29(J, Inst!~ Enol. Vitieul. Yamanashi Univ.

1529-321980)

Changes in the Wild Yeast Flora ofSuflited Grape Musts
SHOJI

GOTO

Progressive changes in the wild yeast flora at the initial stage of incubation ofgrape musts added with various concentrations of S02 were studied qualitatively andquantitatively. Initial total counts of yeasts in the original grape must were 6.5 X 10 5cells/ml and increased rapidly within a short period of time, and after 32 hours fermentation was observed. As early as 4 hours after incubation, total counts of yeastsand the proportion of the five yeast groups (Apiculate, Saccharomyces, Torulopsis,Film, and Others) were affected by the initial levels of free·S02 added. Total countsof yeasts in the must containing 46.7 ppm of free-S0 2 decreased gradually and reach·ed a minimum after 48 hours. After this period, however, total counts of yeasts
increased gradually, and this was due to the increase in Saccharomyces group. Totalcounts in the musts containing 97.5 and 228 ppm of SO 2 decreased rapidly to lessthan 10 cells/ml after 24 and 48 hours. Apliculate yeast group and part of Film andOther yeast groups decreased rapidly in the initial stage of incubation of sulfitedgrape musts, whereas Saccharomyces and Torulopsis yeast groups decreased gradually. Yeasts remainded in the sulfited musts were resistant to S02'

Yeast flora in the process of wine·making has
been a subject of interest for ecologists andenologists. For examples, yeast flora, before
fermentation, during the active fermentation andat the end of fermentation, were reported byOhara and coworkers l~ Peynaud and Domercq 2),I
Minarik 3), and van Zyl and du Pleesis 4). Effects
bf SO 2 on wine yeasts have been reported by
Ohara and coworkers 5 ). These studies have been
carried out with samples taken at long intervals.

! In the present work, wild yeast flora at theinitial stage of incubation of grape musts added}.h
WIt various concentrations of SO 2 was studied
both qualitatively and quantitatively with samples
taken at hourly interval.
Materials and Methods
Grape must Pressed grape must (reducing

sugar 15.8g/100ml, total acid 0.52g/100ml, andpH 3.3) of a white variety of the Koshu wasused. This must (1700mI) was poured into each
2 1 glass bottle and four concentrations of S02(0, 46.7, 97.5, and 228.4 ppm) were added imme·diately. Each bottle was sealed with a glass
fermentation lock and incubated at 23'C.
Total counts of yeasts

The samples ofgrape must were diluted 0 to 10 6 fold with ste·rile water. Medium and methods were the sameas described in a previous report 6~
Grouping of yeast isolates All colonies
appearing on a plate, approximately 10 to 20 colonies, were picked up on YM agar slants. Methods of purification and grouping of isolates werethe same as described in a previous report 6;
Identification of yeast strains

Typicalisolates, 2 to 7 strains, were selected from the30

GOTOJ'-:::::~8~O\\:"-0'oi~~/\x,'C· 502 /..........
228 ppm
X'x/

Results and Discussion
Changes in the total counts of yeasts inthe sulfited grape musts

L...L............L-_--'--4.

o12

24 32

Progressive changes

in the total counts of yeast in each lot are shown

07in Fig. 1. Total counts in the original must (Jot0) at 0 time were 6.5 X 10 5 cells/ml, and after
4 hours slightly increased.

B.5~ 97.5ppm

f1--r......
- -)(--)(=1=1'H...

ofree-S02 were analyzed by the Ripper proced·
ure 9!

II,

48

72

96

After 32 hours, to·tal counts increased to 1.4X108 cells/ml andweak fermentation was observed. In the sulfitedgrape musts, a decrease in the total counts ofI(h)120Fig. 1.

Progressive changes in the total
counts of yeasts in sulfited and nonsulfited grape musts.
Grape musts were prepared by crushing andpressing a white variety of Koshu. Components of grape must were 15.8 g/100 ml ofreducing sugar, 0.52 g/100 ml of total acid,and pH 3.17. Pottasium meta-bisulfite wasused as a source of SOz.

yeasts was observed, after 4 hours.

In the lot

A containing 46.7 ppm of free-S02' total counts
decreased gradually and reached a minimum, 4.7
X 10 2 cells/ml, after 48 hours. After this period, however, total counts increased graduallyand reached 3X10 8 cells/ml after 120 hours.
This increase was due to the proliferation of 5(Saccharomyces) group. In the lots Band C
containing 97.5 and 228.4 ppm of initial 50 2, reo
spectively, total counts decreased rapidly andreached levels less than 10 cells/ml after 24
and 48 hours.

so, 228 ppm

G,taminations were not observed.

=" fjJ1 ~~so, 97.5ppm

~u.

o~

50o

Wild yeasts were not sterilized

completely after 120 hours, but microbial con-200

musts was observed, as shown in Fig. 2.
----IllI__.":~-==-'~~

Yeast species of six yeast groups-----G--L:'I

O-U-o_1272!

o24

During the in-cubation time, reduction in free·S02 in grape

48'1t='96(h)Onehundred and five isolates which were randomly

selected from those samples of the grape musts
I addedwith four concentrations of 50 taken at

9120Fig. 2.

Reduction in free'SOz in grape mustsof a white variety of Koshu.
K Z S Z 0 5 was used as the source of SOz.

2
the stated time were classified into 20 species

belonging to 10 genera. Details of identificationand dishibution of isolates will be reported ina separate paqer.

Tpe yeast species classified

subcultures obtained from each test lot, and a

into the six groups, KA, 5, T, F, R, and 0, aretotal of 105 strains was identified according toshown in Table 1.

the system of "The Yeasls" 7) and to the meth-Group KA (apiculateyeast) consisted of Klo·ods described by. Iiz.llka and Goto·8~.. ·Analysis of SOz
Th.e pottasium meta- bi-The former
was the predominant yeast in the original must

sulfite was used as a source of 50 2 , Total- and(Jot 0).

eckera apiculate and K. corticis.

The latter was isolated at high fre-Wild yeast flora in suflited grape musts
Table 1.
GroupYeast species in the six yeast groups.

YeastGroup

Kloeckera apiculata
K. corticis
Saccharomyces cerevisiae
S. bailii
S. bayanus
S. inconspicuus
S. rosei
Torulopsis stellata
T. magnoliae

KA5T

luency from sulfited must.

~dRo~

enos

-The former was dominant.~

S. bailii was isolat·

at high frequency from sulfited grape musts.

;roup T (Torulopsis)
md T. magnoliae.

Pichia membranaefaciens
P. terricola
Candida valida
Ca. krusei
Rhodotorula rubra
Cryptococcus laurentii
Ca. inositophila
Ca. acutus
Saccharomycodes ludwigii
Metshnikowia pulcherrima

F

consisted of T. stellata

S. inconspicuus.

YeastGroup S was com·

IOsed of S. cerevisiae, S. bayanus, S. bailii, S.
rose~ andfeasts in this group. Both yeasts were isolated">.
0en= 1>'<...0 0.en
0.
0::l0... ...btl:rom sulfited musts. Group F (film yeasts) wasPo.

:omposed of Pichia membranaefaciens, P. terri-LoIs tested 0
Elapsed time 0(hours)~ola, Candida valida, andCa. krusei. Ca. krusei

md P. terricola were predominant yeasts in sui·iited grape musts.

Group R (Rhodotorula) was:omposed of only one species of Rh. rubra. This,east was isolated from the original must. GroupJ (other yeasts) was composed of Saccharomycodes ludwigi~ Metschnikowia pulcherrima, Ca.
lcutus, Ca. inositophila, and Cryptococcus lau·rentii.

310_0ABO ABO A B
122448A B
96

Fig. 3.

Progressive changes in the counts ofwild yeasts in sulfited and non·sulfi tedgrape musts.
o : non-sulfited must, A : 46.7 ppm of initial
free-50 z, B: 97.5 ppm of free-50 z , KA:
Apiculata yeast group, 5: Saccharomyces
yeast group, T: Torulopsis yeast group,
F : Film yeast group, 0 : Other yeastgroup.

Saccharomycodes ludwigii was isolated

it high frequency from sulfited musts.

Ca.

lcutus, descri bed as a new species lO~ was iso-

10% of S, T, and 0, and 20% of F groups.

Inthe original grape must (lot 0), a rapid increase

lated from sulfited must added with 228.4 ppm

in the total counts was observed. This increaseof 50 2 ,was mainly due to the proliferation of group Syeasts and the proportion of groups KA, F, andProgressive changes in the proportion of fiveo decreased simultaneously. The proportion ofProgressive changes in the fivegroup T increased gradually with the reduction

yeast groups, KA, S, T, F, and 0, in grapeof group KA till 24 hours, but it decreased withmusts added with three concentrations of initial

the increase in group S.

free·S0 2 (0, 46.7, and 97.5 ppm) are shown inFig. 3. Group R was included in group O. The,46.7 ppm of S02, after 12 hours, the proportion

proportions of five yeast groups in the originalter 24 hours to less than 10%. but the propor·

grape must (lot 0 at 0 time) were 50% of KA,tion of group S increased gradually.

yeast groups

In lot A containingof group KA decreased rapidly to 15%, and af·
After 48

32

GOTO

hours, total counts of yeasts reached a minimum,
but increased gradually after this time. This
increase in the total counts was due to the pro-when an excellent active starter is used, S02
less than about 50 ppm will be enough to guar·antee safe fermentation.

liferation of group S and the proportion of group
S reached 90% after 120 hours. After 12 hours,
the proportion of group T increased to 40%.
This group, however, decreased rapidly to several percent with the increase of group S after

1) Ohara, Y., Nonomura, H. : Nippon Nogeikaga-

72 hours. Changes in the proportions of groups
F and 0 also tended to change similarly to thatof group T. In lot B containing 97.5 ppm of free·

Vit ie., 10, 69 (1959).
3) Minarik, E. : Vitis, 4, 368 (1964).
4) Van Zyl, J. A., du Pluessis, L. : S. Afr. J.

S02' the' proportion of group KA decreased rapidly to 5% after 24 hours and increased to 15

Agr. Sci., 4, 393 (1961).
5) Ohara, Y., Nonomura, H., Yunome, H. : BuLL

and 20% after 48 and 96 hours again. After 12hours, groups Sand T increased to 35 and 40%, respectively, and the proportions of bothgroups almost did not change thereafter. Pro-Res. Inst. Ferment., Yamanashi Univ., 6,7(1959).
6) Goto, S., Yokotsuka, I. : J. Ferment. TechnoL,portion of groups F and 0 almost did not changeduring the experimental period. In lot C containing 228.4 ppm of S02' progressive changes in
the proportions of five yeast groups were similar to those of lot B.

In the grape must containing about 50 ppm of
free-S02' after 8 or 12 hours, a rapid decrease
in total counts of yeast and reduction in major
wild yeast species were observed. From theabout results, and since generaly in wine-making
a yeast starter was added at about 8 hours,

References

ku Kaishi, 30, 524 (1956).
2) Peynaud. E., Domercq, S. : Am. J. Enol.

55, 417 (1977).
7) Lodder, J. (ed.) : The Yeasts, a Taxonomic
Study, 2nd., North-Holland Pub. Co., Amster·dam-London, p. 1385, 1970.
8) Iizuka, H., Goto, S.: Classification and Iden·
tification Methods for Yeasts, 2nd., Univ. ofTokyo Press, p. 183, 1972.
9) Amerine, M. A., Ough, C. S. :Wine and Must
Analysis, John Wiley & Sons, New York,
p.81, 1973.
10) Goto, S.: J. Gen. Appl. Microbiol., 25, 145(1979).