Growth of Natural Flora during the Fermentation of Inoculated Musts from "Pedro Ximenez" Grapes

Transcription

1 Growth of Natural Flora during the Fermentation of noulated Musts from "Pedro Ximenez" Grapes J. Martinez, C. Millan and J.M. Ortega Department of Mirobiology, Faulty of Sienes, University of C6rdoba, Avda. San Alberto Magno s/n, 144 C6rdoba, Spain Submitted for publiation: February 199 Aepted for publiation: June 199 Keywords: yeast, fennentation, wine, S. erevisiae The growth of naturally ourring miro-organisms in musts from grapes olleted at three degrees ofripeness (unripe, half ripe and ripe) in the Montilla-Moriles (Southern Spain) region during 19and196 was studied. The musts were inoulated with four physiologial raes of Saharomyes erevisiae isolated in the region. Although the inoulated raes of S. erevisiae prevailed throughout the fermentations, there was also a signifiant growth of indigenous raes of S. erevisiae and a less marked growth of other miro-organisms suh as bateria, filamentous fungi and yeasts other than S. erevisiae. Wine makers have traditionally relied on the fermentation of grape musts by yeasts originally present in the grapes or in the ellar. Yeasts of the genera Kloekera, Candida, Pihia, Hansenula, Rhodotorula and Torulaspora grow during the first few days of fermentation and disappear during the later stages, when prevailing Saharomyes erevisiae strains, omplete the proess (fiigo and Arroyo, 196; Benda, 1962; Khayyat et al., 192; Mauriio et al., 196; Fleet et al., 194). As a whole, the non S. erevisiae speies produe a broader spetrum of aroma ompounds (Amerine et al., 192; Benda, 191), whih is desirable in regions with well-established enologial features. The inoulation of musts with seleted indigenous yeasts an offer advantages suh as faster and more ontrolled fermentations and the prodution of wines of more uniform quality provided they prevail over the naturally ourring flora, whose growth is then kept within moderate bounds. Heard and Fleet (19) studied the growth ofnatural flora in inoulated grape musts but failed to determine whether the S. erevisiae strain prevailing at the end of fermentation was that originally inoulated or if, on the ontrary, it was a naturally ourring strain whih took over the proess. n this work we studied the growth ofnatural flora in wines inoulated with four indigenous raes of S. erevisiae isolated in the Montilla-Moriles region by identifiation of their olo- nies in Agar-Bismuth Sulphite medium in order to hek whether the inoulated raes prevailed over the indigenous yeasts of the musts throughout the fermentation. MATRALS AND MTHODS The experiments were arried out on musts obtained from grapes of the Vitis vinifera "Pedro Ximenez" variety olleted at three degrees of ripeness, namely unripe (must ), half-ripe (must ) and ripe (must ), in the 19 and 196 vintages. The diminishing sugar onentration of the musts was determined by density measurements and is listed in Table 1. Although the musts were not sterilized, half their volume was entrifuged in order to redue their suspended solid fration to about 3,% (W N). Sulphur dioxide at a onentration of 12 mg/! and enough tartari aid to adjust the ph to 3,2 were added; these onditions are thought to be those prevailing in the fermentations arried out in the region (Mauriio et al., 196). The yeasts used in the inoula were four physiologial raes of Saharomyes erevisiae, viz. strains erevisiae A (maltose: ferm, aid and gas), erevisiae B (maltose: ferm. aid Assim. + ), hevalieri and apensis (Kreger van Rij, 194). The musts were inoulated with a 2% (V N) inoulum of pure ultures (36-h old) of eah of the seleted yeasts. Raes erevisiae A and B were used to ferment the musts of the 19 TABL 1 Sugar onentration in the musts and aloholi degree reahed in eah fermentation Sugar ontent Year Must (g/l) S. erevisiae raes used erevisiae A erevisiae B hevalieri thanol ontent(% VN) in the wines apensis 19 17, 226,9 276, , 21, 231,9 9, 11, 14,34 9,31 12,3 14, 1,7 12,9 12,69 1,39 12, 13,1 S. Afr. J. nol. Viti., Vot.-4-Nr.2"' ( 12-) 1 19! 31

2 32 Growth of Natural Flora during Wine Fermentation noulated rae: erevisiae A noulated rae: erevisiae B.. en _, 6 A r ~ ::~~ ,-,~--.,r---,~~~~~! Hou rs Hou rs A 9-r-~-~~~~~~~~~~~~~~ noulated rae: apens is noulated rae: hevalieri ':::::- Q) u en _, 6 ':::::- Q) u.. 4-t-~-.~-.~-,-~--,~~.--~T-~~ Hours Hours FGUR 1 Growth of inoulated rae of S. erevisiae (fine lines) and total indigenous yeasts (dashed lines) during the fermentation of the three types of musts: (6) ( D) and (). S. Afr. J. nol. Viti., Vol. 1 No

3 vintage, while raes hevalieri and apensis ated on the musts of the 196 vintage. All fermentations were arried out at 2 C, the most suitable temperature for the fermentations arried out in the region (Moreno et al., 196). Samples were periodially taken until fermentations were ompleted (Table 2). The total number of viable miro-organisms (yeasts, bateria and filamentous fungi) was determined by seeding into YM Agar (Wikerham, 191 ). The numberof S. erevisiae of eah inoulated rae was determined by seeding in Biggy Agar (Difeo). This medium ontains bismuth sulphite, whih yields a brown preipitate with hydrogen sulphide whih allows one to distinguish between olonies aording to their olouration: the more H 2 S they produe and store, the darker they appear (Rupela and Tauro, 194). n our experiments, the darkest olouration was yielded by the strain erevisiae B, followed by hevalieri - whih, in addition, grew more slowly in this medium-, erevisiae A, and apensis. The number of indigenous yeasts other than S. erevisiae was determined by seeding in Yeast Carbon Base (Difeo), ontaining lysine as sole nitrogen soure. None of the four S. erevisiae raes seleted grew in this medium. The indigenous yeasts whih were isolated were identified aording to Kreger van Rij (194). The aloholi ontents of the fermented grape musts were determined by the method of Crowell and Ough (1979). Growth of Natural Flora during Wine Fermentation 33 S. Afr. J. nol. Viti., Vol. 1 No RS UL TS AND DSCUSSON Figure 1 shows the evolution of the growth of the four S. erevisiae raes inoulated and that of total indigenous yeasts throughout the fermentations. The number of yeasts from the inoulum was substantially greater than that of indigenous yeasts throughout the fermentation. The indigenous yeasts grew appreiably during the first few days and therefore probably ontribute to some extent to the bouquet of the end produt. The maximum population of the inoulated yeast as well as the total indigenous yeast was reahed within the first 4-72 hours. As a rule, the maximum onentration was kept for a longer time in must - with the highest sugar onentration - than in must (unripe grapes), whih testifies to a lengthier fermentation in the former. As far as the inoulated raes are onerned, apensis remained suspended for the longest time, followed by hevalieri, erevisiae A and erevisiae B - this was also the order of fermentation rate, from lowest to highest (Millan and Ortega, 19). The indigenous yeast population onsisted mainly of S. erevisiae raes, unable to grow in lysine-agar, together with speies other than S. erevisiae whih do grow in this medium. The latter aounted for a small fration whih, exept at the beginning of the fermentation, never reahed levels of 14 ells/ml (Table 6). Tables 3- list the perent frequenies of the samples where bateria, filamentous fungi and yeasts of different speies were deteted on the lysine-agar medium. Table 6 gives the average numbers of these miro-organisms in all positive samples. Bateria were isolated during the fermentations until the ethanol onentration reahed 1-12% (VN), above whih none was deteted (Tables 3-). Their onentration dereased as the alohol ontent inreased (Table 6). Filamentous fungi were deteted throughout the fermentation (Tables 3-); their onentrations were partiularly high for ethanol ontents between and 2% (VN), and dereased dramatially above 4% ethanol (V N) (Table 6). These results are onsistent with those reported by Garfa Maiquez (197) for Jerez wines. Candida stellata was the indigenous yeast most frequently isolated in all the fermentations (Tables 3-). ts onentration dereased partiularly at ethanol ontents above 6-% (V N) (Table 6). This osmophili yeast is frequently isolated from grapes (Sapis-Domerq and Guittard, 1976; Benda, 191) and ours even more frequently in grapes infeted by Botrytis inerea (Minarik et al., 197; Le Roux et al., 1973). Kloekera apiulata was not deteted in the musts from unripe grapes, though it was indeed isolated with a high frequeny in those from half-ripe and ripe grapes, partiularly in the early stages of fermentation (Tables 3-), whih appears to indiate that this yeast, reported as one of the most ommon at the beginning of spontaneous grape must fermentations (Benda, 191 ), only multiplies in grapes in their final stage of ripening. ts onentration in the wine diminished dramatially at ethanol ontents above 1% (V N) (Table 6). The two Rhodotorula speies isolated, namely Rh. rubra and Rh. glutinis, were deteted only sporadially, with the exeption of the latter in the musts from ripe grapes of the 196 vintage, where it was found to our with higher frequenies. Zygosaharomyes bailii was only isolated in the late stages of fermentation, partiularly in the wines with the highest alohol ontents (must ). This shows their high tolerane to ethanol, whih, aording to Van der Walt (197), is a result of the singular properties of its ytoplasmi membrane. The ourrene of this yeast in grape musts has been reported by various authors. Benda (191) regards it as an induer of veil and turbidity, though it has never been shown to alter wines. Candida guilliermondii was only isolated at the beginning of fermentation, while Pihia membranaefaiens was deteted almost exlusively in the musts from half-ripe grapes of the 196 vintage. ts onentration flutuated up to an ethanol ontent of % (V N), above whih it started to derease sharply. Both yeasts are aerobi and liable to produe detrimental veils on the surfae of wines. Our results show that yeasts naturally ourring in musts - partiularly indigenous raes of S. erevisiae - ontribute signifiantly to the fermentation of inoulated wines and, aording to Benda (191) and Fleet et al. (194), exert a major influene on wine bouquet. However, the inoulated yeasts prevailed over their indigenous ounterparts in all the fermentations; they resulted in fast fermentations (Martinez et al., 19) and partly inhibited the growth of the latter, espeially that of speies other than S. erevisiae - and partiularly those onsidered to be detrimental. Thus, the inoulation of musts with strains of S. erevisiae seleted among indigenous yeasts ourring naturally in the winemaking area an ontribute to a better ontrol of the aloholi fermentation of wines and allow only partial growth of the remaining indigenous yeasts.

4 34 Growth of Natural Flora during Wine Fermentation TABL2 Distribution of samples during the fermentation of the three types of must thanol ontent Number of samples taken (%VN) Must Must Must Total Total TABL3 Frequeny of isolation(%) in the samples grown in Lysine-Agar during the fermentations arried out on musts from unripe grapes Speies Candida stellata Kloekera apiulata Rhodotorula rubra Rhodotorula glutinis 33 Zygosaharomyes bailii 1 Candida guilliermondii 29 Pihia membranaefaiens Filamentous fungi Bateria Unidentified yeasts TABL4 Frequeny of isolation(%) in the samples grown in Agar-Lysine during the fermentations arried out on musts from half-ripe grapes thanol ontent (%VN) Speies C. stellata K. apiulata Rh. rubra Rh. glutinis 11 Zygosaharomyes bailii 17 C. guilliermondii P. membranaefaiens Filamentous fungi Bateria 33 2 Unidentified yeasts S. Afr. J. nol. Vitl., Vol.1 No. 2199

5 Growth of Natural Flora during Wine Fermentation 3 TABLS Frequeny of islolation (%)in the samples grown in Agar-Lysine during the fermentations arried out on musts from ripe grapes Speies C. stellata K. apiulata Rh. rubra Rh. glutinis Zygosaharomyes bailii 7 C. guilliermondii 29 P. membranaefaiens 2 Filamentous fungi , 6 Bateria Unidentified yeasts 14 2 TABL6 Average onentration (.f.u./m/) of bateria, filamentous fungi and yeasts other than S. erevisiae in the samples with positive isolation in Lysine-Agar in the three types of must Speies C. stellata ,34 434,4 K. apiulata ,42 7,66 R. rubra Rh. glutinis ,63 16,6 Zygosaharomyes bailii C. guilliermondii 63 P. membranaefaiens 176,6 329,9 Filamentous fungi 1334,43 22,1 6,6 Bateria 1144,2 746,64 29,6 Unidentified yeasts 937,7 114,7 24, ,6 1,24 222,19,3 497,3 233,3 42,4 1, 2 6 6,3 3,3 266,6 266,6 76,6 212,2 74,9 46,7 12,3 6,1 7,72 12,1 99,24 47, 2,3 64,6 114,4 3,2 13,3 LTRATUR CTD AMRN, M.A., BRG, H.W., KUNK, R.., OUGH, C.S., SNGLTON, V.L. & WBB, A.O., 192. The Tehnology of Wine Making. 4th d. A V Pub!. Co. Westport, Connetiut. BNDA,., ologial investigation of the yeasts flora in the distrit of Franonia. Bayer. Landwirtsh. Jahrb. 39, BNDA,., 191. Wine and Brandy. n Presott and Dunn's ndustrial Mirobiology, 4th ed., A V Pub. Co., Westport, Connetiut. CROWLL,.A. & OUGH, C.S., A modified proedure for alohol determination by dihromate oxidation. Am. J. no/. Viti. 3, FLT, G.H., LAFON-LAFOURAC, S. & RBRAU-GAYON, P., 194. volution of yeasts and lati aid bateria during fermentation and storage of bordeaux wines.app/. nviron. Mirobial. 4, GARCA MAQUZ,., 197. studios sobre las levaduras y otros miro-organismos presentes en los mostos de Jerez. PhD Thesis. Faulty of Sienes. University of Granada. HARD, M.G. & FLT, H.G., 19. Growth of natural yeast flora during the fermentation of inoulated wines. Appl. nviron. Mirobial. SO (3), NGO, B. & ARROYO, V., 196. Los agents de la fermentai6n vfnia en la zona Montilla-Moriles. Rev. Cien. Ap/i.72, KHAYYAT, N., ARROYO, V., SOMAVLLA, J.F. & NGO, B., 192. La spaiia vitiviniola. studio mirobiol6gio. Alimentaria 131, KRGR-VAN RJ,NJ.W., 194. The Yeasts.A Taxonomi Study. 3rd d. lsevier Si. Pub!. B.V. Amsterdam. L ROUX, G., SCHNBRUCH, R., & D BRUN, S., The mirobiology of South Afrian winemaking. V. The miroflora of healthy and Botrytis inerea infeted grapes. Phytophylatia S, 1-4. MARTNZ, J., TOLDANO, F., MLLAN, C. & ORTGA, J.M., 19. Development of aloholi fermentation in non-sterile musts from "Pedro Ximenez" grapes inoulated with pure ultures of seleted yeasts. Food Mirobial. (in press). MAURCO, J.C., MORNO, J., MDNA, M. & TAORTGA, J.M., 196. Fermentation of "Pedro Ximenez" musts at various temperatures and different degrees of ripeness. Belg. J. Food Chem. Biotehnol. 41, MLLAN, C & ORTGA, J.M., 19. Fermentation of "Pedro Ximenez" grape musts by different yeast raes. Correlation between fermented sugars, aetaldehyde, aeti aid and ethanol yielded. Belg. J. Food Chem. Biotehno/. 43 (3), MNARK,.,JUNGOVA,. & MRAUD, M., 197. Frutophili yeasts and their effet on naturally sweet wines. Wein-Wiss. 33, MORNO, J., MAURCO, J.C., ORTGA, J.M. & MDNA, M., 196. Choix de la temperature de fermentation la mieux adapt&: pur!'elaboration des vins blans a partir du epage "Pedro Ximenez" dans le Sud de l'spagne. Conn. Vigne et Vin 2, RUPLA, O.P. & TAURO, T., 194. solation and haraterization of low hydrogen sulphide produing wine yeast. nzyme Mirob. Tehnol. 6, SAPS-DOMRCQ,S. & GUTARD, A., Study of the yeast miroflora of Rosillon. Connaiss. Vigne Vin. 1, VAN DR WALT, J.P., 197. Saharomyes Meyen emend. n: The Yeasts. A Taxonomi Study. 2nd d. J. Lodder(d.). North-Holland Publishing Co. Amsterdam. WCKRHAM, L.J., 191. Taxonomy of yeasts. Tehn. Bull US Dept. Agr. Washington, DC. S. Afr. J. nol. Viti., Vol. 1 No