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Amin et al. Int J Gastroenterol Hepatol Transpl Nutr 2017;2(ii): 4 – 13 ISSN 2455–9393

4

Original Article

Towards managing and controlling food safety based on contamination

with fungi and its mycotoxins Afaf A Amin1, Gulsen S Ahmed1, Hoda H Abo Ghalia2, Amera A Hamed1

ABSTRACT

A significant portion of the agricultural produce in the countries and the world over

become unfit for human consumption due to mycotoxins contamination of grains and

cereals. The main toxic effects are carcinogenicity, genotoxicity, teratogenicity,

nephrotoxicity, hepatotoxicity, reproductive disorders and imunosuppression. This

study was done to identify the molds and aflatoxins that contaminate cereal–based

baby foods and corn–based snacks products. The most frequent fungal genera found in

the samples were Aspergillus, Penicillium, Alternaria, Fusariumand Cladosporium

with frequencies of 41, 16, 10, 8 and 3%, respectively. Additionally, the numbers of

contaminated cereal–based baby foods samples with AFB1, B2, G1 and G2 were 14, 2,

6 and 4%. Also, 34, 14, 18 and 8% of corn-based snack samples respectively. Ten

essentials oils of (cinnamon, cumin, clove, fennel, garlic, lemon grass, marjoram,

peppermint, rosemary and thyme) plants using in combating aflatoxigenic mold A.

flavus growth and its aflatoxins production. The ten essential oils showed notable

inhibitory effects on A. flavus growth and its aflatoxins production. Cinnamon and

garlic essential oils caused complete inhibition to all types of aflatoxins at

concentrations of 60 and 80µl respectively. There were some alterations produced by

cinnamon, garlic and cumin essential oils at sub-lethal inhibitory concentrations such

as abnormal cell shape, leakage of cell wall and the membranous organelles were

disrupted.

INTRODUCTION

Cereals and other crops are exposed to fungal attack in the field

or during storage and this attack may result in mycotoxin

contamination of the corps. The most commonly found of

mycotoxins, namely aflatoxins, which carry potential risks for

humans (Alpsoy, 2010). Aflatoxins are a group of structurally

related toxin metabolites produced by many strains of

Aspergillus flavus, Aspergillus parasiticus and Aspergillus

nomius. Aflatoxin exposure has been linked to impaired growth

(Gong et al., 2002), kwashiorkor (Coulter et al., 1986) and may

also have a role in the modification of the etiology of hepatitis B

(Turner et al., 2000) in African children. Aflatoxin B1 (AFB1)

is one of the most potent toxic carcinogen, teratogen and

mutagen and it is listed as a group I carcinogen by the

International Agency for Research on Cancer (IARC) as it the

cause of human primary hepatocellular carcinoma (IARC,

2002). The liver is the main target organ for aflatoxin toxicity

and carcinogenicity.

The ranges of worldwide regulations for AFB1 and total

aflatoxins were from 0 -30 µg kg-1and from 0-50 µg kg-1,

respectively (FAO, 2004). In the European Union, AFB1 and

total aflatoxins levels in human commodities are regulated with

Maximum Residue Levels (MRLs) that cannot be greater than 2

and 4 µg kg-1, respectively ( European Commission (EC)

Regulations, 2010).

Mycotoxins affecting ground nuts / pea nuts, cereals (maize,

rice, sorghum, wheat, barley and oats), spices (black pepper,

ginger and nutmeg) and chili are considered to be of greater

significance world over for human beings. The exposure to

levels of aflatoxins from nanograms to micrograms per day

occurs through consumption of maize and peanuts, which are

dietary staples in several tropical countries. Therefore, this study

has been performed to evaluate the mycological quality and

aflatoxins contamination based on analysis of AFB1, AFB2,

AFG1 and AFG2 in one hundred randomly collected samples

representatives of cereal–derived products. The risk for the

consumers is also assessed. Thus, there is a need to search for

International Journal of Gastroenterology, Hepatology,

Transplant & Nutrition

1Microbiological Unit, Food Safety and

Nutrition Research Department, National Nutrition Institute, Cairo, Egypt. 2 Botany Department, Women’s College for

Arts, Science and Education, Ain Shams

University, Cairo, Egypt.

Address for Correspondence:

Dr. Afaf Amin

E-mail: [email protected]

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Key words: Aflatoxins, fungi, cereal-derived products, essential oils, risk assessment


5

alternative approaches to store grains/cereals for human

consumption without toxicity problems that are eco-friendly and

not capital intensive. Plant extracts have antifungal and

antitoxins properties under laboratory trails (El-Nagerabi et al.,

2013).

PROBLEM FORMULATION

Food Defense and Crisis Management: The consequences of

unsafe food can be serious and food safety management

standards help organizations identify and control food safety

hazards. As many of today's food products repeatedly cross

national boundaries, International Standards are needed to

ensure the safety of the global food supply chain.

Food safety management system (as HACCP, ISO 22000)

specifies requirements for a food safety, where an organization

in the food chain needs:

• To demonstrate its ability to control food safety hazards in

order to ensure that food is safe at the time of human

consumption.

• To plan, implement, operate, maintain and update a food

safety management system aimed at providing products

that, according to their intended use, are safe for the

consumer.

Developing a Food Defense Plan:

• It is necessary to control and reduce any food safety

hazards, must endorses the implementation of food safety

management systems in retail and food service

establishments dcombine with good basic sanitation, and

other prerequisite programs.

• To effectively communicate food safety issues to their

suppliers, customers and relevant interested parties in the

food chain.

• To ensure that the organization conforms to its stated food

safety policy, the safety management approach to food

safety is based on a detailed examination of every stage in

the production process for an individual food product, with

an objective to identifying where and when hazards could

occur.

• To design effective controls for each hazard:

o Perform a Hazard Analysis

o Decide on the Critical Control Points (CCPs)

o Determine the Critical Limits

o Establish Procedures to Monitor CCPs

o Establish Corrective Actions

o Establish Verification Procedures

o Establish a Record Keeping System

Crisis-management plan: In the case of situations entailing

direct or indirect risks to human health not provided for by the

Regulations, Countries must establish a general crisis-

management plan. As in the case of a serious risk, which cannot

be dealt with under the existing provisions, must immediately

set up a crisis unit, in which the Authority participates by

providing scientific and technical support. The crisis unit is

responsible for collecting and evaluating all relevant

information and identifying the options available for preventing,

eliminating or reducing the risk to human health.

Agriculture and health must move together. This means

policymakers and researchers in both sectors will have to take

off their blinders and look beyond the boundaries of their

traditional areas of action for food safety management.

MATERIALS AND METHODS

Samples collection: One hundred cereal – derived samples,

including 50 samples of cereal – based baby foods (for infants

and young children) and 50 corn – based snacks samples, were

randomly purchased from different supermarkets and small

shops from Great Cairo, Egypt. The samples were purchased

with intact package and analyzed before the expiration date.

• The cereal – based baby foods samples contain one or more

of the following major ingredients: Wheat bran, barley,

malt extract, sugar flakes of corn, cereals (wheat flour, rice

flour, and corn flour), oats, dried fruits (date, raisin, orange

and bananas), honey, chamomile, carrot, sugar, vegetable

oil, vitamins, iron, traces of milk and minerals.

• The corn – based snacks samples, corn is the main

component, in addition to different types of flavors

including cheese, chili, pea nut, tomato, salt, ketchup and

spices.

Culture media:

• Potato Dextrose Agar (PDA) (Difco™ Becton, (Jawetzet

al., 2004)

• CzapekDox Liquid medium (modified) (OXOID) (Smith,

1960) Peptone, bacteriological (HIMEDIA, India) (Straka

and Stokes 1957)

Chemicals:

• Standard aflatoxins, Carouse Chemical Company,

Nitherland.

• HPLC grade solvents and chemicals, Lab-scan Company,

Egypt.

• Reagent kits that used in aflatoxins detection, Bio –

diagnostic Company, Egypt.

Investigated aflatoxins: Four types of aflatoxins were

investigated (B1, B2, G1 and G2).

Essential oils: Ten plants; cinnamon, cumin, clove, fennel,

garlic, lemon grass, marjoram, peppermint, rosemary and thyme

were purchased from the local market as medicinal plants and

used for the preparation of essential oils by using glassware

apparatus for steam distillation. The oils were dried over

anhydrous sodium sulphate, stored in dark sterilized vial at 4°C

until used (Association Official of Analytical Chemistry

(A.O.A.C), 1995).

Mycological analysis: Ten g of each sample were ground and

homogenized with 90 mL Peptone medium for 30 min on a

horizontal shaker. Further serial dilutions to 10-3 were prepared.


6

One milliliter portions of these dilutions were transferred to

sterile Potato dextrose agar medium and incubated at 22 - 25 0C

for 5 days (Egyptian Standards 1793/2005). The number of

colony-forming units per gram (cfu/g) was determined for each

genus. All mycological analysis was done at Food Safety

Department, Microbiology Unit, National Nutrition

Institute, ministry of health, Cairo, Egypt.

Aspergillusflavus was found to be the most frequent mold

isolated from the samples, so pure culture of the isolated

A.flavus was subjected to identification at the Regional Center

for Mycology and Biotechnology (RCMB), Al-Azhar

University (Cairo/ Egypt) to use it in further investigations.

Stock culture of the identified A.flavus was maintained on slants

of potato dextrose agar (PDA) medium, stored at 4 0C and

subculture was made every two weeks.

Preparation of the spore suspension: Spores of A. flavus were

harvested by adding mixture of 10 ml of sterile distilled water

and 0.1% tween 80. The spore suspensions were aseptically

filtered off the mycelia and adjusted with sterile distilled water

to give a final spore concentration of approximately 106 spore/

ml. The numbers of spores were counted using a

haemocytometer, according to Atandaet al. (2007).

Determination of aflatoxins concentration in food samples

by HPLC: The concentrations of aflatoxins (B1, B2, G1 and

G2) in the collected samples were determined by High

Performance Liquid Chromatography (HPLC) in the Central

Laboratory of Residues analysis of Pesticides and Heavy

Metals in Food /Agriculture Research Center (Ministry of

Agriculture), Cairo, Egypt, according to the procedure of

A.O.A.C (2003).

Estimation of daily intake (EDI) of aflatoxin B1 and total

aflatoxins: Dietary intake is the tool for calculating the amount

of ingested any toxin due to the consumption of contaminated

food. Estimation of the daily intake (EDI) by using the

following equation (Codex Alimentarius Commission, 2006):

EDI = Food consumption of the product* × mean of contamination**

Average of body weight***

* Food consumption of the product: Baby = 30g, adolescent and

adult = 50 g

** Mean of contamination in samples by µg/kg

*** Average body weight: Baby = 10 kg, adolescent = 40 kg

and adult = 60 kg

RESULTS AND DISCUSSION

Frequencies of different genera of fungi present in samples

From Figure 1, the samples of cereal- based baby foods and

corn-based snacks are liable to infection with different fungi.

Five genera of filamentous fungi were isolated from samples,

Aspergillus, Penicillium, Fusarium, Alternariaand

Cladosporium. These results were in agreement with Ismail et

al., 2012, who found that Aspergillus, Penicillium, Fusarium

and Cladosporium were the most common genera in baby food

products that mainly made of cereal flour(s).

Figure 1: Frequency (℅) of different genera of fungi present in samples of cereal – based baby foods and corn – based snacks.

Aspergillus (36%) and Penicillium (12%) were the most

prevalent genera. Aspergillusflavus was found in high

percentage (16%) from the total Aspergillus spp. The

manufacture of baby foods is based on dried cereals like corn,

rice, millet, sorghum, wheat, oats and barley with various

additional ingredients such as dried fruits or vegetables which

are very suitable for growing these types of fungi. Fungi

contaminating grains have been conventionally divided into two

groups – field fungi and storage fungi. Field fungi are those that

infect the crops throughout the vegetation phase of plants and

they include plant pathogens such as Alternaria, Fusarium,

Cladosporium, and Botrytis species. Their numbers gradually

decrease during storage, and replaced by storage fungi of

Aspergillus, Penicillium, Rhizopus and Mucor genera that infect

36

46

16

22

12

20

4

16

4

12

24

Cereal-b

ased b

aby foods

Corn -

based sn

acks

Genera of fungi

0

10

20

30

40

50

60

Perc

ent o

f fun

gi

Total Aspergillus spp

Aspergillus flavus

Penicillium spp.

Alternaria spp.

Fusarium spp.

Cladosporium spp.


7

grains after harvesting and during storage (Piotrowska et al.,

2013). Among all the isolated fungal species, 93.2% belonged to

the group of toxigenic fungi. Several toxin-producing

Aspergillus species were reported to dominate on cereals,

especially A.flavus. Also showed that Aspergillus was

recovered in high Frequency, contaminating (46%) of corn -

based snacks samples.

Corn is the main source of contamination in corn snacks plus

some flavors or additives especially chili, spices and peanut.

Rajasinghe et al., 2009, found that the aflatoxigenic A.flavusto

be dominant in spices during storage. The most important

factors which influence the fungal growth and aflatoxins

production are water availability and temperature. In Egypt,

grains stored under high moisture / humidity (>14%) at warm

temperature (>20° c) and/or inadequately dried can potentially

become contaminated. Also initial growth of fungi in grains can

form sufficient moisture from metabolism to allow further

growth and mycotoxin formation, thus we must apply the food

safety management system fom farm to fork.

Monitoring of aflatoxins in cereal - based baby foods and

corn - based snacks samples: Little information on the

occurrence of aflatoxins on baby food exists. The products

screened in this study are generally commercialized in Egypt in

small shops and supermarkets and consumed especially by

children. Thus, more importance to their safety is needed. The

data in Figure 2 concluded that 7 of 50 (14%) cereal – based

baby foods samples were contaminated with aflatoxins B1 and

its concentrations ranged from 0.54 to 2.22 µg/ kg, with a mean

value of 1.38 µg / kg., the numbers of contaminated samples

with B2, G1 and G2 were 1 (2%), 3 (6%) and 2(4%) with mean

value of (0.5), (1.66) and (0.9) µg / kg, respectively.

Figure 2: Number of contaminated cereal- based baby foods

and corn-based snacks samples with aflatoxins B1, B2, G1

and G2.

The high humidity and temperature in tropical and subtropical

areas provide optimal conditions for toxin formation. Improper

storage and less than optimal conditions during transportation,

marketing and processing can also contribute to fungal growth

and increase the risk of mycotoxin production (Sherifet al.,

2009). Also 17 of 50 (34%) corn-based snacks samples,

contained AFB1 in the range of 0.59 - 15.83 μg /kg with a mean

value of 4.43 µg / kg and AFB2 was detected in seven samples

(14%), its concentration ranged from 0.51 to 2.01 μg /kg with a

mean value of 1.9 µg/kg. Sabbagh and Abedi-Tizaki,(2011)

have shown that collected corn from south of Caspian Sea were

contaminated by aflatoxin.

The most contaminated samples of corn – based snacks were

those with chili, peanut and spices flavor than other flavor.

Rajasinghe et al., 2009, found that the aflatoxigenic A.flavus to

be dominant in spices during storage.

Maximum Residue Level (MRL) for afaltoxin B1are seated in

the regulations all over the world but no limits were seated for

B2, G1 and G2. Infant's food must be free from any traces of

aflatoxin B1 and total aflatoxins due to their dangerous effect on

their growing. The European Maximum Level allowed for

aflatoxin B1 in Processed cereal- based foods for infants and

young children is 0.10 µg /kg (European Commission

Regulation, 2010), so that in this case all cereal – based food

samples which contaminated with aflatoxin B1 (7 samples) with

arrange from 0.54 to 2.22 µg / kg are considered to be violated

Because it over passed the European Maximum Level as

showed in Table 1.

Thus according to the present study twelve samples of corn –

based snacks surpassed the established limit (2 µg/kg) for

AFB1with a range from 2.26 to 15.83 µg/kg and with violation

percentage 71 % and ten samples over passed the limit

established for total aflatoxins (4 µg/kg) with a range from 4.18

to 28.37 µg/kg and with violation percentage 59 %. The

carcinogenicity, mutagenicity and acute toxicology of AFB1

have been well documented, the IARC determined it to be a

human carcinogen (group 1A) (Piotrowska et al., 2013). So a

calculation of Estimated Daily Intake (EDI) is very important to

lead up to assume the risk assessment of aflatoxin in consuming

of the studied commodities.

Estimation of the Daily Intake of AFB1 and total aflatoxins

Exposure assessment is to identify the exposures that occur, or

anticipated to occur, in human populations, risk of children from

aflatoxin depends on the degree of exposure to and on the

degree of hazard from individual toxins. A study (Sherif et al.,

2009) investigated aflatoxin exposure in Egyptian children

(n=50, aged 1-2.5 years) by assessing urinary aflatoxin

metabolite. The Provisional Maximum Tolerable Daily Intake

(PMTDI) which equal 1 ng AFB1 /kg body weight for adult and

children without hepatitis B.

Data in Tables 2 and 3 represent the Estimation Daily Intake

(EDI) of aflatoxin B1 and total aflatoxins for consumers in

Egypt due to consuming corn based snakes and cereal based

baby foods, which proved from this study that they were

contaminated. Also showed that consumption of 30 g from the

most contaminated sample (15.83 μg /kg) of corn – based

snacks, by a child (20 kg) leads to an AFB1 daily intake about

23.75 ng AFB1 /kg bw/ day, that is more than 23 – fold higher

than the PMTDI (1 ng /kg bw/day), which subject children to

health risk, in addition, a consumption of 50 g by an adolescent

0

2

4

6

8

10

12

14

16

18

Cereal-Based Baby Foods

Corn-Base

d Snacks

Type of Aflatoxins

No

. of

Co

nat

min

ated

Sam

ple

s

B1

B2

G1

G2


8

(50kg) and an adult (70kg) displayed a daily intake from about 11 to 15 – fold higher than the PMTDI.

Table 1: Number of contaminated samples, mean, minimum, maximum, number of violated samples and percentages of

violation of aflatoxins in samples of cereal- based baby foods and corn-based snacks.

Samples toxin

No. of

contaminate

d samples

Mean

(µg/kg)

Minimum

(µg/kg)

Maximum

(µg/kg)

MRL

(µg/kg)

No. of

violated

samples

Violation

%

Cereals-

based baby

foods

AFB1

AFB2

AFG1

AFG2

Total

7

1

3

2

7

1.38

0.5

1.66

0.9

2.42

0.54

0.5

0.8

0.6

0.54

2.22

0.5

2.98

1.2

6.9

0.1*

-

-

-

0.1

7

-

-

-

7

100

-

-

-

100

Corn- based

snacks

AFB1

AFB2

AFG1

AFG2

Total

17

7

9

4

17

4.43

1.9

3.74

1.6

7.22

0.59

0.51

1.1

0.92

0.59

15.83

2.01

8.14

2.42

28.37

2**

-

-

-

4

12

-

-

-

10

71

-

-

-

59 * EU MRL allowed for aflatoxin B1 in Processed cereal- based foods for infants and young children is 0.10 µg / kg.

**EU MRL in human commodities for aflatoxin B1 is 2μg/kg and total aflatoxinis 4μg/kg.

Table 2: Estimated Daily Intake (EDI) of aflatoxin B1 and total aflatoxins by children, adult and adolescent consuming corn -

based snacks:

Toxin Concentration of toxin µg/kg Consumption

(g/person/day)*

Mean Body weight

(bw)**

EDI (ng/kg

b.w./day)***

AFB1

More contaminated sample

15.83 ug/kg

Children 30 g

Adolescent 50 g

Adult 50 g

Children 20 kg

Adolescent 50 Kg

Adult 70 Kg

23.75

15.38

11.30

Less contaminated sample

0.59 ug/kg

Children 30 g

Adolescent 50 g

Adult 50 g

Children 20 kg

Adolescent 50 Kg

Adult 70 Kg

0.88

0.59

0.42

Total

aflatoxin

More contaminated sample

28.37 ug/kg

Children 30 g

Adolescent 50 g

Adult 50 g

Children 20 kg

Adolescent 50 Kg

Adult 70 Kg

42.5

28.37

20.2


0.59 ug/kg

Children 30 g

Adolescent 50 g

Adult 50 g

Children 20 kg

Adolescent 50 Kg

Adult 70 Kg

0.88

0.59

0.42

*Estimated as the less daily consumption (Content packaging of the product)

**WHO (1983) ***EDI (ng /kg bw./day ) Calculated for each toxin

Table (3): Estimated daily intake (EDI) of aflatoxin B1 and total aflatoxins by babies consuming cereals - based food.

Toxin Concentration of toxin

µg/kg

Consumption

(g/baby /day) *

Mean Body weight

(bw)**

EDI (ng/kg

b.w./day)***

Aflatoxin

B1

High contaminated sample

2.22 ug/kg 30 g

Baby (6 months) 6 kg

Baby (12month) 8 kg

Baby (24 month) 10 kg

11.1

8.32

6.66

less contaminated sample

0.54 ug/kg 30 g


Baby (12month) 8 kg


2.7

2.02

1.62

Total

aflatoxin

High contaminated sample

6.9 ug/kg 30 g


Baby (12month) 8 kg


34.5

25.8

20.7


0.54 ug/kg 30 g


Baby (12month) 8 kg


2.7

2.02

1.62

*Estimated as the less daily consumption (Content packaging of the product).

**WHO (1983) ***EDI (ng /kg bw./day ) Calculated for each toxin


9

Similarly, there is a significant risk for babies (6, 12 and 24

months) who occasionally consumes a cereal – based food (30 g

per day) where, an AFB1daily intake is from 6.66 to 11.1 ng /kg

bw/day, that is more than 6 to 11 fold higher than the PMTDI,

while the consumption of less contaminated sample, the daily

intake is about 2 fold higher than PMTDI.

Australia Market Basket Survey (1992) reported that the

mean dietary intakes of total aflatoxin are 0.15 ng /kg bw per

day for Australians, EU Scientific Cooperation on Questions

Relating to Food Projects (SCOOP), 1996) showed that the

dietary exposure of Europeans to AFB1 ranged from 0.03 to

1.28 ng/kg bw per day.

Results of this study explain the reason behind outbreak of

children diseases especially cancer and liver diseases.

Antimicrobial activity of essential oils on Aspergillusflavus

growth:

Antifungal activity assay: Ten essential oils were tested for

their antifungal effect on the growth of A. flavus isolate on

potato dextrose agar media, using agar well diffusion method at

concentrations 10 and 20 μl for each essential oil. The inhibition

of A.flavus growth was expressed as inhibitory zones (Mean)

and measured in (mm) diameter as illustrated in Figure 3.

Figure 3: The antifungal activity of different essential oils on

Aspergillusflavus growth expressed as inhibition zone

diameter (mm).

As showed in Figure 3, the three essential oils cinnamon, garlic

and cumin were particularly effective against A. flavus growth

exhibiting high diameters of growth inhibition (44.7, 42.3 and

32.7 mm) respectively, at a dose of 10 µl. Mohammadpour et

al. (2012) indicates that Cuminumcyminum L.(cumin) has good

antifungal activity against A.flavus, A.parasiticus and A. niger.

Othman and AL-Delamiy (2012) found that garlic (Allium

sativum) extract is more effective as anti- Aspergillus spp. agent

than black cumin extract.

Determination of Minimum Inhibitory Concentration (MIC)

and Minimum Fungicidal Concentration (MFC) of the more

efficient 3 essential oils against A.flavus: Table 4 provides

(MIC) and (MFC) values and MIC/ MFC ratio, results revealed

that the oils tested exhibited different degrees of antifungal

activity against toxigenic A. flavus. The maximum antimycotic

activity was shown by cinnamon followed by garlic and cumin

essential oil. Cinnamon essential oil exhibited the strongest

antifungal activity, with MIC and MFC values of 0.05 µl/ ml for

each, when cinnamon essential oil was added to A. flavus

culture medium, development was affected from the

concentration of 0.005 μL /mL. Also, Wahegaonkar and

Shirurkar (2013) reported that garlic is the most effective in

inhibiting spore germination of different fungi. While, cumin

essential oil exhibited relatively moderate activity with an MIC

value of 0.38 µl/ ml and 0.66 µl/ ml as MFC value.

Mohmmadpour et al. (2012) reported that C. cyminum L.

essential oil had substantial antifungal activity against A.flavus

PICC-AF39, Aspergillusflavus PICC-AF24. C. cyminumL. oil

killed more than 60% of the spores of the tested Aspergillus

species within 12 hours.

Table 4: MIC and MFC of the more efficient 3 essential oils

against A. flavus growth.

MFC/MIC ratio MFC

(µl/ml)

MIC

(µl/ml)

Essential

oils

1 (microbiocidal) 0.05 0.05 Cinnamon

Greater than 1

(microbiostatic) 0.22 0.11 Garlic

Greater than 1

(microbiostatic) 0.66 0.38 Cumin

Karouet al. (2005) stated that, if the MFC/MIC ratio is greater

than 1, it is considered as microbiostatic, while ratio equal or

smaller than 1 is considered as microbicidal. From the obtained

results we may conclude that cinnamon essential oil has

fungicidal nature on A.flavus, garlic and cumin essential oil have

a fungi static nature on A.flavus.

Effect of different concentrations of essential oils on

aflatoxins Production by Aspergillusflavus in Czapex liquid

media: The biosynthesis of aflatoxins can be inhibited by

essential oils (EOs) extracted from herbal, medicinal and

aromatic plants which are toxic to the fungal growth and

aflatoxin production of A. flavus and A. parasiticus and this has

been suggested by many researchers.

Figure 4: The percentage of inhibitory effect of cinnamon

essential oil on aflatoxins B1, B2, G1 and G2 production by A.

flavus in Czapek medium.

44.7

42.3

32.7

32

24.7

23.3

15.7

15.3

14.3

8.7

90 90

47.2

41.5

29.3

28.5

17.7

17.5

16

10.4

Cin

namon

Garli

c

Cum

in

Thyme

Clo

ve

Marjo

ram

Lemon g

rass

Peppermin

t

Fennel

Rose

mary

Essential oils Concentrations(10and 20µL/ w ell)

0

20

40

60

80

100

Inhi

bitio

n zo

ne d

iam

eter

(m

m)

No growth No growth

60

65

70

75

80

85

90

95

100

105

110

115

120

10 µl 20 µl 40 µl 60 µl 80 µl

Cinnamon essential oil concentrations (µl/50 ml

medium)

Inh

ibit

ion

Pe

rce

nta

ge

% o

f A

fla

tox

ins

Co

nc

en

tra

tio

ns

B1

B2

G1

G2


10

Cinnamon essential oil: Figure 4 demonstrate the effect of

different concentrations (10, 20, 40, 60, 80 µl / 50 ml media) of

cinnamon essential oil on aflatoxins production. The

concentration ofaflatoxinsB1, B2, G1 and G2 in control sample

were 541, 219, 292.3, and 174.7 μg/50 ml media, respectively.

The most effect observed with concentration (20µl /50 ml

media) aflatoxins concentrations B1, B2, G1 and G2 were 1.77,

2.43, 4.73 and 1.1 μg/50 ml media, respectively, which meaning

it reduced by (99.7℅), (98.9℅), (98.9℅) and (99.4℅),

respectively).Moreover the highest inhibition percentage of

aflatoxins production (100℅) was at concentration (40µl /50 ml

media) of cinnamon essential oil. Montes-Belmont and

Carvajal (1998) used the essential oils of

Cinnamonmzeylanicum for the protection of stored maize grains

against A. flavus, it has significant antifungal activity against A.

flavus in culture media and a complete inhibition of A. flavus

growth was observed at 200 ppm of the essential oil.

Also, Figure 5 illustrated that treated concentrations (40, 60, 80

µl / 50 ml media) of cinnamon essential oil gave clear reduction

in the mycelium dry weight, it dramatically depressed to 0.09,

0.01, 0.0 g, with inhibition percentages of (96.1℅), (99.6℅) and

(100℅), respectively.

Cinnamon essential oil concentrations (µl/50 ml medium)

Figure 5: The percentage of inhibitory effect of cinnamon

essential oil (EO) on dry weight of mycelia of

Aspergillusflavus in Czapek medium.

Figure 6: The percentage of inhibitory effect of garlic

essential oil on aflatoxinsB1, B2, G1 and G2production by A.

flavus in Czapek medium.

Garlic essential oil: The greatest inhibition (Figure 6) for the

four types of aflatoxins was observed at (20µl EO /50 ml

media), at concentrations (40, 60, 80 µl EO / 50 ml media) there

were a complete inhibition and no aflatoxins detected, the

inhibition percentage was 100℅ for all types of aflatoxins.

Allicin, thiosulfonate and other compounds of garlic showed

fungistatic activity against A.spp. such as A. flavus, A.

fumigatus, A. terreus and P. chrysogenum (Kumar and Jain,

2010).

Also, Figure 7 illustrated that the dry weight in treated media

with different concentrations were reduced .This mean there was

a great effect of garlic essential oil on the mycelium growth of

A.flavus and this result was in agreement with Wahegaonkar

and Shirurkar (2013) who reported that garlic is most effective

in inhibiting spore germination of different fungi.

Garlic essential oil concentrations (µl/50 ml medium)

Figure 7: The percentage of inhibitory effect of garlic

essential oil on dry weight of mycelia of Aspergillusflavus in

Czapek medium.

Cumin essential oil: The concentration (10µl EO /50 ml media)

gave clear reduction in the aflatoxins concentration B1, B2, G1

and G2 (Figure 8).

The complete inhibition of aflatoxin production (100℅) was at

(80 µl EO /50 ml media). Singh and Upadhayay (1991)

showed that Cuminaldehyde, an active principle from the oil of

Cuminum cerium Linn, has antifungitoxic activity against A.

flavus.

Figure 8: The percentage of inhibitory effect of cumin

essential oil on aflatoxinsB1, B2, G1 and G2 production by

A. flavus in Czapek medium.

0

20

40

60

80

100

10 µl 20µl 40 µl 60µl 80 µl

Inhib

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6065707580859095

100105110115120

10 µl 20 µl 40 µl 60 µl 80 µl

Garlic Essential Oil Concentrations

(µl / 50 ml medium)

Inh

ibit

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Pe

rce

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% o

f

Afl

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B1

B2

G1

G2

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20

40

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10 µl 20µl 40 µl 60µl 80 µl

Inhib

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of

myce

lium

dry

we

ight

6065707580859095

100105110115120

10 µl 20 µl 40 µl 60 µl 80 µl

Cummin Essential Oil Concentrations

(µl / 50 ml medium)

Inh

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Pe

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% o

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Afl

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B1

B2

G1

G2


11

The lowest mycelium dry weight was obtained at concentration

80 µl EO /50 ml media, as showed in Figure 9. This means that

the increasing concentrations of cumin essential oil caused a

significant reduction in the aflatoxin concentration and in the

mycelium growth of Aspergillusflavus. Karbinet al. (2009)

found that Cuminumcyminum essential oils possess a remarkable

inhibiting the growth of Aspergillusflavus.

Cumin essential oil concentrations (μl/50 ml medium)

Figure 9: The percentage of inhibitory effect of cumin

essential oil on dry weight of mycelia of Aspergillusflavus in

Czapek medium.

Examination under the Transmission Electron Microscope

(TEM)

Stained sections were examined with a Transmission Electron

Microscope (JEOL 1010, Japan) at the Regional Center for

Mycology and Biotechnology (RCMB), Al-Azhar University.

Figure 10: TEM Micrographs of mycelial T.S. of A.flavus

under normal conditions (untreated) and under essential oils

treatment.

(A) Untreated mycelia (control) (X= 25000),

(B) Cinnamon oil-treated mycelia (X= 25000),

(C) Garlic oil-treated mycelia (X= 25000),

(D) Cumin oil-treated (X= 25000), mycelia

Figure 10 & 11 shows that the cell membrane is a very

important target of the essential oil components, the surfaces of

the hyphae and conidia treated by oils became rough in contrast

to the control group. The cell wall (CW) exhibited degenerative

changes; it was disrupted and became rough and villi form.

Subsequently, the cell wall became very thin and even seemed

to disappear in some hyphae. The membrane-disruptive activity

of essential oil components may be closely associated with the

interference with enzymatic reactions of the membrane, such as

respiratory electron transport, proton transport, and coupled

phosphorylation steps (Khosravi et al., 2011). The oils passed

not only through the cell wall but also through the plasma

membrane and then interacted with membranous structures of

the cytoplasmic organelles. The marked action of oil

components might have conferred lipophilic properties and the

ability to penetrate the plasma membrane (Knobloch et al.,

1989). It is believed that lipophilic properties of oils may assist

in penetration of cell membrane and, accumulation of

polysaccharides in stress condition may lead to rupture of

plasmalema in fungal cells.

Figure 11: TEM Micrographs of A.flavus spores under

normal conditions (untreated) and under different essential

oils treatment.

(A) Untreated spore (control) (X= 25000),

(B) Cinnamon oil-treated spore (X= 25000),

(C) Garlic oil-treated spore (X= 25000) and

(D) Cumin oil-treated spore (X= 25000).

A.flavus spores treated with the essential oils were

illustrated, the normal morphology of the fungal conidia was

disturbed, moreover, the conidial cell wall was found to possess

a sheath (sh) with spinuloseechinulations, while the cytoplasmic

membrane was shrunk, and their materials concentrated at the

center of conidia. Additionally there was partial disappearance

of cell wall and the cytoplasm had small pigmented deposits and

vacuoles (V). Fernanda et al. (2012) reported that strong

antimicrobial activity could be correlated with essential oils

containing high percentage of monoterpenes, eugenol, cinnamic

aldehyde, and thymol. Finally there was disorganization of

cytoplasmic contents accompanied by intensive degradation and

lysis of the nucleus and mitochondria.

0

20

40

60

80

100

10 µl 20µl 40 µl 60µl 80 µlInhib

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12

CONCLUSION AND RECOMMENDATION

The current results revealed that cereal – based baby foods and

corn – based snacks were contaminated by fungi. Many of these

fungi are capable of producing mycotoxins. Contamination of

such foods (especially those for babies) is a matter of health

hazard for human consumption. However their safety can be

insured and improved greatly by using quality raw materials. It

was noticed that the contamination occurs for cereal grains

before, during or after harvesting, during drying process, or

even during food production and this contamination could be

also due to long-term storage, marketing under non-hygienic

conditions of the food products. So, we suggest that monitoring

fungal contaminations as well as mycotoxins should be carried

out periodically and the procedures to prevent mould

contamination should be developed. Due to health and economic

consideration, natural plant essential oils may provide an

alternative method to protect food from fungal contamination.

So, this study aims at evaluate the use of essential oils from

cinnamon, garlic and cumin as antifungal agents to be suitable

for applications on the food industry. They can be used as

growth inhibitors of Aspergillusflavus and its aflatoxins

production. The main reason for their suitability is their natural

origin, which consumers find comforting and low risk.

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