Infection and Population of Aspergillus chevalieri on Dried-stored

Tropical Spices

K. Nurtjahja

, C. F. Zuhra

, H. Sembiring

, A. Bungsu

, J. Simanullang

, J. E. Silalahi

B. N. L. Gultom

, Sartini

Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Jln. Bioteknologi no.

1, Medan, North Sumatera, Indonesia 20155

Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Jln. Bioteknologi

no. 1, Medan, North Sumatera, Indonesia 20155

Faculty of Biology, Medan Area University, Jln. Kolam no. 1 Medan, North Sumatera, Indonesia 20223

Keywords: Aspergillus flavus, Fungal Population, Genes, Spices, Toxigenicity.

Abstract: Fourty five samples of 10 dried stored-spices (black pepper, candle nut, cinnamom, cloves, coriander,

cordamom, cumin, nutmeg, star anise, white pepper) obtained from 8 to 9 retailers at 5 traditional markets in

Medan, North Sumatera was investigated for infection by Aspergillus chevalieri (formerly Eurotium

chevalieri). Population of the fungal species was enumerate using dichloran 18% glycerol agar medium.

Moisture content of each spices and fungal characteristics were also observed. Results showed each dried-

stored spices has different moisture content, candle nut and nutmeg have the lowest moisture content (4.2

and 8.1 %) and white pepper and cinnamon have the highest moisture content (14.4 and 12.8 %) consecutively.

All spices observed infected by A. chevalieri, the infection occurred predominantly on coriander followed by

nutmeg and white pepper with fungal population 4.58, 4.41 and 4.02 (log CFU g

-1

) respectively.

1 INTRODUCTION

Molds infection on agricultural commodities

particularly on dried stored spices is one of the most

common problem during storage. The infection is

caused by molds that survive during pre and post-

harvest handling (Stankovic et al. 2006; Toma and

Abdulla, 2013). Pre-harvest handling is the main

inoculum for causing contamination of diverse strains

of molds on spices in the field (Kneifel and Berger,

1994). Poor handling practices of drying,

transportation and storage can incrase fungal

infection.

Xerophilic mold are able to grow at water activity

) ≤ 0.85 (Pitt and Hocking, 2009), the other fungal

species have been reported to grow at a

value 0.64 –

0.75 (Butinar et al. 2005). Aspergillus chevalieri

(formerly called Eurotium chevalieri) characterized

by having yellow cleistotecia, uniseriate conidial

head and yellow orange hyphae. Hubka et al. (2013)

reported that A. chevalieri is one of xerophilic and

xerotolerant molds that cause predominant spoilage

on nut, dried beans, spices etc. [Samson et al. 1995;

Pitt and Hocking, 2009). The ability to grow at low

or equilibrium relative humidity (ERH) makes E.

chevalieri increase a

value and allow other toxigenic

molds such as Aspergillus and Penicillium to grow.

The aim of the present study was to investigate

infection and population of Aspergillus chevalieri

isolated from dried stored spices in retailers on

traditional markets in Medan, North Sumatera.

2 MATERIALS AND METHODS

2.1 Sample Collection

Fourty five composite samples (200 g for each

sample) of 10 kinds of stored-dried spices i.e. black

pepper, candle nut, cinnamom, cloves, coriander,

cardamom, cumin, nutmeg, star anise and white

pepper were collected from 8 to 9 retailers at 5

different traditional markets. Each of the sample were

packed in steril polyethylene bag and stored in

refrigerator (-4 ºC) for further use.

Nurtjahja, K., Zuhra, C., Sembiring, H., Bungsu, A., Simanullang, J., Silalahi, J., Gultom, B. and Sartini, .

Infection and Population of Aspergillus chevalieri on Dried-stored Tropical Spices.

DOI: 10.5220/0010100101170120

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

117-120

ISBN: 978-989-758-449-7

117

2.2 Morphology Colony

Colony characteristics were observed

macroscopically from cultures grown for 7 days

(29±2 ºC) at dichloran 18% glycerol agar (DG18) and

malt extract agar (MEA) medium. Microscopic

morphology made using light microscope, Olymphus

CH2 Japan.

2.3 Determination Population of

A. chevalieri

Population of A. chavalieri on each spice was

determined using dilution method according to Pitt

and Hocking (2009). Each sample (200 g) was

ground for 30 seconds using blender (Model RT-04,

Taiwan). Triplicate plates were made for each

dilution. Each plates were incubated for 7 days at

29±2

C. All A. chevalieri colonies were counted as

colony forming unit (CFU g

-1

) of sample. Each single

separate of the colony was isolated and cultured on

czapex yeast extract agar (CYA) or CYA+20%

sucrose (CYA20S) and identified according to

procedure Pitt and Hocking (2009).

2.4 Moisture Content Analysis

Spice moisture content was determined according to

oven drying method [4]. Fifty gram of ground sub

sample stored-dried spices were put in aluminum foil

dish and dried in oven at 110 ºC for 24 h and

reweighed, three replicates per sample. Moisture

content was calculated using the following formula:

– M

)

Moisture content (% wet basis) = ---------------- × 100

(1)

= initial weight, in grams of test portion

= final weight, in grams of dried test portin

3 RESULTS AND DISCUSSION

Dried-stored spices sold by retailers in traditional

markets commonly packed separately in small plastic

container or plastic bag. Each sample of the spices

studied was presented in Table 1.

3.1 Spice Moisture Content

Moisture content of each dried-stored spices was

presented in Figure 1. The spices has different

moisture content (4.2-12.8%), except white pepper

has moisture content (14.4%) higher than

recommended by International food standard that

stated safe moisture level that has to be achieved for

spices is 12-14%.

Table 1: Ten species of dried-stored spices commonly sold by retailers at traditional markets.

No. Spices (scientific name) English name Used parts

1. Piper nigrum L. black pepper seeds

2. Aleurites moluccana L. candle nut kernels

3. Cinnamomum zeylanicum Blume cinnamon bark

4. Syzygium aromaticum L. Cloves flowers

5. Coriandrum sativum L. coriander seeds

6. Amomum cardamomum L. cordamom seeds

7. Cuminum cyminum L. Cumin seeds

8. Myristica fragrans Houtt. nutmeg kernels

9. Illicium verum Hook. star anise fruit

10. Piper nigrum L. white pepper seeds

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

118

Figure 1: Moisture content (% wet basis) of dried-stored spices obtained from retailers in traditional Markets.

3.2 Morphological Characteristics of

A. chevalieri

Aspergillus chevalieri, formerly Eurotium chevalieri,

is characterized forming a yellow cleistotecia

(teleomoph) in DG18 medium. Previuos study by

Hubka et al. [6] reported that the ascospores A.

chevalieri were smooth. Andrew and Pitt [9]

described the ascospore with prominent crests like

pulley wheels, with two prominent, narrow,

longitudinal flanges. Conidial head (anamorph)

uniseriate with blue or yellow orange.

Figure 2: Population of A. chevalieri (CFU g

-1

) at different dried-stored spices isolated on DG18 medium at ambient

temperature (29±2 ºC).

14,4

12,3

8,1

10,6

11,8

11,2

11,1

12,8

4,2

11,1

0 5 10 15 20

White pepper

Star anise

Nutmeg

Cumin

Cordamom

Coriander

Cloves

Cinnamon

Candle nut

Black pepper

Moisture content (% wet basis)

Spices

3,57

3,44

3,95

4,58

3,66

3,04

4,41

4,02

A. chevalieri population (log CFU g

-1

)

Spices

Infection and Population of Aspergillus chevalieri on Dried-stored Tropical Spices

119

3.3 Population of A. chevalieri

All spices studies were infected by A. chevalieri

with different population (Figure 2). Coriander was

the most infected (4.58 log CFU g

-1

) and star anise

was the less (2 log CFU g

-1

). The presence of A.

chevalieri

in substrate with low moisture content levels was

studied at physic nut, pepper and garden thyme

(Hashem and Alamri, 2010), nutmeg (Dharmaputra

et al. 2015). Yazdani et al. (2009) reported that

genus Eurotium sp. was obligately xerophilic that

growth at a

range 0.93-0.68. Low moisture level on

spice (in Figure 1) not reduce the fungal population

(in Figure 2). However, star annise with moisture

higher content (12.3%) than nutmeg (8.1) and

coriander (11.2%) infect by low population of A.

chevalieri. The presence of antifungal avtivity of

star anise (Illicium verum) on A, niger was

previously studied by Yazdani et al. (2009). We

assumed that the growth of A. chevalieri was

inhibited by antifungal in the spices.

4 CONCLUSIONS

Dried-stored spices sold by retailers in traditional

markerts were infected by A. chevalieri. Among of

the spices, coriander was the most infected followed

by nutmeg kernels and white pepper. Reducing the

mold growth is required to prevent deterioration of

the spices during storage.

ACKNOWLEDGEMENTS

The study was funded by Sumatera Utara

University, contract DRPM Reseach no.

67/UN5.2.3.1/PPM/KP-DRPM/2018.

REFERENCES

Andrews, S, Pitt, JI. (1987). Further studies on the

water relations of xerophilic fungi, including

some halophiles. Journal of General

Microbiology 133: 233-238.

Butinar, L. Zalar, P. Frisvad, J. Gunde-Cimerman,

N. (2005). The genus Eurotium-members of

indigenous fungal community in hypersaline

waters of salterns. FEMS Microbiol Ecol. 51:

155-166.

Codex Alimentarius, International Food Standard\,

Joint WHO/FAO. (2017). Code of practice for

the prevention and reduction of mycotoxins in

spices. CAC/RCP 78-2017.

Dharmaputra, OS. Ambarwati, S. Retnowati, I.

Nurfadila, N. (2015). Fungal infection and

aflatoxin contamination in stored nutmeg

(Myristica fragrans Houtt.) at various stages of

the delivery chain in North Sulawesi province.

Biotropia 22 2: 129-139.

Hashem, M. Alamri, S. (2010). Contamination of

common spices in Saudi Arabia markets with

potential mycotoxins-producing fungi. Saudi

Journal of Biological Sciences 17: 167-175.

Hubka, V. Kolařík, M. Kubátová, A. Peterson,

SW. (2013). Taxonomic revision of Eurotium

and transfer of species to Aspergillus.

Mycologia 105 4: 912-937.

Kneifel, E. Berger, E. (1994). Microbial criteria of

random samples of spices and herbs retailed on

the Austrian market J. Food. Prot. 57: 893-901.

Pitt, JI. Hocking, AD. (2009). Fungi and Food

Spoilage. Springer. New York (US), 3

Edition.

Samson, RA. Hoekstra, ES. Frisvad, JC. Filtenborg,

O. (1995). Introduction to Food-Borne Fungi.

Utrecht: Centraalbureau voor

Schimmelcultures.

Stankovic, N. Komic, L. Kocic, B. (2006).

Microbiological correctness of spices on sale

health food stores and supermarket in Nis. Acta

Fac Med Naiss 23 2: 79-84.

Toma, FM. Abdulla, NQF. (2013). Isolation and

identification of fungi from spices and

medicinal plants. Research Journal of

Environmental and Earth Sciences 5 3: 131-

138.

Yazdani, D. Rezazadeh, Amin, GH. Abidin, Z.

Shahnazi, S. Jamalifar, H. (2009). Antifungal

activity of dried extract of anise (Pimpinella

annisum L.) and star anise (Illicium verum

Hook.f.) against dermatopjyte and saprophyte

fungi. Journal of Medicinal Plants 8 5: 1-6.

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

120