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Asian Pac J Trop Med 2014; 7(Suppl 1): S238-S243

Contents lists available at ScienceDirect

Asian Pacific Journal of Tropical Medicine

journal homepage:www.elsevier.com/locate/apjtm

Document heading

Screening

doi: 10.1016/S1995-7645(14)60239-X

and study of antifungal activity of leaf litter actinomycetes

isolated from Ternate Island, Indonesia
*

Arif Nurkanto , Heddy Julistiono

Research Center for Biology, Indonesian Institute of Sciences (LIPI), Jl. Jakarta-Bogor KM 46 Cibinong, West Java-16911, Indonesia

ARTICLE INFO

ABSTRACT

Article history:
Received 20 Feb 2014
Received in revised form 1 Mar, 2nd revised
form 7 Mar, 3rd revised form 12 Mar 2014
Accepted 26 Apr 2014
Available online 19 Aug 2014

Objective: To characterize abundance of leaf litter actinomycetes from Ternate Island and to
assess the antifungal activity of actinomycetes isolates against Candida albicans, Saccharomyces
cerevisiae (S. cerevisiae), and Aspergillus niger.
Methods: Actinomycetes were isolated from leaf litter of Durio species, Syzygium aromaticum,
Piper betle, Myristica fragrans, or Pandanus species and unknown plants. Actinomycetes isolates
were cultured in a liquid medium. Bioactive compounds were extracted and tested against fungal
using Beury-Kirby method with modification. Minimum inhibitor concentration and cell leakages
were conducted. Actinomycetes that produced the highest antifungal activity were indentified
using molecular sequence data in 16S rRNA gene.
Results: Out of 50 selected isolates, two isolates MG-500-1-4 and SR-2-2 has highest activity
against S. cerevisiae. Concentration of material containing nucleic acids, proteins, Ca+ and K+
ions and morphological observations indicated that extracts of MG-500-1-4 and SR-2-2 caused
cell leakage and invagination of S. cerevisiae cells. Based on 16S rRNA gene identification, MG500-1-4 and SR-2-2 isolates are similar to Streptomyces misakiensis and Streptomyces tricolor
respectively.
Conclusions: Ternate Island contains interesting biodiversity of actinomycetes that has potential
use in agriculture, fisheries, and human health to reduce problem of fungal pathogen.

Keywords:
Ternate island
Wallace biogeographic
Actinomycetes
Antifungi

1. Introduction
Ternate Island (Moluccas) is one of eastern Indonesian
islands located in W allace biogeographical region.
T hese regions are meeting-point of the biota of two
major geographical regions: A ustralia and O riental
region. Ternate has a large number of species that are

unique to the area and is recognized as a biodiversity

hotspot[1]. The diversity of microbes of Ternate Island is
expected to be more higher than that of plants. This is
based on the prediction of ratio of unique species and
vascular plant of 6:1[2]. Despite the richness of fauna and
flora of Ternate Island, there is only a few information
about microbial diversity. M icrobial diversity of the
island is, therefore, intriguing to study. Actinomycetes
*Corresponding author: Arif Nurkanto, Research Center for Biology, Indonesian
I nstitute of S ciences ( LIPI ) , J l. J akarta- B ogor KM 46 C ibinong, W est J ava- 16911 ,
Indonesia.
E-mail: arif.nurkanto@lipi.go.id
Foundation Project: Supported by Research Center for Biology, Indonesia Institute of
Sciences (Grant. No. 3400.003.002/028).

belong to a microbial group that is still interesting

to explore until now due to their potency to produce
active substances against drug resistance microbes [3].
In agriculture, actinomycetes are promising to be used
for biocontrol against fungal pathogen of plant [4], and
bacterial pathogen in aquaculture [5]. F ish pathogenic
fungi is also a problem in aquaculture[6]. Fungal diseases
candidiasis and aspergillosis are infections caused by
fungi, leading to increasing morbidity and mortality[7].
Thus, actinomyctes having activity against harmful fungi
are useful not only for agriculture and aquaculture but
also for human health.
In an ecosystem, leaf litter is an important source of
microbes including actinomycetes [8] . M oreover, it is
also reported that actinomycetes could be antagonist
to decomposer fungi including fast growing genera
Mucor, Penicillium, Trichoderma and moderately slowgrowing genera Cladosporium and Mortierella [9]. T he
aim of this research is to obtain information about
actinomycetes abundance from leaf litters in T ernate

Arif Nurkanto and Heddy Julistiono/Asian Pac J Trop Med 2014; 7(Suppl 1): S238-S243

S239

Island and to reveal their potential activity against yeast

disc were measured after 24 h.

(S.

2.2.3. Identification and phylogenetic analysis

I dentification and phylogenetic analysis were
conducted just for active actinomycetes isolates. Isolated
strains were cultivated in 5 m L of A ctino medium N o.
1 broth ( ISP 2 ) . P ellets were collected during early log
phase growth. DNA extracted was conducted with GES
method. A bout 0 . 5 L DNA from extraction process
was used as a temple for polymerase chain reaction
( PCR ) amplification of an approximately 1500 base
segment of the 16 S r DNA gene. T he PCR primers were
20 F ( 5 - GATTTTGATCCTGGCTCAG - 3 ) and 1500 R ( 5 GTTACCTTGTTACGACTT - 3 ) . T he resulting PCR products
were purified with polyethylene glycol precipitation[10].
T he purified PCR products were sequenced on an ABI
3130 Genetic Analyzer (Applied Biosystems Inc., Foster,
C alifornia ) with the B ig D ye T erminator version 3 . 1
sequencing method. T he sequencing primer used was
primer 20F (5-GATTTTGATCCTGGCTCAG-3). The sequences
were then assembled with BioEdit.
T he 16 S r DNA sequences inferred from the 16 S r DNA
sequences determinated as described above were
aligment with 16S rDNA sequences from selected species
from several related genera. Sequences of the reference
strains were obtained from NCBI gene bank and were
chosen based on a hight similarity rank with the strain
from the actinobacteria along with a sequence to serve
as an outgroup. Approximately 800 bases were included
in the phylogenetic analysis, which was performed with
the Clustal X 8.13 version and NJ plot win 95 computer
program.

Candida albicans (C. albicans), Saccharomyces cerevisiae

cerevisiae) and filamentous fungi Aspergilus niger (A.
niger).
2. Material and methods
2.1. Sample site of actinomycetes isolation

Leaf litter samples were obtained from Ternate Island,

mainly in G amalama M ountain, N orthern M oluccas,
Indonesia. Sites for sampling had been taken according

to the altitude and canopy of plantation ( monoculture)

or vegetation (heterogenic mixture ) of 5 species: Durio
species, Syzygium aromaticum (S. aromaticum),
Piper betle (P. betle), Myristica fragrans, or Pandanus
species and others (mixture of unknown plant species)
respectively.
2.2. Isolation of actinomycetes

2.2.1. Fermentation and extracts preparation

A ctinomycetes isolates were cultured in a liquid
medium as follows. A loopful of mature slant culture was
inoculated into a 50 mL culture tube containing 10 mL
Actino medium No. 1 (Daigo, Japan) of the seed medium
consisting of 5 g polypeptone, 3 g yeast extract and 1 L
water, p H 7 . 2 . T he inoculated tubes were shaken on a
rotary shaker (130 r/min) at 28 C for 7 d. About 3 mL of
the seed culture were transferred into a 500 m L flask
containing 250 mL of the production medium consisting
of glucose with same composition. The inoculated flasks
were shaken on a rotary shaker (130 r/min) at 28 C for 7
d. Then, 250 mL of ethyl acetate: methanol (4:1) was added
to the flasks and they were shaken for 1 h. Mycelium was
removed by filtration and centrifugation. The resulting
supernatant without water was evaporated until dry
powder. The powder extracts were resolved using acetone
and used for bioassay.
2.2.2. Antifungal assay
T hree strains of fungi/yeast tested in this screening
were C. albicans NBRC 1594, S. cerevisiae NBRC 10217 and
A. niger. Fungi and yeasts were cultured on fresh yeast
malt agar. The inhibitory effect of the extract obtained
from leaf litter actinomycetes was tested by a modified
Beury-Kirby method with paper discs (5 mm diameter).
Sterile paper discs were placed on the surface of assay
plates, and 20 L of extract were pipetted onto each disc
and incubated at 35 C. The inhibition zones around each

2.2.4. Minimum inhibitor concentration (MIC)

MIC value was determined based on broth micro
dilution method[11,12]. Fungi were cultivated in Sabouraud
broth at 35 C, respectively until microbial concentration
reached approximately 1 10 5 CFU /m L to 5 10 5 CFU /m L .
We conducted 3 times repetition. Agar diffusion method
for confirm MIC value was done. W e used commercial
antibiotic, such as antibacterial and antifungal, to
compared the results.
2.2.5. Fungal cell leakages: protein, nucleic acid and ion
C ell leakage analysis was carried out based on
measurement of content of material absorbing at 260
nm or 280 nm according to Lunde and Kubo[13], and ion
+
+
K and C a according to P rashar method [14], with some
modifications. Y east grew up in S aboroad medium for
24 h. A bout 10 m L suspension was added with T ween80 and centrifuged at 3 500 r/min for 20 min. Supernatant

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Arif Nurkanto and Heddy Julistiono/Asian Pac J Trop Med 2014; 7(Suppl 1): S238-S243

was discarded, pellets were washed twice with phosphate

buffer. Cell in phosphate buffer were treated with control
(0 MIC), 1 MIC and 2 MIC. Solvent without extract were used
for control treatment. T reatment 1 MIC and 2 MIC used
in this research mean one time and twice concentration
of extract, respectively. After treatment, incubation was
conducted for 12 h. P ellets cell and supernatant were
separated using centrifugation (3 500 r/min for 20 min).
Pellets cell was then observed using scanning electron
microscope ( SEM ) ( after SEM procedure preparation ) .
Nucleic acid and protein leakages were investigated using
UV / VIS ( S himadzu, J apan ) at 260 nm and 280 nm. I on
+
+
C a and K leakages were detected in supernatant
using atomic absorption spectroscopy ( GBC S cientific
Equipment, Sens AA).

Abundance of leaf litter actinomycetes from various type of ecosystem and

different altitude.

Samples code GPS coordinate Altitude (m) pH Substract or vegetation

MG.10

0047462 N

17

7.0

Durio sp.

LGN

0046 N

250

7.0

S. aromaticum

---

250

6.8

Colony abundance
(CFU/g)

104
46.004.95
0.000.00
1.750.35

250
514

P. betle L
6.8 P. betle L
6.5 Myristica fragrans

11.5014.14

MG.500

0047001 N

MG.750

0047252 N

742

6.9

S. aromaticum

75.009.80

MG.1 000.1

0047764 E

1 000

6.8

Hetero-culture

1.250.35

MG.1 250

0047705 N

1 253

6.0

Pandanus sp.

27.5019.80

MG.1 500

0047881 N

1 463

6.9

Hetero-culture

85.007.07

12721118 E
12720749 E
12720864 E
12720686 E

MG-10-1-L-3

MG-10-1-L-4
MG-10-1-L-5

MG-10-1-L-6

MG-10-1-L-7
MG-10-1-L-8

MG-10-1-L-9
MG-500-1-1

MG-500-1-2
MG-500-1-3

MG-500-1-4

MG-1500-1-1
MG-750-1

MG-500-2-1

SR-1-2

Table 1

12721325 E

MG-10-1-L-2

SR-1-1

altitude ranging from 17 to 1463 m above sea level with

variation substract or vegetation (Table 1).

SR-2

MG-10-1-L-1

MG-500-2-4

Nine leaf litter samples were collected from different

SR-1

Isolates

MG-500-2-3

3.1. Abundance of leaf litters actinomycetes

12720969 E

Antifungal activity of metabolite produced by selected actinomycetes isolates.

MG-500-2-2

3. Results

12718072 E

Table 2

31.8022.50

3.2. Screening of antifungal activity of actinomycetes

W e then selected 50 actinomycetes isolates based

on morphotype characters ( color of colony, pigment

production, spore chain) and different ecosystem source.
These isolates were cultivated for extracts preparation.
Extracts obtained were used for screening of bioactivity
against A. niger, C. albicans and S. cerevisiae by using
paper disk method, the resluts are shown in Table 2.

MG-1250-2-1

MG-1250-2-2
MG-1250-2-3

MG-1250-2-4

MG-1500-1-2
MG-1500-1-3

MG-1500-1-4
MG-1500-1-5

MG-1500-1-6

MG-10-1-L-10
MG-1250-2-5

MG-1250-2-6

MG-1250-2-7
SR-2 -1
SR-2 -2

MG-100-1-S-1
MG-100-1-S-2

MG-100-1-S-3
MG-500-2-5
MG-500-2-6

MG-500-2-6
MG-500-1-5

MG-500-1-6

MG-500-1-7
MG-500-1-8

MG-500-1-9

MG-500-1-10

MG-500-1-11

MG-500-1-12

C.albicans
-

Clear zone diameter (mm)

A. niger
11.5
15.0
13.0
17.0
-

S. serevisiae
30.0
20.5
-

MIC from extracts produced by MG-500-1-4 and SR-2-2

were observed (Table 3). MG-500-1-4 has high activity
against S. cerevisiae; about 128 times than commercial
antifungal ( nystatin and kabicidin ) . H owever, antifilamentous fungal activity of MG-500-1-4 isolate is less
than that of nystatin and kabicidin and not active against
C. albicans. Antifungal activity of SR-2-2 isolate is lower
than that of MG-500-1-4, nystatin and kabicidin.

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Table 3

Microbe tested

Extracts (g/mL)
MG-500-1-4 SR-2-2

C. albicans
S. cerevisiae
A. niger

0.5

128

>128

>128

Comercial antifungal (g/mL)

Nystatin
Kabicidin
32

32

64

64

16

64

3.3. Effects of extract of MG-500-1-4 and SR-2-2

isolate on S. cereviseae cells integrity
Since S. cereviseae is more sensitive, we focused more

on S. cereviseae response treated with the active extracts.

Nucleic acid and protein content in supernantan of yeast
cells treated with MG - 500 - 1 - 4 ( F igure 1 ) or SR - 2 - 2
( F igure 2 ) increased significantly compared to control.
Control, 1 MIC (0.5 g/mL) and 2 MIC (1 g/mL) were treated
for MG-500-1-4 extract. Treatment for SR-2-2 extract in
this investigation was control, 1 MIC (128 g/mL) and 2 MIC
(256 g/mL).
0.07

260 nm

0.06

280 nm

20

Optical density (OD)

Ca

15
10
5
0

0 MIC

1 MIC

2 MIC

Figure 3. Ca+ and K+ leak from S. cereviseae after treatment with MG-500-1-4

extract.

25
20

Ca

15
10
5
0

0.05

0 MIC

1 MIC

2 MIC

Figure 4. Ca+ and K+ leak from S. cereviseae after treatment with SR-2-2

0.04

extract.

In relation with membrane cell damage in S. cerevisiae

0.03

cells treated with active extracts, morphological change

was also observed by using SEM (Figure 5 and 6).

0.02
0.01
0

Total ion leakages (g/mL)

fungi.

25

Total ion leakages (g/mL)

MIC value of most active compound and commercial antifungal against three

0 MIC

1 MIC

2 MIC

Figure 1. Protein and nucleic acid leak from S. cereviseae after treatment with
MG-500-1-4 extract.
1.6
1.4

Optical density (OD)

1.2

260 nm

280 nm

Figure 5. Morphological observation using SEM from S. cereviseae after

treatment with MG-500-1-4 extract (A: 0 MIC/control; B: 1 MIC; C: 2 MIC); 5 000
magnification.

0.8

0.6

0.4
0.2
0

0 MIC

1 MIC

2 MIC

Figure 2. Prorein and nucleic acid leak from S. cereviseae after treated with
SR-2-2 extract.

In addition, we evaluated ion leakages (Ca and K ) in

S. cereviseae treated with MG-500-1-4 or SR-2-2 extract.
+
+
A s presented in F igure 3 and 4 , content of C a and K
+

ion in supernatant of treated cells was higher than that

of control. T here was no significant difference of ions
concentration between 1 MIC and 2 MIC treatment. These
data support our suggestion that the extracts altered cell
membrane and caused cell leakage.

Figure 6. Morphological observation using SEM from S. cereviseae after

treatment with MG-500-1-4 extract (A: 0 MIC/control; B: 1 MIC; C: 2 MIC); 5 000
magnification.

3.4. Indentification of MG-500-1-4 and SR-2-2 isolates

Two isolates, MG-500-1-4 and SR-2-2, were identified

based on 16S rRNA gene. Both of them belong to Streptomyces
genera. MG-500-1-4 has similarities with Streptomyces
misakiensis (S. misakiensis) (99%) and SR-2-2 has similarities

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Arif Nurkanto and Heddy Julistiono/Asian Pac J Trop Med 2014; 7(Suppl 1): S238-S243
0.005

15

SR-2-2

40

67

64

Streptomyces lohii
67

Streptomyces globisporus
Streptomyces avidinii
Streptomyces virginiae
Streptomyces colombiensis
51
Streptomyces sporoverrucosus
99
Streptomyces sporoverrucosus

79

44

100
86

79
78

100

63

34

Streptomyces tauricus
Streptomyces tauricus
Streptomyces umbrinus
Streptomyces ederensis
Streptomyces kunmingensis
Streptomyces avermitilis

Streptomyces coelicolor
Streptomyces nodosus

Streptomyces albovinaceus

63

Streptomyces rimosus
Streptomyces aburaviensis
Streptomyces tsukiyonensis

90

47
99

Streptomyces aureus

44

84

67

Streptomyces griseus

99

98

17

32

S. tricolor

Streptomyces anulatus
Streptomyces rubiginosohelvolus
Streptomyces tanashiensis
Streptomyces anulatus
12
Streptomyces californicus
100 Streptomyces globisporus
74
Streptomyces roseochromogenus
Streptomyces celluloflavus

84

41
57

92

Kitasatospora kifunensis
Kitasatospora azatica
Streptomyces purpeofuscus

30

76

S. misakiensis
MG-500-1-4

Kitasatospora arboriphila
Figure 7. Phylogenetic positions of selected actinomycetes isolates based on partial sequences of 16S rDNA.
The tree was constructed by Neighbour-Joining method.

with Streptomyces tricolor (S. tricolor) (99%) (Figure 7). MG500-1-4 has bootstrap value 76 and SR-2-2 has bootstrap
value 84 . I solates that collected in this research were
deposited at Indonesia Culture Collection as open access
isolates.
4. Discussion
T he results indicated that there were no specific

correlation between colony abundance of actinomycetes and

the altitude. No actinomycetes were isolated in monoculture
leaf litter of S. aromaticum. The absence of actinomyctes
living on leaf of S. aromaticum, might be caused by toxicity
of the plant substances against actinomycetes shown by

Cai and Wu[15]. Ecosystem with heterogen vegetation gave

the most abundance of actinomycetes. Complex nutrient
came from different sources of leaf litter in an ecosystem
with different plants and may ensure a better condition for
actinomycetes growing.
In our study, there were only 10% of actinomycetes isolates
have activity against S. cerevisiae and A. niger. No extracts
could inhibit C. albicans. However, some of them have the
highest activity especially against S. cerevisiae. MG-500-1-4
and SR-2-2 are isolates that have highest activity. These
two isolates were then selected for extracts scaling up and
further investgation.
N ucleic acid and protein content in supernatant of
yeast cells treated with MG-500-1-4 or SR-2-2 increased
significantly compared to control. However, in cell treated

Arif Nurkanto and Heddy Julistiono/Asian Pac J Trop Med 2014; 7(Suppl 1): S238-S243

with MG - 500 - 1 - 4 , there was no significant difference

between 1 MIC and 2 MIC treatments. Higher content of
nucleic acids and protein in supernatant indicated cell
leakages and cell wall alteration[13,16].
This visual observation on yeast cells treated with toxic
substance may be helpful to understand cells damage
mechanism. In this research, we observed oval shaped
cells with apparent smooth cell surfaces on untreated
yeast. After exposure to MIC of MG-500-1-4 and SR-2-2
extracts 1 or 2 times, invaginations of cells were observed.
The changes in morphology of the yeast cells in response
to polyene antifungal or osmotic pressure are consistent
with the findings of other researchers[17,18]. They reported
invagination of plasma membrane upon examining the
freeze fracture images and outside envelope.
Ternate has high abundance of leaf litter actinomycetes.
Several isolates produced bioactive substances against fungi.
Two bioactive extracts of MG-500-1-4 and SR-2-2 have
highest activity against S. cerevisiae. MG-500-1-4 extract
inhibited S. cereviseae better than nystatin and kabicidin
did. Treatment with these two extracts effected nucleic acid,
protein and ion leakages. Morphological change of cells after
treatment with MG-500-1-4 and SR-2-2 extracts were also
observed. Based on 16S rRNA gene identification, MG-5001-4 and SR-2-2 isolates have similarity with S. misakiensis
and S. tricolor respectively.
Conflict of interest statement
We declare that we have no conflict of interest.
Acknowledgements
Funding to carry out the project was provided by regular
governmental annual fund every fiscal year from Research
Center for Biology, Indonesia Institute of Sciences (Grant No.
3400.003.002/028). Authors would like to thank Professor Ibnu
Maryanto for managing the Ternates expedition and Dian
Matrika Widyasih for assistance during laboratory analyses.

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