1.

Introduction

Agriculture crops suffer considerable losses by the invasion of pests, viz., insects, molds,

rodents, bacteria, etc., during their storage. Nearly 30–35% of agricultural commodities are lost

from farm to fork (Kumar et al., 2007). Such enormous losses may become a serious problem

in the near future in view of the continuous growing population of the world. Molds are the

opportunistic agents of ubiquitous nature having potent capability to spoil the food

commodities, at any stage of production and storage, rendering them unfit for human

consumption. Loss of food commodities due to storage pests is a major reason of food crisis

particularly in tropical countries. According to FAO, food borne molds and their toxic

metabolites render quantitative and qualitative losses ofnearly 25% of agricultural food items

throughout the world (Pittet, 1998; Singh et al., 2010). Mold infestation results in reduction of

grain quality, change in colour and texture, increase in free fatty acids, reduction in nutritional

value (Shukla et al., 2009). Mycotoxins produced by some mold strains have many times

resulted to famine even in developed countries (Wagacha & Muthomi, 2008).

Mycotoxins are a group of highly toxic secondary metabolites of fungi capable of causing

disease and death in human and other animal. Thus Mycotoxins are insidious poisons. Cereals

and grains are major mycotoxin vectors because they are consumed both by humans and

animals (Singh et al., 2010). Mycotoxins occur in small amount in the food; however, their

continuous intake even in trace amounts can result in accumulation in the organisms. The most

dangerous of these toxins include the aflatoxins produced by Aspergillus flavus and Aspergillus

parasiticus. Aflatoxins have currently assumed significance due to their deleterious effect on

human being. A variety of tissue and organ such as the liver, kidney, nervous system and

1
 gastrointestinal system have been reported to be affected by aflatoxin. In plants, aflatoxins

inhibit seed germination, seedling growth, root elongation and chlorophyll and carotenoid

synthesis and retard protein and nucleic acid and synthesis of some enzymes (Shukla et al.,

2009). About 4.5 billion people living in developing countries are chronically exposed to

uncontrolled amounts of toxins. Aflatoxicosis is recently recognized as the 6th amongst 10 most

important health risks by the WHO for developing countries (Prakash et al., 2010). Aflatoxin

B1 and B2 and ochratoxins produced by Aspergillus flavus and Aspergillus ochraceus are some

common example of mycotoxins. Aflatoxin B1 has been classified as potent human carcinogen

by the international Agency for Research on Cancer. Several disease outbreaks of aflatoxicosis

in humans and animals have been reported because of the consumption of aflatoxin

contaminated food and feed (Dubey et al., 2010). In addition to fungal and mycotoxin

contamination, lipid peroxidation due to the chain reaction of free radical oxidation in food

items is another major problemfor food industries because the oxidized lipid imposes an

undesirable influence on humans (Deba, Xuan, Yasuda, & Tawata, 2008). Aflatoxins have

been also reported to enhance the generation of free radicals (ROS) (Choy, 1993; Prakash, et

al.,, 2012). Hence, prevention of fungal growth, aflatoxin secretion as well as lipid

peroxidation by using a single measure will be a novel and economical strategy to combat food

losses during storage and transit.

Several synthetic additives and preservatives are effectively used in management of post

harvest losses but their continuous application may cause the development of fungal resistance as

well as residual toxicity (Holly and Patel, 1995). Synthetic preservatives are also responsible for

the origin of partially reduced form of oxygen such as superoxide (O2−) hydrogen peroxide

(H2O2) and hydroxyl radicals (OH−) which are highly reactive molecules causing oxidative

2
diseases by damaging the proteins, lipids and DNA and also responsible for the stimulation of

aflatoxin biosynthesis (Jayashree and Subramanyam 2000). Attempts to control post harvest

losses, practices have been carried out by different physical and chemical treatments. Physical

treatment include heat treatment viz- heat therapy, low temperature storage and radiations.

Farmers often thermally disinfect grains by directly exposing the commodities in sunlight. This

is an effective and repeatable technique that is suitable for areas of the world where the

climate is hot and dry. Low temperature storage which is widely used has too limitations. Firstly

some parasitic troubles such as red blotches, pitting, membrane stain etc. develop on the

commodities. Secondly, some fruit rotting fungi develop well even at low temperature. More

over, refrigeration is impracticable in several countries due to its high cast. During the past few

decades, application of synthetic pesticides to control agricultural pests has been a standard

practice. The wide spread indiscriminate use of chemical preservatives has resulted in the

emergence of resistance microorganisms leading to food borne disease. The use of synthetic

chemicals as pesticides and fumigants have made a great contribution in the management of such

losses but has also raised a number of ecological and medical problems due to residual toxicity,

carcinogenicity, teratogenicity, hormonal imbalance, spermatotoxic, impotency, infertility,

abortions etc (Kumar et al., 2007). To reduce these problems, there is a need to adopt strategies

that are accessible, simple in application, non toxic to humans and plants and that have

sustainable broad spectrum fungitoxicity. As a consequence, microbiologists are paying serious

attention to investigate alternative means of food preservatives to minimize the fungal

contaminations and to improve the quality of foods. Among various alternatives, botanical

antimicrobials being biodegradable and ecofriendly are catching attention of scientist worldwide

(Dubey et al., 2010). Botanical pesticides have a very high level of safety for humans, animals,

fish and other non target organisms principally because they have been reported to act by

3
different modes of action than most organic chemical pesticides, which attack metabolic system

shared by both pest and non pest organisms. Research and development cost of biological from

discovery to marketing is reported to be less compared to chemical pesticides. Plants are an

important source of useful chemicals, facts has been recognized from ancient times. Our

ancestors were quite successful in exploring and exploiting this natural treasure. Therefore, the

use of safe, low toxicity botanical pesticides is now emerging as one of the prime means to

protect crops, their products and the environment from pesticide pollution, a global problem. In

recent years, considerable attention has been directed to the research and application of

alternative sources of pesticides (botanical pesticides/phyto pesticides) in place of synthetic

pesticides. Many plant extracts and essential oils may be an alternative source of stored-product

insect control agents (Shukla et al., 2011) because they constitute a rich source of bioactive

chemicals. There has been a renewed interest in botanical pesticides because of several distinct

advantages. Botanicals, being natural derivatives are biodegradable, so they do not leave toxic

residues or by products to contaminate the environment (Burt, 2004; Bakakli et al., 2008). For

many years plants secondary metabolites have been neglected in science. Gradually, recognition

of the important role of these compounds has increased particularly in terms of resistance to pest

and diseases more than 30,000 of these secondary metabolites have been reported from plants so

for, but since only approximately 5- 10% of all higher plants have been analyzed

phytochemically in detail, the overall total number of secondary compounds will probably

exceed 1,00,000 (Prakash 2013). Aromatic herbs are widely claimed to have broad- spectrum

antimicrobial activity. Among natural products, essential oils (EOs) of higher plants and their

components are gaining interest as food additives and widely accepted by consumers because of

their relatively high volatility, ephemeral nature and biodegradability. The term ‘essential oil’ is

thought to derive from the name coined in 16th century by the Swiss reformer of medicine,

4
Paracelsus von Hohenheim; he named the effective component of a drug Quinta essential

(Guenther,1978). The essential oils are one of the major secondary metabolite having broad

antimicrobial, antifungal and antimycotoxin action. The therapeutic use of essential oils and their

combinations comprising more than one fungitoxic ingredients may also provide a solution for

the rapid development of fungal resistance which is currently noticed in case of different

prevalent antifungal therapeutics (Srivastava et al.,2008). Essential oils (also called volatile or

ethereal oils; Guenther,1978) are aromatic oily liquids obtained from plant material (flowers,

buds, seeds, leaves, twigs, bark, herbs, wood, fruits and roots). They can be obtained by

expression, fermentation, enfleurage or extraction but the method of steam distillation is most

commonly used for commercial production of essential oils. Carvacrol, innamaldehyde, citral,

thymol and limonene are some major bioactive compounds of some essential oils which are

recommended as food additives by European commission with no harm to human health (Burt,

2004).

α-terpineol is a naturally occurring monoterpene alcohol that has been isolated from a

variety of sources such as cajuput oil, pine oil. There are three isomers, alpha-, beta-, and

gamma-terpineol. Terpineol is usually a mixture of these isomers with alpha-terpineol as the

major constituent. α-terpineol has a pleasant odor similar to lilac and is a common ingredient in

perfumes, cosmetics, and flavors.

Therefore, the present investigation has been undertaken to find out the efficacy of α-

terpineol against the growth and toxin production by test fungus Aspergillus flavus. In addition to

this free radical scavenging activity of α-terpineol was also determined so as to recommend it in

control of post harvest deterioration of some food commodities and enhancing their shelf life.

5
2. Material and Methods

2.1 Chemicals and equipments

All the chemicals viz. chloroform, methanol, sodium sulphate, tween-80, toluene,

isoamyl alcohol, Potato dextrose broth (PDB), SMKY (Sucrose 200 g; MgSO4·7H2O, 0.5 g;

KNO3, 0.3 g and yeast extract, 7 g; 1 L distilled water), 2,2-diphenyl-1-picrylhydrazil (DPPH),

α-terpineol, were procured from Hi-Media Laboratories Pvt. Ltd., Mumbai, India. The major

equipments used were hydro-distillation apparatus, (Merck Specialities Pvt. Ltd., Mumbai,

India), centrifuge, UV transilluminator (Zenith Engineers, Agra, India) and spectrophotometer

(Systronics India Ltd., Mumbai, India).

2.2 Determination of minimal inhibitory concentration (MIC) of α-terpineol against A.

flavus

The minimal inhibitory concentration (MIC) for A. flavus was determined by broth

dilution method as reported earlier (Shukla et al., 2009). Different concentrations of the α-

terpineol was dissolved in 0.5 ml acetone and incorporated to 9.5 ml PDB broth to achieved

concentration (0.25 to 5.0 µl/ml). Thereafter 50 µl spore suspension of A. flavus containing 106

spores/ml was inoculated in treatment as well as control sets. The tubes were incubated at 30°C

for a week. The lowest concentration that did not permit any visible growth of fungus was taken

as MIC.

2.3 Antiaflatoxigenic activity of α-terpineol

The antiaflatoxigenic efficacy of α-terpineol with toxigenic isolate Aspergillus flavus

using SMKY broth (sucrose, 200 gm; magnesium sulphate, 0.5gm; potassium nitrate, 0.3 gm;

yeast extract, 7gm; distilled water, 1000 ml; pH, 5.6±0.2) as nutrient medium (Shukla et

6
al.,2009). Requisite amounts of α-terpineol were taken separately in 100 ml Erlenmeyer flask,

dissolved in 0.5 ml acetone an added to 24.5 ml SMKY medium in flasks to achieve the various

concentration between (0.25 to 5.0 µl/ml). The control sets were prepared without PEA. The

flasks were inoculated with 50 µl spore suspension of the toxigenic isolate of Aspergillus flavus

(106 spores/ml) and incubated at 27 ± 2°C for 10 days. The percent mycelial inhibition of treated

and control cultures was calculated by the mean value. The content of each flask was filtered

(Whatman No. 1) and mycelium was oven dried at 100°C till their weight remained constant for

biomass determination. The filtrate of control and treated sets was extracted with 20 ml

chloroform in a separating funnel to determine the aflatoxin B1 production. The chloroform

extract was subsequently evaporated till dryness on water bath at 70°C. The residue left after

evaporation was re-dissolved in 1 ml chloroform and 50 µl of chloroform extract was spotted on

TLC plate (20 × 20 cm of silica gel). The plate was then developed in toluene: isoamyl alcohol:

methanol (90: 32 : 2 v/v) solvent system proposed by Kumar et al (2007). The developed plates

were air dried and observed under UV light (360 nm) according to association of Official

Analytical Chemists Methods. An initial, visual identification of aflatoxin B1 was made by

comparing the colour and intensity of fluoroescence of the sample and standard spots. The spots

of aflatoxin B1 on the TLC plate were subsequently removed by scraping the TLC plate,

dissolved in 5 ml cold methanol, centrifuged at 3000 rpm for five minutes to separate the

aflatoxin from the silica. The absorbance of the supernatant was measured at 360 nm using a

spectrophotometer and the amount of aflatoxin B1 was calculated by the equation –

DM
aflatoxin B1 content (µg/l) =  1000
E l

Where, D = absorbance,

7
 M = molecular weight of aflatoxin (312)

E = molar extinction coefficient of aflatoxin B1 (21,800) and

l = path length (1 cm cell was used).

2.4 Antioxidant activity

Free radical scavenging activity of the α-terpineol was measured by recording the extent

of bleaching of the purple-colored DPPH solution to yellow. Different concentrations (25 to 250

μl/ml) of the samples were added to 0.004% DPPH solution in methanol (5 ml).After a 30 min of

incubation at room temperature, the absorbance was taken against a blank at 517 nm using

spectrophotometer. Scavenging of DPPH free radical with reduction in absorbance of the sample

wastaken as a measure of their antioxidant activity following Sharififaret al., (2007). IC50,

which represented the concentration of the α-terpineol that caused 50% neutralization of DPPH

radicals, was calculated from the graph plotting between percentage inhibition and concentration.

I% = ( Ablank–Asample /Ablank )×100

where, Ablank is the absorbance of the control (without α-terpineol), and Asample is the

absorbance of the α-terpineol.

2.5 Statistical analysis

Antifungal, antiaflatoxigenic, and antioxidant experiments were performed in triplicate

and data analyzed are mean ± SE.

8
3. Results

The Minimum inhibitory concentration (MIC) of α-terpineol against Aspergillus flavus was

found to be 1.75 μl/ml.

Figure 1. Minimum inhibitory concentration (MIC) of α-terpineol against Aspergillus flavus.

9
Table 1 Effect of α-terpineol on mycelial biomass and Aflatoxin B1 production by A. flavus in
SMKY medium.

Concentration α-terpineol

(µl/ml)

MDW AFB1

CNT 499.5 ± 32.5 569.5 ± 35.1

0.25 253.5 ± 11.5 377.8 ± 22.9

0.50 153.5 ± 09.5 320.6 ± 11.4

0.75 89.0 ± 02.0 188.9 ± 28.6

1.00 30.6 ± 01.5 211.7 ± 40.1

1.25 20.2 ± 0.8 28.6 ± 5.7

1.50 9.5 ± 1.5 00.00

1.75 00.00 00.00

2.00 00.00 00.00

Conc.= Concentration (µl/ml); MDW Mycelial dry weight (mg); AFB1 Aflatoxin B1 content
(µg/L) Value are mean (n =3) ± SE.

It is evident from the Table 1 and Figure 2 that α-terpineol inhibited AFB1 production in a dose

dependent manner. A corresponding decrease in mycelia dry weight as well as aflatoxin B1

production was recorded on increasing the concentration. In control set, 569.5 ± 35.1 μg/l AFB1

was secreted. The α-terpineole caused complete prevention of AFB1 secretion in SMKY broth at

10
1.50 μl/ml, lower than the concentration of minimum inhibitory concentration (MIC) against

Aspergillus flavus.

Figure 2. Fluorscent spot of aflatoxin B1 inhibited by α-terpineol.

Result of radical scavenging assay of α-terpineol depicted that the α-terpineol showed free

radical scavenging activity in dose dependent manner and its IC50 value was 66.6 μl/ml.

11
Table 2. Antioxidant activity of α-terpineol through DPPH free radical scavenging activity.

α-terpineole

Conc. (µl/ml) % inhibition

25 33.05 ± 0.21

50 38.87 ± 0.23

75 55.26 ± 0.31

100 61.56 ± 0.46

150 80.14 ± 1.07

IC50 66.6

100

80 alpha-terpineol
% radical scavenging activity

60

40

20

0
0 20 40 60 80 100 120 140 160

Concentration (µl/ml)

12
4. Discussion

The findings of the present investigation reveal the α-terpineol as suitable candidate for

formulation of plant based food additives preservatives against biodeterioration of food items

from the storage mold, lipid peroxidation as well as their contamination from aflatoxin. The

MIC value of α-terpineol compound was determined against the toxigenic strain of A. flavus by

the broth dilution method using PDB broth medium. This method offers better opportunity to

compound to come in close contact with fungal spores as both of them are homogenously

distributed inside the medium as has been earlier reported by Kalemba and Kunicka, (2003).

The study of MIC of the fungitoxicant against the storage pests is necessary to find out their

minimum amount to check the fungal population and it would be helpful in unnecessary

wastage of pesticides. The minimum inhibitory concentration (MIC) of α-terpineol against the

toxigenic strain of A. flavus was found lower than that of earlier reported essential oils of Cicuta

virosa (Tian et al., 2011), Cinnamomum jensenianum (Tian et al., 2012), Nigella sativa oil (El-

Nagerabi et al., 2012) and some components 1,8- cineole (Shyukla et al., 2009), Linalool and E-

citral, Carvone (Mishra et al., 2013). Both mycelial biomass and AFB1 production exhibited a

statistically significant declining trend with increasing concentration of the α-terpineol. The α-

terpineol checked aflatoxin synthesis by the toxigenic strain of A. flavus at a concentration

lower than the concentration for mycelia inhibition suggesting that the components have

different modes of action for antifungal activity and aflatoxin inhibition as has been earlier

reported by Rasooli & Abyaneh (2004). The lack of sporulation in fungal mycelia below the

minimum inhibitory concentration of the α-terpineol may be also a reason of their antiaflatoxin

activity at lower concentrations. Such type of connection between secondary metabolite

production and ability to reproduce by sporulation has also been suggested earlier in some

13
microorganisms (Prakash et al., 2012). Thus, the results of the present investigation show quite

similarity with the earlier hypothesis of Tian et al. (2011) that the inhibition of AFB1 synthesis

attributed by reduced fungal growth as well as inhibition of carbohydrate catabolism in A. flavus

by acting on some key enzymes. Being the lipophilic nature of α-terpineol it have ability to

disrupt the plasma membrane; enter inside the cell and disrupt the different cellular organization

particularly with the mitochondrial membrane and coagulation of some cell constituents by the

denaturation of protein has also been suggested as the mode of action of essential oils and their

components by the some workers (Cowan, 1999; Nogueira et al., 2010).

Oxidation of fatty acid during the prolong storage condition is consider as a one of the

reason responsible for the spoilage of foodstuff. Therefore, the antioxidant activity of α-

terpineol was evaluated by the prominent methods viz., DPPH assay (free radical scavenging

activity). During DPPH radical assay, DPPH solution is mixed with a substance that has the

capacity to donate hydrogen atom and gives rise to reduced form 1,1 diphenyl-2-

picrylhydrazine molecule (non radical). The extent of the antioxidant activity is visually

observed in the loss of violet color the loss, converting it to yellow colour and measured in

terms of the decrease in absorbance at 517nm. The α-terpineol having antioxidant activity has

capability to neutralize the DPPH free radical possess dual function in protection of food items

from the undesirable harmful effect of free radical (Kartal et al., 2007). However, the

antioxidant activity of α-terpineol was found to be superior than the earlier reported EOs of

Stachys inflate (Ebrahimabadi et al. 2010), Cistus ladanifer, Citrus latifolia, Cupressus

lucitanica, Eucalyptus gunnii (Guimaraes et al. 2010), Ocimum gratissimum (Prakash et al.

14
2011a) and Zanthoxylum alatum (Prakash et al. 2011b) and components methyl cinnamte; α-

pinene; β-caryophyllene (Mishra etal ., 2012).

Hence, the findings of the present investigation demonstrate the possible application of α-

terpineol as plant-based preservatives for the improvement of shelf life of agricultural

commodities by molds, aflatoxin contamination as well as from the harmful effect of lipid

peroxidation and other free radical-mediated processes during postharvest processing. However,

further testing of efficacy of the formulation of the α-terpineol as fumigant on the food systems

through microencapsulation technologies need to be carried out before possible large-scale

application.

15
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