Effect of Acetobacter aceti on the growth of different yeast ...

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Nature and Science 2010;8(10)

Growth of Different Yeast Strains During Fermentation of Soursop
(Annona muricata) Juice as Influenced by Acetic acid Bacteria
(Acetobacter aceti)

Sunday P. Ukwo1, Chidi F. Ezeama1 and Nyaudoh U. Ndaeyo2*
1Department of Food Science and Technology, Michael Okpara University of Agriculture, Umudike, P.M.B. 7267,
Umuahia, Abia State, Nigeria.
Department of Crop Science, Faculty of Agriculture, University of Uyo, Akwa Ibom State, Nigeria.
[email protected]

Abstract: A study was conducted to investigate the growth of different yeast strains as influenced by Acetic acid
bacteria (Acetobacter aceti) during fermentation of soursop juice. Preliminary studies were carried out to identify
the endogenous species of yeast responsible for natural fermentation of soursop juice and the effect of exogenous
acetic acid and pH on growth and tolerance rate of the different yeast isolates. The growth rate of different isolates
was monitored for 7th days with and without Acetobacter aceti (AAB). Soursop juice was inoculated with the
different yeast isolates Saccharomyces cerevisiae (SC), Hansenula anomala (HA), Canadida tropicalis (CA) and a
brewery strain Saccharomyces pastorianus (SP). Results showed a reduction in maximal cell concentration with
samples treated with Acetobacter aceti. The inhibition of growth rate was higher on non-Saccharomyces yeasts HA
and CA while the brewery strain SP indicated a higher tolerance to Acetobacter aceti in the fermenting juice.
Addition of 0.5ml v/v of exogenous acetic acid resulted in a drop in pH from 6.8 to 4.1 and caused a reduction in
cell count while 1.0ml v/v acetic acid resulted in a drop of pH from 6.8 to 3.7 and showed complete inhibition of
growth of all the yeast isolates. When the medium was adjusted by 1N hydrochloric acid to pH 4.1 and 3.7 all the
yeast isolates showed the same growth as the control (pH 6.8). [Nature and Science 2010;8(10):285-291].
(ISSN: 1545-0740).

Key words: fermentation, soursop juice, yeast, acetic acid bacteria.

1. Introduction

According to Battcock and Azam-Ali

The development of fermentation
(1998), other micro-organism during fermentation
technologies has been lost in the mist of history and
have the potential to influence the performance of
has been regarded as one of the oldest methods of
yeast at various stages of fermentation process. He
food processing as well as preservation for decades
asserted that apart from their “Killer Strains” of
and virtually every culture has it as part of its diets
yeast, bacteria like acetic acid and lactic acid bacteria
(Viljeon and Heard, 1999). Today, alcoholic
posses a tremendious effect on the activities of yeast
fermentation is quite indispensable for the production
vis-à-vis the final products. According to Gafner
of alcoholic beverages including wine from tropical
(2006), most of the off –flavour encountered during
fruits such as orange, grapes, pineapple, banana,
fruit juice fermentation comes from acetic acid which
guava, soursop and sugar cane. Fermented foods
are either produced by undersirable yeasts or acetic
according to Food and Agricultural Organization
acid bacteria during fermentation. Studies has also
(FAO) have played important role in improving food
shown that any condition that affect the growth of
security, enhancing livelihood and increased the
yeast cells during fermentation process posses a
nutritional status of the final products (Steinkraus
negative effect on the final fermentation products.
According to Ayugo (1999), the amount of acetic

Yeasts as a group of micro-organism has
acid produce by desirable yeast is between the range
been quantitatively and commercially exploited as a
of 0.04-0.2g/l and this amount of acetic acid
fermentative species needed to carry out alcoholic
according to Reed and Nagodawithana, (1991),
fermentation and this has urged many scientist to
produced by yeasts are dependent on fermentation
study the factors governing its growth, survival and
temperature and composition of the medium.
biological activities at different ecosystem (Heard

Although the important role of undissociated
and Fleet 1985).
form of acetic acid on yeast cells have been
It has been established that the growth of
investigated, literature on the successfully induced
yeast during fermentation depends on the media
fermentation where both yeasts and acetic acid
composition (substrate), the initial level of pH,
bacteria like Acetobacter aceti are inoculated
temperature and dissolved oxygen (Ruiz et al, 2004).
together during fermentation are either non-existing


Nature and Science 2010;8(10)

or are lacking and hence this study is directed toward
Champion Breweries, Uyo and maintained in YPD-
that. The objective of this work is to assessed the
agar and stored at the same temperature.
growth rate of different yeast isolates singly and in
Acetic acid bacteria was isolated from a
the presence of Acetobacter aceti during fermentation
fermented palm wine dreg. A 0.1ml of dreg was
of soursop juice. The effect of exogenous acetic acid
seeded on glucose-yeast extract containing 100g
and pH on the growth and tolerance rate of different
glucose, 10g of yeast extract, 10g of CaCO3 and 20g
yeast isolates is also investigated.
of agar in 1 litre and incubated aerobically at 28±20C

for 2-3 days (Lisdiyanti et al., 2001) colonies were
Materials and methods
randomly picked and subcultured using the same
2.1 Sample


Fresh and fully ripe soursop (Annona

muricata L.) fruits were purchased form Akpana-
2.4 Preparation
Ndem market in Uyo, Southern Nigeria. The fruits

Yeasts inoculum were prepared by
were washed with sterile water and further sterilized
inoculating each species of yeasts identified into
with 2% sodium hypochlorate solution. Under a
25ml YPD without Agar and water to prepare the
sterile condition, the fruits were hand peeled, decored
inoculum was substituted by soursop juice to
and deseeded. The pulp was blended using an
condition the yeast and were allowed for 24 hours at
electric blender (National, model MX-795N,
28±20C before inoculum.
Malaysia). Sterile water was added at the ratio of 1:2

Acetobacter aceti inoculum was from a 24
(w/v, pulp/water) to facilitate blending process and
hours culture that grew on slant into 1% D-glucose,
make filtration process easier. The pulp was filtered
0.5% ethanol 0.3% acetic acid, 1.5% bactopeptone
using a sieve and Muslin cloth. The juice collected
and 0.8% yeast extract and the pH adjusted to 3.4
was pasteurized at 650C for 30 minutes and allowed
using citric acid (Lisdiyanti et al., 2001) and stored
to cool.
for 24 hours before inoculation.

Preliminary Microbiological Studies
2.5 Fermentation

A preliminary experiment was carried out to

Fermentation was carried out by inoculating
identify the endogenous species of yeast mostly
1% of the actively growing yeast cells (5.0 x
responsible for the natural fermentation of soursop
105cfu/ml) into 500ml sterile soursop juice in 1 litre
juice as outlined by Ezeama (1999). This was done
conical flask capped with cotton wool. Fermentation
to ensure that species of yeasts to be identified were
was carried out at room temperature (28±20C) for 7
used for the fermentation studies. Healthy soursop
days without agitation.
fruit was washed with sterile water, the skin was

To study the effect of Acetobater aceti on
peeled off and the pulp (mesocarp) squeezed out
yeast performance and fermentation rate, 1% of
under aseptical condition and the juice collected. The
actively growing cell (1.0 x 106 cfu/ml) was
soursop juice so collected was allow to undergo
inoculated into the fermenter. Samples were taken
natural fermentation for 3 days. The fermenting
daily and analysed for the determination of yeast
sample was then collected for this study.
growth. All determinations were carried out in

triplicate and each value used in figure and tables
2.3 Micro-Organisms

were the arithmetic mean.

Yeast strains used for this study was isolated

from soursop juice. A 0.1ml of the juice prepared
Enumeration of Yeasts Growth
after 10 fold serial dilution was seeded on PDA

The growth rate of yeasts were determined
(Potato infusion 200g/l, Dextrose 20g/l and Agar
by the plate count technique (Kapsopoulou et al.,
15g/l) at pH 5.6±0.2 at 200C was fortified with
2005). Samples were taken at 24 hours intervals and
0.25g/l of chloramphenicol to inhibit bacterial
serially diluted and spread-inoculated (0.1ml) on
growth. The plates were incubated aerobically at
plates of YPD agar containing the following per litre:
room temperature (28±20C) for 3-5 days for colony
Yeast extract 10g, peptone (oxoid), 20g glucose
development. Discrete colonies were streaked on
(BDH) 20g and agar 20g and was fortified by 0.25g/ℓ
fresh YPD-Agar (Yeast extract 10g/l peptone 20g/l,
of chloramphenicol. The plates were incubated at
glucose 20g/l and agar 20g/l) (Kapsapoulou et al.,
room temperature (28 ± 20C) for 3-5 days.
2005) and incubated for 3-5 days the isolate stored at

40C. The ability to utilize certain sugar for growth
and assimilation was use for identification (Lee et al.,
2006). A pure culture of brewery stain of
Saccharomyces pastorianus was obtained from


Nature and Science 2010;8(10)

Effect of exogenous Acetic acid and ph on
media with various concentrations of acid were added
the growth and tolerance rate of yeast
swirled and incubated aerobically at room
temperature for 5 days followed by observation for
This was a pure culture experiment. The
growth and the level of tolerance of the isolates to
method outlined by Bechem et al (2007) was adopted
acetic acid and pH.
with minor modification. To investigate the growth

and tolerance rate of different yeast isolates to
3. Results

exogenous acetic acid, and pH, the inoculum used for
Characterization and Identification of
fermentation experiment in section 3.5 were grown
Yeast Isolates
on a synthetic media prepared in 5 places as follows:
The results obtained from the study
1.0g peptone (oxoid), 10.0g of glucose (BDH), 1.0g
identified three (3) endogenous species of yeast
of yeast extract and 2.0g of agar all in 100ml into
mostly responsible for the natural fermentation of
which 0ml, 0.5ml and 1.0ml of exogenous acetic acid
soursop juice. The ability of the different yeast
were added to each of the media while the last two
isolates to utilizes certain sugars for growth was
their pH were adjusted with IN HCl to pH of level of
further used for identification as indicated in the
4.1 and 3.7 before autoclaving. A 0.1ml of the
Table 1 below.
inoculum were transferred into each plate and the

Table 1: Biochemical, assimilation tests and morphology of 3 yeast isolates obtained from soursop

+ + +
+ + +
- - +
+ + +
+ + +
+ + +
- - -
+ + +
+ + +
Methanol -
Cell Morphology
Smooth, spherical cremish Whitish to cream colony
Elevator and cremish
Cell shape
Round, oval with thick cell Cylindrical, spherical hat
Enlongated cell with
wall ellipsoidal
shape or Saturn shape
spores. True Mycelum
Saccharomyces cerevisiae
Hansenula anomala
Candida tropicalis
+ positive
- negative

Effect of Acetic Acid Bacteria (Acetobacter aceti) on Yeast Growth
They include; Saccharomyces cerevisiae (SC) Hansenula anomala (HA) and Candida tropicalis (CA). The
growth pattern of Saccharomyces yeast isolates which include Saccharomyces cerevisiae (SC) and a brewer strain
Saccharomyces pastorianus (SP) inoculated into soursop juice without Acetobacter aceti (AAB) was characterized
by a very high concentration of cells which reached 8.5 and 8.4 log (cfu/ml) for SC and SP respectively as indicated
in Figure 1. The non-Saccharomyces yeast isolates; Hansenula anomala (HA) and Candida tropicalis (CA)
inoculated into a similar medium without Acetobacter aceti (AAB) showed a similar growth pattern (Figure 2).
Furthermore, the cells concentration of Saccharomyces cerevisiae (SC) showed higher viability and decreased much
more slowly to a level of 7.0 log (cfu/ml) after 7 days of fermentation.
However, the growth of yeast isolates inoculated together with Acetobacter aceti (AAB) was characterized
by lower concentration of cells as indicated in Figures 1, 2, 3. There was no exponential phase of fermentation. The
non-Saccharomyces yeast, HA and CA showed a higher sensitivity to the presence of AAB and were therefore
strongly inhibited (Figure 2) while the Saccharomyces yeast SC and SP showed minimal cell concentration with SP
giving 6.7 log (cfu/ml) which was the highest compared (Figure 1) to the other.
However, strain of Saccharomyces pastorianus showed a high tolerance to the presence of Acetobacter aceti during
fermentation especially when inoculated together with other yeasts as indicated during the mixed culture


Nature and Science 2010;8(10)


Nature and Science 2010;8(10)


Nature and Science 2010;8(10)

Effect of Exogenous Acetic acid and pH on growth and tolerance rate of Yeast Isolates
The addition of 0.5ml v/v of acetic acid to the test medium resulted in pH reduction from 6.8 to 4.1. The
cultivated yeast isolates showed that Saccharomyces cerevisiae (SC) and Saccharomyces pastorianus (SP) exhibited
a comparable slight decrease in growth and cell counts a little lower than that of the control medium while that of
the non- Saccharomyces yeasts Hansenula anomala (HA) and Candida tropicalis (CA) exhibited a much lower cell
count and decrease in growth.
This shows that acetic acid at 0.5 ml or 0.5% concentration can slightly inhibit growth of yeast by causing
loss of viability after inoculation. The medium added with 1.0 ml v/v of acetic acid resulted in pH reduction from
6.8 to 3.7. There was no growth at all in all the test medium containing the yeast isolates after 5 days (Table 2).

Table 2: Yeast growth and tolerance rate as affected by exogenous acetic acid and pH

Yeast Count cfu/ml
Sample pH
0ml v/v (acetic acid)
3.0 x 105
3.2 x 105
3.2 x 105
3.1 x 105
0.5ml v/v (acetic acid)
2.8 x 105
2.3 x 105
5.0 x 104
4.0 x 104
1.0ml v/v (acetic acid)
pH 4.1 with IN HCl
3.1 x 105
3.3 x 105
3.0 x 105
3.1 x 105
pH 3.7 with IN HCl
3.2 x 105
3.2 x 105
3.2 x 105
3.0 x 105
N.D =
Values are means from 3 determinations

To investigate the effect of acetic acid on
could therefore be as a result of oxidation of ethanol
growth reduction and inhibition of yeast isolates was
to acetic acid (Battcock and Azam-Ali, 1998). The
not from low pH caused by adding acetic acid,
acetic acid causes a reduction in maximal cell
experiment were carried out by adjusting the pH of
concentration of yeast cells during fermentation
the synthetic media by IN HCl to the same pH level
process (Phowchinda et al, 1995). It is likely that, the
(pH 4.1 and 3.7). Results showed that the growth and
undissociated acetic acid produced by Acetobacter
rate of tolerance of yeast isolate were not different
aceti diffused into yeast cells and caused decrease in
compared to the control medium (Table 1). This
the pH of cytoplasm which may have inhibited the
indicates that the low pH did not inhibit the growth of
activities of key enzymes. The higher cell count
yeast isolates but acetic acid as reported by
observed in this mixed culture of SP with HA and
Phowchinda et al., (1995).
CA with Acetobacter acid may be due to relative

tolerance of SP to AAB as was found by Limtong,
4.0 Discussion

(2000) that some strains of Saccharomyces yeasts
This study has shown a rapid growth of
showed higher tolerance rate to acetic acid than non-
yeast cells during fermentation followed by decrease
Saccharomyces yeasts.
in cells concentration after 5 days to a level of 5.5,
In the findings of the effect of exogenous
5.8, and 5.6 log (cfu/ml) for SP, HA and CA
acetic on yeast growth and tolerance rate of yeast
respectively. This could probably be due to
isolates, addition of 1.0ml v/v of acetic acid resulted
combined influence of alcohol and anaerobic
in pH reduction from 6.8 to 3.7, there was no growth
condition caused by yeast growth. Furthermore, the
in all the test medium indicating strong inhibition at
cells concentration of Saccharomyces cerevisiae (SC)
this concentration of acetic acid. These findings
showed higher viability and decreased much more
agrees with the report of Bechem et al. (2007) on
slowly to a level of 7.0 log (cfu/ml) after 7 days of
tolerance rate of different isolates of Saccharomyces
fermentation. A similar result was observed by
spp on acetic acid solution and that of Ferrari et al.,
Kapsopoulou et al., (2005) and Mora et al., (1990)
(1992) on the inhibitory effect of acetic acid on
during their individual studies on growth and
xylose fermentating yeasts. When the pH was
fermentation characteristics of wine yeasts.
adjusted by IN HCl , to 4.1 and 3.7, there was normal
Acetobacter aceti has shown that it is
growth compared to the control medium. This shows
responsible for oxidizing ethanol to acetic acid in
that the pH of the medium did not inhibit the growth
wine and the effect of this acid has also been reported
of isolates but acetic acid influence
to have an inhibitory effect on yeast growth and

metabolism during fermentation processes. The
inhibitory effect of Acetobacter aceti on yeast growth


Nature and Science 2010;8(10)

5.0 Conclusions

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