level, dme shape, mucus), the clur and the pigment prductin were bserved. Grwth n liquid medium (2 % Glucse-yeast-peptne brth after 2-3 d f incubatin at 25 "C). The film frmatin, aspect f the film, ring frmatin and sediment frmatin were bserved.

5 650 M.E. Trnadij et al Sexual characteristics 3. RESUL TS Asci and ascspres frmatin and interfertility f the yeasts. The presprulatin media yeast malt brth and yeast malt agar were used, incubating at 25 "C fr 1 r 2 d. Frm the cultures grwth in the presprulatin media, cultures in different sprulatin media were made (the fllwing media were tested befre cnfirming that they did nt prduce ascspres: Grdkwa agar, McClary's acetate agar, Kleyn's acetate agar. Vegetable juice agar. Crn me al agar, Ptat glucse agar, and Starkey's ethanl agar), incubating at "C and at 20 "C and bserving the prductin f ascspres at 3, 7, 14, and 21 d f incubatin. In rder t investigate the interfertility f the yeasts and t prve that it did nt de al with haplid types f heterthallic species, the strains studied which did nt prduce any ascspres in the previusly mentined media were used. T d s, strains were taken fur by fur (strains f a same previusly identified species) and were inculated in the afrementined sprulatin media in rder t bserve if there was ascspres frmatin Bichemical characteristics Assimilatin f glucse, lactse, maltse, raffinse, cellbise, galactse, sucrse, trehalse, ribitl, L-srbse, D-xylse, L-arabinse and insitl; fermentatin f glucse, lactse, galactse, sucrse, maltse and melibise; nitrate assimilatin; urease. In rder fr a better identificatin, in srne strains the grwth in a medium withut vitamins, and in presence f 100 ppm f cyclheximide were studied Study f srne prperties f technlgical interest Accrding t the methds described by Besançn et al. [3], n each islate the fllwing technlgical prperties were studied: grwth in 5, 10, 15 and 20 % f salt; assimilatin f lactse at 0.5 %; assimilatin f lactic acid at 0.5 %; assimilatin f tributyrin; assimilatin f cadaverine, histamine, putrescine and tyramine. Table 1 shws the evlutin f the cunts in OGYE agar during the manufacturing and ripening, and the percentage f yeasts at the different sampling times. Cunts in OGYE agar increased ne lg unit in curd and anther lg unit during the first week f ripening, remaining stable in cheese f tw and fur weeks f ripening. Frm this pint nwards, cunts decreased until the end f ripening. The percentage f yeasts in the islates carried ut remained fairly cnstant thrughut the prcess, nly falling at the last sampling time (l6-week-ld cheese). Of the 188 islated yeast strains, 60 were identified as Getrichum candidum (Galactmyces getrichum), 58 as Candida lambica (Pichia fermentans), Il as C. krusei (Issatchenkia rientalis), 4 as C. liplytica (Yarrwia liplytica), 2 as C. ingens (Dipdascus ingens), 1 as C. catenulata, 1 as C. pintlpesii (Arxizyma telluris), 1 as C. clliculsa (Trulaspra delbrueckii), 19 as Kluyvermyces lactis, Il as K. marxianus, 10 as Saccharmyces unisprus and 3 as Trichsprn beigelii. Seven strains were nt identified at species level in a reliable way. Table II shws the distributin f the islated species in the sampling pints. G. candidum (32 % f the islated species) dminated in 1- and 2-week-ld cheese. Amng the typical yeasts, a clear predminance f anascsprgenus nes was bserved; C. lambica (31 % f the islated species) was the predminant species in milk and in 4- and 8-week-ld cheese, being C. krusei dminant in l ô-week-ld cheese. Of the sprgenus yeasts, K. lactis, K. marxianus and S. unisprus predminated. Table III shws the assimilatin and the fermentatin f tested sugars by the islated species. Table rv shws the mrphlgical, cultural and sexual characteristics f the strains. The prperties f technlgical interest f these strains are shwn in table V.

6 Yeasts f Armada gat's milk cheese DISCUSSION 00 Cunts f mulds and yeasts thrughut the ripening prcess f Armada cheese were similar t thse bserved in Valdeteja gat milk cheese [19]. Hwever, they were higher than thse bserved in ther Spanish gat milk cheese varieties as Majrer [12], Ibres [26], and Cendrat dei Mntsec [27]. "E ::l U C'l <') ci ci +1 0\ \0 -.0 Vl ci +1 Vl <') <') Accrding t ur data, the xytetracycline is nt effective against bacteria at 100 %. Of the 274 strains islated frm OOYE agar during the manufacturing and ripening f Armada cheese, 188 were identified as yeasts, 30 as mulds, and 56 were bacteria. The mulds and yeasts represented nly 80 % f the ttal strains islated. Getrichum candidum was ne f the main yeast species during the manufacturing and ripening f Armada cheese, being dminant in the earliest stages f the ripening. The majrity f the strains f G. candidum islated frm Armada cheese and frm.the milk used in its making mrphlgically belng t the type 1 described by Guéguen and Jacquet [17], nly 9 strains culd be cnsidered smewhat clser t type 2 f Guéguen and Jacquet as they shwed a whitish and dusty aspect. G. candidum is present n the surface f many cheeses [16] where the high levels f fatty acids fund are mainly due t the actin f their enzymes. This species can exert a prtectin against the develpment f undesirable mulds f the Mucr genus [6,9, 00 w >--.S C/J 'i:: ::l U In ur study we islated Candida lambica at high prprtins in milk, curd and cheese at different stages f ripening which indicates that it can easily adapt t the Aw, ph and salt cncentratin cnditins fund in this cheese and it is highly cmpetitive. BariIIer and Schmidt [2] islated anascsprgenus yeasts at high levels frm agricultural explitatins (silages, water, sil, and s n) with C. lambica being particularly imprtant. Hwever, these authrs did nt islate this species in milk nt even during the ripening f the cheeses.

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11 656 M.E. Tmadij et al. They regarded it as cntaminant flra, which in cheese is seen as being displaced by ther species better adapted t this fd, such as K. lactis and its asexual frm Candida sphaerica. In the evlutin f C. lambica thrughut the ripening prcess f Armada chee se nly a slight drp in ne- and twweek-ld chee se was bserved where G. candidum predminated, and in sixteenweek-ld chee se where it is hardly present and is surpassed by C. krusei. It is unusual t islate C. lambica as the main yeast species frm chee se, althugh it is usually fund in nt quite negligible percentages in srne studies [7, 37] C. lambica was als islated in French gat's cheeses such as Crttin de Chavignl and Puligny Saint-Pierre [29], althugh at nt imprtant levels. Mrever C. lambica is the imperfect state f Pichia fermentans, which was fund in butter milk, kefyr and cheese [1]. In additin, in the review by Fleet [Il] the perfect state P. fermentans is reprted as a frequently fund species in dairy prducts. Other species f the genus Candida islated in Armada chee se were C. liplytica, C. krusei, C. ingens, C. catenulata, C. pintlpesii and C. clliculsa. C. krusei was als islated by Sala Trepat and Burgs [35] in Cabrales cheese, by Deiana et al. [8] in gat's cheese frm different places in Sardinia and by Cke and Brazis [5] frm raw milk, pasteurized milk and different milk prducts, cheese being included. Nüüez et al. [32] als described this species {Candida krusei was previusly denminated Pichia membranaefaciens) as being dminant in the inner f Cabrales cheese during ripening. C. catenulata was frequently bserved in Pnt-L' Evêque cheeses, representing 24 % f the islates in this cheese varietie [4]; this species was als islated by Nitgedagt and Hargt [31] frm Brie and Camembert cheeses. C. pintlpesii was als identified by Nahabieh and Schmidt [29] frm gat's cheese at a percentage f 15 % f the ttal islates, nevertheless these authrs pint ut its presence t be unusual. C. liplytica can play an imprtant rle in the arma develpment f the cheese increasing the vlatile fatty acid cntent [ID, 33], its presence in different types f cheeses has been widely demnstrated [10, 25, 30]; Nahabieh and Schmidt [29] islated this species in French gat's cheeses in its sexual and asexual frms in prprtins f 13 % f islatins, being the dminant species in Crttin de Chavignl cheese thrughut the ripening prcess. K. lactis was islated in imprtant percentages in ne-week-ld cheese, representing apprximately 28 % f the islated yeasts at this sampling pint. This species apprximately made up 10 % f ail islated yeasts in Armada cheese. K. marxianus represented 6 % f all islated yeasts in Armada cheese, with its presence being utstanding in ne-week-ld cheese. The imprtance f K. lactis and K. marxianus in dairy prducts, because they are able t metablize lactse, is weil knwn. The reprted data cnfirm that pint. Srne authrs [10] have attributed t K. lactis the ability t act n Penicillium rquefrti by stimulating the prductin f metilketnes. Gergantas [15] als attributes it a stimulating effect n the prductin f acid thrugh lactccci. Saccharmyces unisprus was the nly islated species f the Saccharmyces genus and cnstituted 5 % f ail the identified yeasts frm Armada cheese. This species was als identified by Nüûez et al. [32] frm Cabrales cheese, making up 3 % f the islates btained in this cheese. Trichsprn beigelii is a species which always crnes frm cntaminatins. In ur study it represented 1.5 % f islates carried ut. Ntiûez et al. [32] islated this species in milk destined fr the making f Cabrales cheese. Nahabieh and Schmidt [29] islated it in very lw prprtins, similar t urs, in gat's cheese. In srne cheeses, hwever, it makes up the main species; in Kpanisti cheese, fr example, Karninarides and Anifantakis [22] identified as T. beigelii 61 f the 86 yeast strains islated.

12 Yeasts f Armada gat's milk cheese 657 With regard t the bichemical and technlgical characteristics f the yeasts islated frm Armada cheese, nly the K. lactis and K. marxianus strains were able t assimilate and ferment lactse which in agreement with Cke and Brazis [5] leads us t cnclude that the mst part f the species we islated are casual cntaminants and nt 'selfsame flra' f chee se and milk. The K. lactis and K. marxianus strains pssibly cntribute, tgether with the lactic acid bacteria, t the degradatin f lactse which takes place in the early stages f the ripening prcess f this cheese [13]. The 90 % f the strains identified (the G. candidum, K. lactis, K. marxianus, C. lambica, C. krusei, C. liplytica, C. catenulata and C. pintlpesii strains) shwed the ability t assimilate lactic acid. These species culd actively participate in the cnsumptin f lactic acid and be respnsible fr the fall in D and L lactic acid cntents bserved in Armada cheese frm the first week f ripening [13]. G. candidum seems t play a rle in the liplysis in Armada cheese. Accrding t Sidebttm et al. [38] G. candidum prduces 2 lipases: lipase A (nt specifie) and lipase B (specifie fr esters f cis-6-9 fatty acids) with a preference fr C 18 : 1 (leic acid) [21], althugh it als frees t a lwer extent saturated fatty acids. In view t the prfile f free fatty acids bserved in Armada cheese thrughut ripening [14], G. candidum seems t play an imprtant rle in the degradatin f the lipids in the first stages f ripening. In this cheese, the rati f the main free fatty acids CI6/CI8:1 is very lw (0.5) and favurable t C 18 : 1 in the first weeks f ripening when G. candidum predminates, which agrees with the lipase B activity f G. candidum. This rati increases up t 0.85 as G. candidum decreases frm the secnd mnth f ripening. The rest f yeasts frm Armada chee se d nt seem t take part in the lipid hydrlysis which takes place thrughut the ripening prcess f this chee se. Of ail the typical yeast strains islated, nly 9 (4 frm C. liplytica, 2 frm C. ingens and 3 frm T. beigelii) were able f hydrlyzing the tributyrin. Of the 181 strains identified, 13 (60 frm G. candidum, 58 frm C. lambica, Il frm C. krusei, 4 frm C. liplytica, 2 frm C. ingens and 1 frm C. pintlpesii) assimilated the fur bigenic amines tested. Ail the identified strains assimilated at least ne f these amines. Ali the G. candidum strains islated shwed a gd grwth at 5 % NaCl. G. candidum has been knwn fr a lng time t be particularly sensitive t salt with a ttal inhibitin at cncentratins f 5-6 % [36]. In this sense these strains have an exceptinal behaviur. The islated yeasts d nt seem t be very resistant t salt and nly a few f them are capable f grwing at NaCI cncentratins higher than 5 %. Only 12 strains f K. lactis and 4 f K. marxianus were able t grw at a 15 % NaCI cncentratin. The resistance t high salt cncentratins is regarded as being a very imprtant characteristic in chsing starter cultures fr cheese making [3]. ACKNOWLEDGEMENTS This study was financially supprted by the Junta de Castilla y Len (the Reginal Gvernment). Authr M E Trnadij was awarded a grant frm the Cnsejerfa de Agricultura y Ganaderfa f the Junta de Castilla y Len. REFERENCES [1] Barnett J.A., Payne R.W., Yarrw D., Yeasts: characteristics and identificatin, 2nd. ed., Barnell J.A., Payne R.W., Yarrw D. (Eds.), Cambridge University Press, Cambridge, UK, [2J Bariller C, Schmidt J.L., Cntributin à l'étude de l'rigine des levures du frmage de Camembert, Lait 70 (1990) [3] Besançn X., Smet C., Chabalier c., Rivemale M., ReverlJel J.P., Ratmahenina R., Galzy P., Study f surface yeast flra f Rquefrt cheese, Int.J. Fd Micrbil. 17 (1992) 9-18.

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