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Author 1 OKAGBUE Richard N.

Author 2
Author 3

Title A Note on the Leaving Activities of

Yeasts Isolated from Nigerian Palm Oil
Keywords

Description Journal of Applied and Bacteriology

Category Microbiology
Publisher

Publication Date 1988

Signature
Journal Applied Bacferioloyy 1988.64, 23S240

A note on the leavening activity of yeasts isolated from

Nigerian palm wine

R . N . 0 K A G B U E Department of Microbiology, Ahmadu Bello University, Zaria, Nigeria

Received 10 July 1987, revised 19 October 1987 and accepted 23 October 1987

O K A G U UR~. N
, . 1988. A note on the leavening aclivity of yeasts isolated from
Nigerian palm wine. Journal oJ'Applied Bacreriology 64, 235-240.
The role of the yeast flora of Nigerian palm wine in the leavening activity of the
beverage was investigated by subjecting organisms from the wine to dough-raising
tests. Those with appreciable leavening activity were identified as Saccharomyces
cerevisioe and Canditia spp. They produced maximum dough volumes in 3-4 h at
37C. The study has provided experimental evidence that yeasts contribute to the
leavening activity of palm wine and has identified strains which have potential
utility in commercial bread baking.

Palm wine is an alcoholic beverage produced by
natural fermentation of the sap of various palms
(Elacis guincensis and Raphia spp.). Consider-
able work has been done on the basic micro-
biology of the beverage and the literature has
been reviewed (Okafor 1978). The predominant
micro-organisms include a few genera of
yeasts (mainly Saccharomyces) and a more
heterogeneous bacterial flora of the genera
Micrococcus, Streptococcus, Leuconostoc,
Lacfohucillus, and also acetic acid bacteria.
Knowled e of the microbiology of the wine has
!-
been use ul in attempts to devise suitable
methods for its preservation (Okafor 1975a;
Uwaifo & Bassir 1982).
In recent years the potential of palm wine and
its microflora as a substrate or agents, respec-
tively, of industrial fermentations, has attracted
considerable interest. For example, the wine has
been used successfully as a base for vinegar pro-
duction. In addition, yeasts isolated from the
beverage have been tested for their potential
uses as brewery and winery yeasts and as pos-
sible sources of single cell protein (Rajagopal
1977; Amanchukwu e f al. 1986; Onyeajuwa e f
al. 1986; Opara e f al. 1986).
In addition to its traditional use as a bever-
Present address: Department of Microbiology, Uni-
versity of Nigeria, Nsukka.
age, Nigerian palm wine is used as a leavening
agent especially when imported active dried
baker's yeast is in short supply. It has been
stated that yeasts are responsible for the leav-
ening activity (O'Reilly-Wright 1964), but there
seems to be no experimental evidence. The
purpose of this study was to isolate palm wine
yeasts, to assess their leavening activity, and to
identify positive strains.
Materials and Methods
C O L L E C T I O N OF PALM W I N E A N D
I S O L A T I O N OF YEASTS
Samples of palm wine were collected in sterile
25 ml screw-capped bottles on five different
occasions from local markets at Nkpor and
Lokoja in Anambra and Kwara States of
Nigeria, respectively. O n each occasion, the
sample was shaken vigorously by hand within
1 h of collection and plated on triplicate potato
dextrose agar (PDA) plates which were held at
room temperature (28" f 2C) and brought to
the laboratory at Zaria (usually within 6 1 0 h
of collection, depending on the source of the
sample). Incubation of the inoculated plates was
continued for a maximum of 48 h. Representa-
tive colonies (confirmed to be yeasts by
microscopy) were purified on PDA plates,
 R. N . Okagbue
stored in the refrigerator on slopes of the same
medium and subcultured every 2 months.
S C R E E N I N G A N D SELECTION OF
FERMENTATLVE YEASTS
A total of 15 yeasts was isolated. They were
screened for fermentative activity in yeast
extract glucose broth with Durham's tubes.
Arter incubation at room temperature
(28" 2C) for 48 h ihe presence of gas was
taken as an evidence of a reasonably high rate
of fermentative activity.
brand), 20 g; cane sugar, 2 g; and yeast suspen-
sion, 15 ml, was prepared. The mixture was
poured slowly into a clean 100 ml measuring
cylinder which had been smeared on the inside
with a thin layer of groundnut oil to aid deposi-
tion of the dough at the bottom of the cylinder.
After reading the initial volume of the dough in
the cylinder, it was incubated at 37OC and sub-
sequent volumes recorded at regular intervals.
Each yeast strain was tested four times.
I D E N T I F I C A T I O N OF P A L M W I N E Y E A S T S
W I T H LEAVENING ACTIVITY

P R O P A G A T I O N A N D ASSESSMENT O F Morphological and physiological tests were

1,EAVENING ACTIVITY O F YEASTS
The yeast strains selected on the basis of their
fermentative ability were propagated in the fol-
lowing way. A loopful of each strain was sus-
pended in 1-2 ml of sterile distilled water in a
test-tube and 0.1 ml amounts of the suspension
were spreid on quadruplicate PDA plates
which were incubated at room temperature for
24 h. The lawns of cells which developed on the
agar plates were scraped off and suspended in
tap water to give a standard turbidity; a 10-fold
dilution of the suspension had an absorbance of
0.14 at 550 nm (WPA spectrophotometer, Cam-
bridge, UK). A commercial brand of baker's
yeast (Engedura; Gist-Brocades, Delft, Holland),
preserved earlier on a PDA slope, was propa-
gated on agar plates and used to prepare a sus-
pension using the same methods.
Leavening activity of the yeasts was tested by
modifica.$on of the cylindtr method described
by Staples (1973). For each strain a smooth
dough mixture containing wheat flour (Ideal
used. Details of the tests and identification
tables are given by Barnett et al. (1983). Initially,
the yeasts were separated into two groups;
ascospore-formers and non-ascospore-formers.
A commercial yeast, Engedura (Saccharomyces
cerevisiae), was used as a control in various
physiological tests to aid in the identification of
the ascospore-forming strains.
Results and Discussion
Initial studies with the Engedura yeast showed
that 90% of the final dough volume (usually
attained in 4 h), was reached in 3 h. Conse-
quently, leavening activities of the palm wine
yeasts were monitored up to a maximum time
of 3.5 h.
Table 1 shows the performance of the yeasts
in the dough-raising tests. The leavening abil-
ities of some of the palm wine strains (la, Ic, 3a
and 5a) were comparable with that of Engedura.
It is interesting that each of the five samples
examined yielded at least one yeast which

Table 1. Leavening activity of ~ a l mwine yeasts

Dough volumes (cm3)at intervals (min)*
Leavening
Strain no. 0 30 60 90 120 150 180 210 ability (%)t
Engedura (control) 29 30.0 32.5 41.5 56 68 77 86
I 29 30.5 32 35.5 42 59.5 62.5 69
la 29 29.6 39 51.6 62.6 73.3 77.8 81
Ib 29 29.6 35.3 46.3 56 64 72.6 78
Ic 29 32.0 40.6 49.6 65 73.3 79.6 83-7
2 29 30.0 36 41.5 46.6 62 66.5 72.5
2c 29 30.0 35.0 47.0 50.5 61.5 68 74
3a 29 30.4 35 41 51.3 67 76 83.5
5a 29 31.5 35 39 52.3 ND 74 81.5
* The data are averages of quadruplicate determinations.
t Computed with the dough volume at 210 min using that of Engedura as control (lOOOh).
ND, Not determined.
 Leavening activity of palm wine yeasts 237
Table 2. Identification of palm wine yeasts with leavening activity
Palm wine yeasts
--- --- -

Engedura:
Succharamyces Saccharomyces Saccharomyces Candida
Assimilation cereoiaiae cereoisiae cereoisiae Candido sp. Candida sp. curiosa
or reaction (control) (la) (lb, lc, 3a, 5a) (1) (2) ( 2 4 .. ..
- ,
Glucose
Galactose
Maltose
Sucrose
Lactose
Melibiose
Rafinose
Cellobiose
1,-mannose
warabinose
Me-a-u-glucoside
11-glucono-b-lactone
1,-mannitol
Erythritol
Xylitol
Cilrate
Gluconate
DL-lactate
2-keto-glucohate
Inulin
Starch
Nitrate
Lysine
Cadaverine
Growth in 0-01%
cycloheximide
Growth In 50%
glucose
Growth at 30C
35C
37C
42C
Growth without
niacin
Growlh without
pyrido?ine
Growth without
thiamine
Starch formation
Urea hvdrolvsis
+ +, Assimilation and fermentation positive; - -, assimilation and fermentation negative; + -, assimilation
positive, fermentation negative.
NT, Not tested.

exhibited leavening activity under the condi-
tions employed in this study. Thus, the study
provides experimental evidence that the yeast
flora of palm wine contributes to its leavening
activity. The strains isolated could be studied
further with a view to their possible use as com-
mercial baker's yeasts.
During the identification of the yeasts (see
Table 2) five were found to produce spherical
ascospore (ranging from one to four per ascus)
on acetate agar (Barnett et a / . 1983), while the
remaining three did not form ascospores Most
of the physiological reactions of the ascospore-
forming strains were similar to those of Enge-
dura. O n the basis of the observed
characteristics the new strains were identitied as
S. cerevisiae. Four of them did not ferment
galactose but this is a variable characteristic of
strains of S. cerevisiae (Barnett et al. 1983).
Saccharomyces spp. are usually present in
 R. N . Okagbue
palm wine, but variations have been observed in
the species occurring in different samples of the
beverage. For example, S. cerevisiae was identi-
fied by Bassir (1962) but not by Okafor (1972).
Thus, strains of S. cereoisiae such as those found
in this study would participate in the leavening
activity of palm wine only if they are present in
the sample involved. Saccharomyces exiguus, a
species which was not isolated in this study, has
been reported in palm wine (Okafor 1972), and
is known to be the yeast responsible for the
leavening action involved in San Francisco sour
dough French bread process (Sugihara et a / .
1971). Conceivably, the species would play a
role in the leavening activity of any sample of
palm wine in which it is present.
The non-ascosporogenous strains from this
study were identified as Candida species. Their
leavening activities appeared to be lower (up to
210 min) than those of the strains of S. cereui-
siae. At 240 min. the maximum dough volumes
were comparable with those of S. cereoisiae (not
shown in Table 1). The occurrence of Candida
spp. in palm wine has been reported (Okafor
1972) and the present results indicate that
actively fermenting species or strains would con-
tribute to the leavening activity of the beverage.
One of the wine samples yielded a combination
of Canrlida sp. and S . cerevisiae as the potent
agents ( l c and 2c). This suggests that synergistic
action of yeasts may be involved in the leav-
cning action of palm wine. In contrast, the
active strains from another sample were only
Candida spp. (1 and 2). In an analogous situ-
ation, Ekundayo (1969) has implicated a strain
of ' ~ a n d i d a sp. in alcoholic fermentation
involved in the production of 'pito', a local fer-
mented beverage. Apparently, pure cultures of
relevant strains of Candida sp. could be devel-
oped and used commercially in fermenting
foods and beverages.
In addition to the yeasts, bacteria also form
part of the microflora of palm wine. Leuconostoc
and Zymomonas, among other bacteria, have
been identified in palm wine samples, albeit
infrequently (Okafor 1975b).
Uwaifo & Bassir (1982) showed that Leuco-
nosfoc mesenfernides may be present in the wine.
The species has been shown to have leavening
activity (Mukherjee el (11. 1965) and Zymomonas
is fermentative (Swings & de Ley 1977). If
present in any palm wine sample, they would be
expected to contribute to gas formation associ-
ated with the leavening activity of the beverage.
In general, this study has shown that palm
wine yeasts, namely S. cerevisiae and Candida
spp. are wholly o r partially responsible for the
leavening activity of the beverage, and has pro-
vided strains which can be developed further for
possible commercial applications.
I thank Miss M. Miranda, Department of Food
Science and Technology, University of Califor-
nia, Davis, for determining the assimilation pat-
terns of the yeasts on 2-ketogluconate and
cadaverine.
References
AMANCHUKWU, S.C., OKIWKWASILI, G.C. & OBAFEMI,
A. 1986 Evaluation of Schizosaccharornyces ponrbe
isolated from palm wine for single cell protein pro-
duction using hydrocarbon feedstocks. In Abstracts,
14th Annual Conference, Nigerian Society for
Microbiology, pp. 7-8, University of Calabar,
Calabar, Nigeria.
BARNET,J.A., PAYNE, R.W. & YARROW, D. I983
Yeasts: Characteristics and Idenrificorion. Cam-
bridge: Cambridge University Press.
BASSIR, 0. 1962, cited in FAPARUSI. S.I. 1969 Effect of
pH on the preservation of palm wine by sulfite.
Applied Microbiology 18, 122-123.
EKUNDAYO, J.A. 1969 The production of pito, a
Nigerian fermented beverage. Journal of Food Tech-
nology 4, 217-225.
MUKHERJEE, S.K., ALBURY, M.N., PEDERSON, C.S., VAN
VEEN,A.G. & STEINKRAUS, K.H. 1965 Role of Leu-
conostoc n~esenteroides in leavening the batter of
idli, a fermented food of India. Applied Micro-
biology 13,227-23 1.
OKA~K N., 1972 Palm wine yeasts from parts of
Nigeria. Jo~rrnalofthe Science of Food ond Agricrrl-
ture 23, 1399-1407.
OKAFOR,N. 1975a Preliminary microbiological
studies on preservation of palm wine. Journal of
Applied Bacteriology 38, 1-7.
OKAFOR, N. 1975b Microbiology of Nigerian palm
wine with particular reference to bacteria. Journal
of Applied Bacteriology 38, 81-88.
OKAFOR, N. 1978 The biochemistry and microbiology
of palm wine. Advances in Applied Microbiology 24,
237-256.
ONYEAJUWA, V.U., NWOBODO, E.N. & ODIBO,F.C.
1986 Beer production from sorghum using palm
wine yeasts. In: Abstracts, 14th Annual Conference,
Nigerian Society for Microbiology, p. 12, University
of Calabar, Calabar, Nigeria.
OPARA,A.A., UTSALO,S.J. & BASSEY, J. 1986 Pro-
duction of fruit wine from pineapple using local
palm wine yeast. In Absrracts, 14th Annual Con-
jerence, Nigerian Society for Microbiology, pp.
23-24, University of Calabar, Calabar, Nigeria.
O'REILLY-WRIGHT, E. 1964 The Students' Cookery
Book. p. 140. Oxford: Oxford University Press.
RAJAGOPAL, M.V. 1977 Production of beer from
cassava. Journal of Food Science 42,532-533.
STAPLES,D.G. 1973 An Infroduction to Microhioloqy,
pp. 65--74 London: Macmillan.
SUGIHARA, T.F., KLINE,L. & MILLER,M.W. 1971
Microorganisms of the San Francisco sour dough
bread process. 1 . Yeasts responsible for the leav-
ening action. Applied ~Microhioloqy21,456458.
SWINGS,J. & V E LEY, 1977 The biology of Zymo-
monns. Bncterioloqical Reviews 41, 1-46.
UWAIFO, A.O. & BASSIR,0. 1982 Preservation of palm
wine with n-octyl gallate. Nigerian Journal o j
Microhioloqy 2,97-107.