characterization of killer yeasts from nigerian traditional

Adesokan I. A.

PJST Vol. 8, 2013

13

The Polytechnic Journal of Science and Technology

PJST. Vol. 8. 2013

ISSN: 1115 – 8336

© 2013 Journal of Science & Technology Polytechnic Ibadan, Nigeria

Characterization of Killer Yeasts from Nigerian Traditional Fermented

Alcoholic Beverages

Adesokan I. A. Department of Science Laboratory Technology,

The Polytechnic, Ibadan, Nigeria.

Email address: [email protected]

Abstract

In this study the killer phenotypes among yeasts isolated from Nigerian traditional

alcoholic beverages was investigated using methylene blue medium. The ethanol

tolerance of the yeast isolates was also determined. A total of 62 yeasts were identified

and found to belong to 16 different species and 10 genera. Saccharomyces cerevisiae was

isolated from all the beverages and had a percentage occurrence of 33.33% in burukutu. It

was closely followed by members of the genus Candida with 26.67% occurrence in the

same beverage. Other yeasts isolated were S. chevalieri and S. uvarum. The viable counts

of the yeasts ranged between 2.0x107 and 9.5x105 cfu/ml while the pH was between 3.6

and 5.4. Candida tropicalis and C. castelli were the most ethanol tolerant by having a

growth of 1.5% in broth with 10% ethanol. All the strains of S. cerevisiae tested exhibited

killer activity. Other killer yeasts isolated were members of genus Candida (except C.

fructus), Pichia ohmeri, Schizosaccharomyces japonicum, S. chevalieri, Geotrichum

candidum and Kluveyromyces apiculata. The non-killer yeasts isolated were S. uvarum,

Sch. pombe, Rhodolorula graminis, K. apiculata and Pichia membranefaciens. P.

membranefaciens and R. graminis were neutral while others were sensitive. Although all

the strains of S. cerevisiae tested were killer yeasts, strain PW1 was especially ethanol

tolerant and therefore seems to be a potentially useful strain.

Introduction

Fermented foods and beverages constitute

a very important component of the

people’s diet in Africa. There are several

different fermented products, which have

been reported. Many fermented foods

serve as main course meals and such food

include gari, fufu and ogi. Others are

highly prized food condiments. Those that

are food condiments are usually made

from the fermentation of protein rich

seeds. Some of these are “iru” from Africa

locust bean; Ugba from Africa oil bean

and ogiri from melon seeds (Odunfa and

Oyewole, 1998).

There are five different types of

traditional alcoholic beverages that are

well known in Nigeria. These are

burukutu, pito, sekete, which are prepared

from malted maize sorghum grain,

mailto:[email protected]

Characterization of Killer Yeasts from Nigerian Traditional Fermented Alcoholic Beverages

PJST Vol. 8, 2013 14

agadagidi from ripe plantain pulp and

palm-wine from sugary sap of palm tree

(Elaeis guineensis) and raphia palm

(Raphia hookeri) (Sanni and Lonner,

1993). These beverages apart from

serving as inebriating drinks are also

important in fulfilling social obligations

such as marriage, naming and burial

ceremonies. Their production is as a result

of mixed, spontaneously generated

fermentation with yeasts and lactic acid

bacteria as the predominating

microorganisms. However, the yeasts

flora is mainly responsible for the alcohol

content of the beverages (Sefa-Dedeh et

al., 1999).

The production by yeasts of exotoxins

with antimicrobial activity mediated by

specific cell wall receptor on susceptible

microorganisms is a relatively common

phenomenon (Polonelli and Morace,

1986; Polonelli et al., 1991). Exotoxins

(generally protein or glycoprotein) that are

able to kill susceptible cells belonging to

the same or congeneric species have been

defined as killer toxins. Killer yeasts are

toxin-producing fungi that are immune to

the activity of their own killer toxins.

After the original description of the

phenomenon in Saccharomyces cerevisiae

reported by Bevan and Makower (Yap et

al., 2000), the attention of increasing

number of investigators has been focused

in the killer effect, which might represent

a model of biological competition

somewhat related to that of bacteriocin

among bacteria (Musmanno et al., 1999;

Schmitt and Breinig, 2006; Buzzini et al.,

2007).

Various yeasts species have been reported

in traditional fermented alcoholic

beverages in Nigeria (Sanni and Lonner,

1993) while the killer activities of these

different yeasts species have been scarcely

reported. Therefore, the objective of this

present study is to provide information on

the killer, sensitive and neutral

phenotypes among the yeast isolated from

Nigerian traditional fermented alcoholic

beverages.

Materials and Methods

Sample collection

Palm wine, burukutu and pito used in this

study were from the local producers

within Ibadan metropolis and agadagidi

was from Ile-ife. Four samples of each

beverage were collected separately in

sterile containers and kept at 4oC before

analysis. Sensitive strain Candida

glabrata Y55 and killer strains P. kluyveri

UW2 and Kluyveromyces UW1 were

obtained from the culture collection of

Department of Biology, University of

Western Ontario, Canada.

Isolation and identification of yeasts Isolation was carried out on samples of

each of these alcoholic beverage collected

at various interval. One ml of suitable

dilutions of each sample was poured into

sterile Petri dishes and sterile molten

(45oC) Potato Dextrose Agar (PDA)

containing 30µg/ml streptomycin was

poured into the Petri dishes. The plates

were purified by repeated streaking on

PDA. The purified cultures were streaked

onto PDA slants, incubated at 25oC for 48

hours. The yeast isolates were then

identified by physiological and

biochemical tests according to the scheme

described by Kreger-Van Rij (1987).

Adesokan I. A.

PJST Vol. 8, 2013

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Viable counts and pH measurement

The viable count was determined using

the pour plate technique. Suitable

dilutions of each sample were plated in

duplicates and colony forming units was

obtained on PDA after incubation at 25oC

for 72 hours. The pH of the samples was

determined using a pH meter.

Determination of killer activity

Killer phenotype was determined

according to the method of Guitierrez et

al. (2001). The assay medium was yeast

extract peptone dextrose (YEPD) agar

containing 0.03g/l methylene blue and

buffered to pH 4.5 and 3.5 with 0.2M

citrate-phosphate buffer. All isolates were

transferred to plates seeded with sensitive

strain Candida glabrata Y55 and

incubated at 25oC for 48-96 hours. The

tested strain was designated a killer yeast

if the streaked strain was surrounded by a

region of bluish-stained cells or by a clear

zone of growth inhibition bounded by

stained cells. To test the sensitive and

neutral phenotypes, killer strains

Kluyveromyces sp UW1 and Pichia

kluyveri UW2 were streaked onto plates

seeded with each of the non-killer isolates.

If the killer strain was surrounded by a

clear zone of inhibition or by a region of

bluish-stained cells, then the tested strain

was designated as sensitive. If there was

no clear zone of inhibition or a region of

bluish-stained cells then the tested strain

was designated as neutral. That is a

sensitive strain was inhibited by the killer

strain while a neutral strain was not.

Ethanol tolerance

Ethanol tolerance was measured as the

ability of the yeast to grow in yeasts

extract peptone dextrose (YEPD) broth

with increasing content of ethanol 5%,

10% and 15%. After 72 hours of

incubation, growth was measured by

turbidity increase at 650nm with camspec

M105 spectrophotometer (Sefa-Dedeh et

al., 1999).

Results

From the four different samples of each

alcoholic beverages analyzed a total of 62

yeast isolates were identified. The yeasts

were found to belong to 16 different

species and 10 different genera (table 1).

The most frequently encountered yeast

which was found in all beverages was

Saccharomyces cerevisiae. Also S.

chevalieri was found in all the beverages

except in palm wine while S. uvarum was

isolated from burukutu and pito. The

members of the Genus Candida isolated

were Candida valida, C. krusei, C.

intermedia C. tropicalis and C. castelli.

Each of these yeasts was isolated from

one beverage except C. valida which was

found in both burukutu and pito. The

viable counts of the yeasts from the

beverages ranged between 2.0X 107 to

9.5X108 cfu/ml while the pH was between

3.6 and 5.4 (table 2). The ethanol

tolerance of the yeasts (figure 1) showed

that Candida tropicalis and P.

membranefaciens had the highest growth

of about 1.5% in YEPD broth with 10%

ethanol. Whereas S. cerevisiae BK2,

PT1,PW1, G. candidum, Sch. japonicum,

Sch. pombe and R. graminis had a growth

above 1.0% in YEPD broth with 10%

ethanol. Furthermore, Candida valida, S.

cerevisiae BK2, PW1, S. chevalieri, R.

graminis, C. castelli and C. fructus grew

in all the 3 ethanol concentration used.


PJST Vol. 8, 2013 16

However, P. kluyveri UW2 and

Kluyveromyces UW1 grew only in YEPD

broth with 5% ethanol. Meanwhile, all the

yeasts that grew in YEPD broth with 15%

ethanol had a value below 0.5% except C.

intermedia which was above 0.5%. The

killer activity of the yeast isolates are

presented in table 3. All the strains of S.

cerevisiae tested and the members of

genus Candida isolated (except C.

fructus) exhibited killer activity. Other

killer yeasts isolated were P. ohmeri, Sch.

japonicum, S. chevalieri, G. candidum and

K. africanus. The non-killer yeasts

isolated were S. uvarum, Sch. Pombe, R.

graminis, K. apiculata and P.

membranefaciens. P. membranefaciens

and R. graminis were neutral while others

were sensitive.

Table 1: Distribution of yeast species found in the alcoholic beverages

Species Bukurukutu No %

Pito No %

Agadagidi No %

Palm wine No %

Candida valida 4 26.67 1 6.67 - - - - Saccharomyces cerevisae

5 33.33 3 20.00 5 26.32 3 23.08

Saccharomyces chevalieri

1 6.67 3 20.00 1 5.26 - -

Pichia ohmeri 2 13.33 3 20.00 - - 2 15.38

Geotrichium candidum

1 6.67 - - - - - -

Saccharomyces uvarum

1 6.67 1 6.67 - - - -

Schizosaccharamyces japonicum

1 6.67 1 6.67 - - 1 7.69

Kluyveromyces africanus

- - 2 13.33 2 10.53 - -

Schizosaccharomyces pombe

- - - - 1 5.26 - -

Candida krusei - - - - 4 21.05 - - Candida intermedia - - - - 1 5.26 - - Kloeckera apiculata - - - - 1 5.26 1 7.69 Rhodolorulagraminis - - - - 3 15.78 2

15.38 Candida tropicalis - - - - - - 1 7.69 Candida castelli - - - - - - 1 7.69 Pichia membranefaciens

- - - - - - 1 7.69

Torulaspora delbruekii

- - - - - - 1 7.69

- - 1 6.67 1 5.26 - -

Adesokan I. A.

PJST Vol. 8, 2013

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Table 2: Yeasts viable counts and pH of the beverages

Beverages pH Colony forming unit/ml

Agadagidi 3.6* 2.0 x107

Burukutu 3.6 5.3x107

Palm wine 5.4 9.5x108

Pito 3.8 7.2x108

*Values are means of four determinations.

Discussion

In this study yeasts were isolated from

four different Nigerian traditional

fermented alcoholic beverages. All the

isolated yeasts in this study have been

reported with exception of C. valida.

Sefa-Dedeh et al. (1999) characterize

yeasts in traditional brewing of pito in

Ghana and identified 21 strains belonging

to 8 genera as Saccharomyces cerevisiae

(8), Candida tropicalis (4), Kloeckera

apiculata (2), Hansenula anomata (2)

among others. Theivendirarajah and

Chrystopher (1986) reported three general

of yeasts in palmyrah palm wine. They

stated the organisms of significant are

those belonging to the genus

Saccharomyces, because of their

numerical predominance and superior

fermentative ability. In the present study

members of the genus Saccharomyces had

the highest percentage of occurrence and

were isolated from all the alcoholic

beverages studied. This showed that the

member of this genus is the most

predominant and the result is in agreement

with the observation of earlier works

(Ekundayo, 1969; Theivendirarajah and

Chrystopher 1986; Demuyakor and Ohta,

1991; Sanni and Lonner, 1993).


PJST Vol. 8, 2013 18

Table 3: Killer activity of yeasts isolated from Nigerian traditional fermented

alcoholic beverages

Species Sensitive yeast in the lawn

(indicator organism)

Killer activity

Pichia ohmeri Candida glabrata +

Candida fructus Candida glabrata -

C. krusei Candida glabrata +

C. valida Candida glabrata +

C. tropicalis Candida glabrata +

C .intermedia Candida glabrata +

Schizosaccharomyces pombe Candida glabrata -

Sch. Japonicum Candida glabrata +

Saccharomyces cerevisiae

AG19

Candida glabrata +

S. cerevisiae BK2 Candida glabrata +

S. cerevisiae PT1 Candida glabrata +

S. cerevisiae PW1 Candida glabrata +

S. uvarum Candida glabrata -

S. chevalieri Candida glabrata +

Geotrichum candidum Candida glabrata +

Kluyveromyces africanus Candida glabrata +

Kloeckera apiculata Candida glabrata -

Rhodolorula graminis Candida glabrata -

Pichia membranefaciens Candida glabrata -

Torulaspora delbruekii Candida glabrata -

Adesokan I. A.

PJST Vol. 8, 2013

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PJST Vol. 8, 2013 20

Next in terms of numerical number are the

members of the genus Candida. The

Candida isolated are C. valida, C. krusei,

C. intermidia, C. tropicalis, C. castelli and

C. fructus. All these members have been

reported by previous studies (Sanni and

Oso 1988). The only exception is Candida

valida.

The yeast viable counts and pH range

reported in this work was significantly

different from values reported by Sanni

and Lonner (1993). A pH range of 3.4 to

5.4, and yeast viable counts of 8.4x104 to

6.2x106 cfu/ml was reported by these

workers. The unpredictability of the

associated yeast flora of traditional

alcoholic beverages in sub-Sahara Africa

could be due to spontaneous nature of the

fermentation, sources and type of

ingredients used.

The growth of the test isolates at different

ethanol concentration over a period of

time indicates their capacity for ethanol

tolerance (Sefah-Dedeh et al., 1999) even

though the alcoholic content of

traditionally brewed beverages is between

1.5-6% (Odunfa and Oyewole, 1998).

Although ethanol tolerance is not directly

related to ethanol production capability

(Sanni and Onilude, 1998), such

information could be important for

selection of potential strains of yeasts that

can be employed for optimization of

processing of the traditional alcoholic

beverages with higher ethanol content.

The dependence of killer activity on pH

was first observed by Woods and Bevan

(1986). Since then, the optimum pH for

killer activity has been defined for a

number of killer toxins, with the majority

of them having an optimum pH between

4.2 and 4.7 (Pfeiffer and Radler, 1984). It

was observed that all the strains of S.

cerevisiae tested exhibited killer activity

at pH 3.5 and 4.5. However, the killer

activity of C. castelli, C. tropicalis and G.

candidum was not detectable at pH 3.5

(result not shown).

All the strains of S. cerevisiae isolated

were killer yeasts and they showed a very

strong ethanol tolerance. S. cerevisiae

PW1 and BK2 grew at 15% ethanol

concentration whereas PT1 and AG19 did

not grow at all. Sefa-Dedeh et al. (1999)

reported that S. cerevisiae I was the most

ethanol tolerant while K. africanus was

the least. Several studies have shown

different applications of killer yeasts for

biotyping, wine fermentation, potential

antifungal activity (Perez et al., 2001;

Sangorin et al., 2002; Schmitt and

Breinig, 2002; Buzzini et al., 2007).

Although all the strains of S. cerevisiae

tested were killer yeasts, strain PW1 was

especially ethanol tolerant and therefore

seems to be a potentially useful strain.

Further investigation could be carry out to

determine the strong and weak killer

phenotypes among the yeasts so as to pick

the one with best toxin production ability

and whose toxin has a broad spectrum of

activities against many yeasts.

Adesokan I. A.

PJST Vol. 8, 2013

21

ACKNOWLEDGEMENT

The author gratefully acknowledge the

technical support and positive criticism by

Prof. A.I. Sanni of Department of

Microbiology, University of Ibadan,

Nigeria in the course of supervising this

project and Prof. M.A Lachance of

Department of Biology, University of

Western Ontario, Canada for kind

donation of some yeast isolates used in

this research work. The technical support

of Dr S.T. Ogunbanwo of Department of

Microbiology, University of Ibadan,

Nigeria was also appreciated.

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