The Potential Methanolic Extract of Coffee Leaves (Coffea Canephora

L.) in Inhibiting Storage Fungi and Yeast

Anggi Audina

, Kiki Nurtjahja

and Albert Pasaribu

Department of Biology, Universitas Sumatera Utara, Medan, Indonesia

Department of Chemistry, Universitas Sumatera Utara, Medan, Indonesia

Keywords: Antifungal activity, Coffee leaves, Storage fungi, Minimum inhibitory concentration

Abstract: Robusta coffee (Coffea canephora L.) leaves contain alkaloids, flavonoids, and phenolics that antifungal

activity. This study aims to determine the ability of robusta coffee leaf extract in inhibiting six spesies of

postharvest fungi and yeast (Aspergillus candidus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus

niger, Aspergillus oryzae, Aspergillus tamarii, Penicillium sp., Candida albicans and yeast). The leaves were

obtained from farmers at Berastagi, Karo Regency, North Sumatra. The extract was obtained by maceration

using methanol. Antifungal activity was determined using disc diffusion method at concentrations 20, 40, 60,

80 and 100%. The minimum inhibitory concentrations were determined by dilution method at concentrations

20, 40, 60, 80, 85, 90, and 95%.The positive control used was ketoconazole 2% and negative control dimethyl

sulfoxide 10%. Results showed coffee leaves extract has anti-fungal activity againts storage fungi and yeast

which were characterized by an inhibition zone from 9.27 to 0.62 mm. The most effective extract at

concentration 100% with the highest inhibition 9.27 mm againts A. oryzae. Whereas, the minimum inhibitory

concentration was at 85% againts A. oryzae and C. albicans.

1 INTRODUCTION

Storage fungi that contaminate agricultural products

become a serious problem particularly on tropical

countries. The ability of the fungi to grow at low

moisture content might increase their population

during storage and it cause spoilage and mycotoxin

contamination (Pitt and Hocking 2009; Bala 2017).

Aspergillus and Penicillium were storage fungi that

commonly found contaminate food and foodstuffs

during storage (Pitt and Hocking 2009).

The used of chemical substances such as

phosphine and ethylene oxyde gas (EtO) as fumigants

were effective to inhibit fungal growth, however, the

substances are carcinogen when inhaled and it leaves

harmful chemical residues. Coffee leaves contain

caffeine, alkaloids, saponin, flavonoid, phenolic,

terpenoid and tannin (Hasanah 2017).

Nartowicz (1979) reported that caffeine was the

most predominant compound in coffee. This

compound inhibit A. flavus, A. parasiticus,

Penicillium citrinum and P. urticeae. The other

components such as phenol, trigonelline and

chlorogenic acid were reported has antifungal activity

(Fardiaz, 1995; Karou et al. 2005)). The potential of

coffee leaves extract (Coffea canephora) in inhibiting

C. albicans was reported by Erisha (2017) and Putri

(2017). The purpose of the recent study was to

investigate robusta coffee leaves extract (Coffea

canephora) in inhibiting storage fungi and yeast.

2 MATERIALS AND METHOD

2.1 Preparation of Fungal Isolates

The experiment was conducted from April to October

2019 at Microbiology and Biotechnology Laboratory,

Faculty of Mathematics and Natural Sciences,

Universitas Sumatera Utara. All fungi isolates used in

this experiment were culture collection of

Microbiology and Biotechnology Laboratory,

Faculty of Mathematics and Natural Sciences,

Universitas Sumatera Utara. All fungal isolates were

previously isolated from dried-stored spices that sold

by retailers at traditional markets in Medan, North

Sumatera. The fungal isolates were were sub-cultured

in potato dextrose agar (PDA) and incubated at 29

for 5 days.

534

Audina, A., Nurtjahja, K. and Pasaribu, A.

The Potential Methanolic Extract of Coffee Leaves (Coffea canephora L.) in Inhibiting Storage Fungi and Yeast.

DOI: 10.5220/0010612500002775

In Proceedings of the 1st International MIPAnet Conference on Science and Mathematics (IMC-SciMath 2019), pages 534-537

ISBN: 978-989-758-556-2

2.2 Extraction of Coffee Leaves

As much as ten kilogram of fresh coffee leaves

(Coffea canephora) obtained from subsistence

farming at Berastagi, North Sumatera. All of the fresh

leaves were air dried for 4 days and powdered using

RT 04 Mill Powder Tech. Co LTD Taiwan at 25.000

rpm for 30 second and 2000 g simplicia obtained were

extracted in methanol 1:2 (w/v,). Homogenization

was conducted every 1×24 hour for 3×24 hours. The

suspension was filtered using Whatman filter paper

no.1, the residu obtained was add by fresh methanol

several times until clear filtrate was obtained. Filtrate

then was air-dried by rotavapor 100 rpm at 50

The extract concentrations made were 100, 80, 60, 40

and 20%, dimethyl sulfoxide 10% used as a solvent.

2.3 Determination of Fungal Spore

Density

All isolates of fungi subculturing on potato dextrose

agar plates (5 days at 28°C) in petri dish (diameter 9

cm). Each petri was added by 10 ml distilled water

containing 0.05% Tween 80. The spores then were

harvested using small brush. The spore suspension

used was made 10

/ml.

2.4 Determination of Inhibition of

Coffee Leaves Extract on Storage

Fungi and Yeast

Well diffusion method was used in this experiment.

Paper disc (5 mm in diameter) containing 10 µl

extract concentration was placed in petri dish (9 cm

in diameter) containing 15 mL potato dextrose agar

plate. Each fungal isolate was tested by inoculating

agar plug. Ketoconazole 2% and dimethyl sulfoxide

10% were used as positive and negative control

respectively. All plates were incubated at 27°C for 5

days. Three replicates were used for each

concentration.

2.5 Determination of Minimum

Inhibitory Concentration

Various extract concentrations (20, 40, 60, 80, 85, 90

and 95% (w/v) were made. Ketoconazole 2% and

dimethylsulfoxide 10% were used as positive and

negative contol respectively. One mililiter of each

extract was mixed homogenously with 1 ml spore

suspension (10

/ml), Serial dilution made was 10

-1

-2

, 10

-3

and 10

-4

One mililiter of each dilution was

pour plate in petri dish containing PDA medium. All

plates were incubated at 27°C for 5 days, Three

replicates were made for each concentration. The

mycelial growth on each concentration was observed.

3 RESULTS AND DISCUSSION

3.1 Inhibition of Coffee Leaves

Methanolic Extract on Mycelial

Growth of Storage Fungi and Yeast

Coffee leave extract potential to inhibit mycelial

growth of storage fungi and yeast (Table 1). In

general, the effect concentration of coffee leave

extract on fungal growth showed each fungal species

has different response. High extract concentration is

followed by the increase of inhibition area. Among

storage fungi, Aspergillus oryzae was the most

inhibited followed by A. tamarii, and A. niger.

Whereas, A. candidus and yeasts were the less

sensitive (< 5 mm).. In our research found that yeast

particularly C. albicans begin to inhibit at 20%

extract, The highest inhibition (2.93 mm) on yeast

occured at 100% extract. We assumed that cell wall

of asexual yeast might resist on the extract exposure,

as previous study by Jawetz et al. (2005) reported that

yeast cells with asexual chlamidospores have resist on

antifungi.

3.2 Minimum Inhibitory Concentration

of Coffee Leaves Methanolic

Extract on Mycelial Growth of

Postharvest Fungi

Minimum inhibitory concentration of the extract to all

fungal species was shown in Table 2.

As shown in

Table 2 each fungal species have different minimum

inhibitory concentration.

The Potential Methanolic Extract of Coffee Leaves (Coffea canephora L.) in Inhibiting Storage Fungi and Yeast

535

Table 1: Inhibition zone (mm) of coffee leaves methanolic extract (%) againts storage fungi and yeast

Fungal isolates

Extract concentration (%) / inhibition zone (mm)

DMSO

(

Ketoconazole

(K+)

20 40 60 80 100 Average

Aspergillus candidus 0 4.21 0.62 0.84 1.40 1.70 2.57 1.42

A. flavus 0 6.60 1.80 2.90 3.70 3.70 4.90 3.40

A. fumigatus 0 5.70 1.90 3.10 3.40 4.40 5.40 3.64

A. niger 0 9.13 2.40 3.10 5.60 6.60 7.60 5.06

A. oryzae 0 10.8 5.10 6.30 7.10 8.70 9.27 7.29

A. tamarii 0 9.60 4.10 6.20 7.80 6.80 8.10 6.60

Candida albicans 0 3.20 2.00 2.13 2.20 2.40 2.73 2.29

Penicillium sp. 0 8.87 0.80 2.10 3.80 5.50 6.50 3.74

Yeast 0 3.53 2.17 2.36 2.40 2.50 2.93 2.47

K - negative control, K + positive control

Table 2: Minimum inhibitory concentration methanolic extract of coffee leaves on mycelial growth of storage fungi and yeast

Extract

(%)

Fun

al species/Minimum inhibitor

concentration of coffee leave extrac

candi

dus

flavus

fumigatus

niger

oryzae

tamarii

Candida

albicans.

Penicillium

sp.

yeast

K- + + + + + + + + +

K+ - - - - - - - - -

100 + -* -* + - + - + +

95 -* + + + - + - + +

90 - + + + - + - + +

85 - + + + -* + -* + +

80 + + + + + + + + +

60 + + + + + + + + +

40 + + + + + + + + +

20 + + + + + + + + +

K (-) spore suspension as negative control

K (+) ketoconazole as positive control

+ mycelial growth occurred

- no mycelial growth

* minimum inhibitory concentration

In compare to ketoconazole that kill all fungal

mycelia, the presence of extract at 100%

concentration was potential to kill A. flavus, A.

fumigatus, A. oryzae and C. albicans. The extract has

no effect both on Penicillium sp. and yeast. The other

fungal species require minimum inhibitory

concentration to inhibit their grow such as A.

candidus (95%), A. oryzae (85%), C. albicans (85%).

Previous study by Handayani and Purwoko (2008)

showed similar results that the presence of coffee leaf

extract inhibit fungal mycelia.

4 CONCLUSION

Methanolic extract of coffee leave inhibit the growth

storage fungi and yeast. However, the inhibition and

minimum inhibitoory concentration was different

depend on the fungal species.

ACKNOWLEDGEMENT

The research was supported by TALENTA-Sumatera

Utara University, Grant no. 322/UN5.2.3.1/

PPM/KP-TALENTA USU/2019.

IMC-SciMath 2019 - The International MIPAnet Conference on Science and Mathematics (IMC-SciMath)

536

REFERENCES

Bala, B.K. 2017. Drying and storage of cereal grains, 2

ed. Willey Blackwell. Inc Minnesota. United Kingdom

Erisha, A.O. 2017. Kemampuan daya hambat ekstrak

kering beku daun kopi robusta (Coffea canephora)

terhadap pertumbuhan Candida albicans. Universitas

Jember. Jember.

Fardiaz, S. 1995. Antimicrobial activity of coffee (Coffea

robusta) Extract. ASEAN Food Journal, 10: 103–106.

Handajani, Purwoko. 2008. Aktivitas ekstrak lengkuas

(Alpinia galanga) terhadap pertumbuhan cendawan

Aspergillus spp. penghasil aflatoksin dan Fusarium

moniliforme. Biodiversitas, 9:161-164.

Hasanah, M., Maharani B., Munarsih E. 2017. Daya

Antioksidan Ekstrak dan fraksi daun popi robusta

(Coffea robusta) terhadap pereaksi DPPH (2,2-Difenil-

1-Pikrilhidrazil). IJPST, 4: 42-29

Jawetz, E.J.L, Melnick, Adelberg E. 2005. Mikrobiologi

Kesehatan. Penerbit Buku Kesehatan. Jakarta.

Karou, D., Savadogo A., Canini A., Yameogo S.,

Montesano C., Simpore J., Traore A.S. 2005.

Antimicrobial activity of alkaloids from Sida acuta.

African Journal of Biotechnology, 4(2): 195-200.

Nartowicz, V.B., Buchanan R.L., Segall S. 1979. Aflatoxin

production in reguler and decafeinated coffee beans.

Journal Food Science. 42: 446-447

Pitt, J.I., Hocking A.D. 2009. Fungi and Food Spoilage.

New York (US): Springer.

Putri, R.J.Y. 2017. Daya hambat ekstrak daun kopi robusta

(Coffee robusta L.) terhadap pertumbuhan C. albicans

secara in vitro. Universitas Jember. Jember.

The Potential Methanolic Extract of Coffee Leaves (Coffea canephora L.) in Inhibiting Storage Fungi and Yeast

537