The Antigungal Activity of lato (Caulerpa racemosa) against the Aspergillus Flavus present

in Corn

Chapter I

I. Introduction

Based on the Mediterranean Journal of Chemistry 2015, 3(6), 1093-1099 of

Alexandra Wilson, Many various kinds of fungi can abundantly be found all around us

especially in soil and air, contaminated grains on stored food commodities and agriculture

products. Aspergillus strains especially Aspergillus Flavus are the most frequent grains

molds producing carcinogenic aflatoxins . Aspergillus flavus is a saprophyte fungus with

pathogenic property for both plants and animals . Secretion of hydrolytic enzymes by the

fungus causes strong affinity to colonize in various foods . These toxins usually contaminate

agricultural products including corn . Growing corn can be tough. Drought can stress the

plants, and when plants are in a stressed weakened condition, they are susceptible to a mold,

or fungus, called aspergillus flavus.

A. flavus produces aflatoxin, a toxic and carcinogenic compound dangerous to

animals and humans (Public Affairs Specialist, NIFA). Aspergillus rot, caused by the

fungus Aspergillus flavus, is observed as a yellowish-green mold growing on corn kernels

(Susan Jongeneel, University of Illinois). Characteristics of A. flavus associated with

pathogenicity and niche specialization include secondary metabolite production, enzyme

elaboration, and a sophisticated oxylipin host crosstalk associated with a quorum-like

development program (September 2011, Annual Review of Phytopathology, Vol. 49: 107-

133),

Ethanolic and lipid soluble extracts from a marine algal species (C. racemosa) from
the coast of Tacloban City, Philippines were evaluated for antibacterial and antifungal

activity against pathogenic microbes. Lato has an active antifungal activity against fungi

(Seno, G.M.M.). Grapefruit-seed extract (GSE) contains bioactive flavonoids (e.g.,

hesperetin, resveratrol, and naringenin) and has been shown to possess anti-microbiological

effect against bacteria and fungus (S-H Brorson, 2007).

In this study, the bioactive flavaniods present in essential oil from Lato that is

extracted from the ramuli, stipe, and holdfast of Lato can cure the Aspergillus Flavus present

in corn.

II. Objectives

The study on the antigungal Activity of lato (Caulerpa racemosa) against the

Aspergillus Flavus affecting in Corn will be conducted to:

A. Know the reaction of essential oil from Lato to Dish cultured Aspegillus flavus

B. Test the significant antifungal effect of Lato essential oil to Aspergillus flaavus.

. Hypotheses

In this study, it isbhypothesized that:

(a) The essential oil from lato has no reaction to Dish cultured Aspergillus Flavus

(b) The essential oil from lato has no signifanct antifungal effect to Aspergillus flavus
IV. Scope and Limitation

The study was conducted only to determine the antifungal effect of essential oil from

Lato on Aspergillus flavus present in corn. The treatment to be use is Lato that contains

active flavenoids that can kill the said fungi.The essential oil will be extracted from the

ramuli, stipe, and holdfast of Lato . In this study, commercial fungicide will be used as

positive control. The Aflatoxin B1producing strain of A. Flavus will be isolated from a corn

and will be obtained from a laboratory. The fungal strain cultures will be reconstituted in

sterile water and inoculated in a corn meal agar for 10 days at 26 deg. Celsius. The study was

only conducted at the laboratory.

V. Significant of the study

The antifungal effect of essential oil from Lato might be the key to solve the problem

in production of corn to farmers. Corn is the second most plentiful cereal grown for human

consumption, and many cultures around the world have lived on this grain. Corn is a

versatile crop, and everything on a corn plant is useable. No part of the corn is wasted. Corn

is cooked used as food and as a vegetable or starch in cooking. Corn is also used as

pharmaceutical, insecticides, etc. (Gibson, 2002).

VI. Definition of terms

Operational Conceptual
Antifungal Effect It is the effect of dill oil to It is destroying or inhibiting the

vanish Aspergillus Flavus in growth of fungi.

corn.
Aspergillus Flavus (A. Flavus) It is a fungus that cause Is a saprotrophic fungus with a

diseases to the corn. cosmopolitan distribution..

Corn It is a plant that has A. Flavus The grain of a cereal grass is the

that needs to be treated. primary crop of the region.

Essential oil It is a natural oil that comes A natural oil obtained by

from dill and has the distillation and having the

properties of dill that can cure characteristics of the plant or the

the Aspergillus Flavus other source which it is

extracted.
Flavanoid It is a component of lato that Any large class of plant

is active that can kill pigments having structure based

Saprolegnia. It possesses on similar to that of flavone.

antifungal activity.
Fungi It is the classification of A. Any one of the group of related

Flavus that cause diseases in plants that have no flowers and

corn. that live on dead or decaying

animals.
Hesperetin It is a flavonoid that can kill Is flavanone glycoside found in

Saprolegnia. It possesses citrus fuits.

antifungal activity.
Lato Is a type of sea weeds that Is typically characterized by

contains bioactive flavonoids possessing short erect branches

 and can kill Saprolegnia. bearing crowded determinate

ramuli with short stalks and oval

or spherical tips.
Naringenin It is a flavonoid that can kill It is the predominant flavanone

Saprolegnia. It possesses in grapefruit.

antifungal activity.
Resveratrol It is a flavonoid that can kill A polyphenol compound found

Saprolegnia. It possesses in certain plants and in red wine

antifungal activity. that has antioxidant properties

and has been investigated for

possible anticarcinogenic

effects.
Saprotrophic Fungi An organism that can cause Any organism, esp. a fungus

diseases in corn. that lives and feeds on dead

organic matter.
Data Collection In this study it will test the is the process of gathering and

antifungal activities of lato measuring information on

against Aspergillus Flavus. targeted variables in an

established systematic fashion,

which then enables one to

answer relevant questions and

evaluate outcomes.
Chapter II

I. Review of Related Literature

Lato (Caulerpa racemosa)

This is the most variable Caulerpa species, but is typically characterized by

possessing short erect branches bearing crowded determinate ramuli with short stalks and

oval or spherical tips. The arrangement and shape of the ramuli differ in the various varieties.
(Plant resources of South-East Asia No. 15(1).

Caulerpa racemosa that grows on sandy substrate in calm and turbid waters tend to

have long erect branches while those growing on rocky wave-exposed waters have strong

short erect branches. (Plant resources of South-East Asia No. 15(1).

As a human uses the fronds are consumed fresh as a salad or eaten mixed with

spices. In the Philippines it is used as a fish feed and in traditional medicine to lower blood

pressure and to treat rheumatism. (Plant resources of South-East Asia No. 15(1).)

Aspergillus flavus

Aspergillus flavus is a saprotrophic and pathogenic (Machida, 2010) fungus with a

cosmopolitan distribution (Ramirez, 2012). A. flavus infections can occur while hosts are

still in the field (preharvest), but often show no symptoms (dormancy) until postharvest

storage and/or transport. In addition to causing preharvest and postharvest infections, many

strains produce significant quantities of toxic compounds known as mycotoxins, which,

when consumed, are toxic to mammals (Agrios, 2005). A. flavus is also an opportunistic

human and animalpathogen, causing aspergillosis in immunocompromised individuals

(Amaike. 2005).

One of the major source of aflatoxin is A. flavus. Aflatoxins are toxic metabolites

produced by certain fungi in/on foods and feeds. They are probably the best known and most

intensively researched mycotoxins in the world. The occurrence of aflatoxins is influenced

by certain environmental factors; hence the extent of contamination will vary with

geographic location, agricultural and agronomic practices, and the susceptibility of

commodities to fungal invasion during pre-harvest, storage, and/or processing periods

(Anon, 1989).

Aspergillus flavuscan be recognized by a gray-green or yellow-green mold growing

on corn kernels in the field or in storage (Figure 1). Plant stress due to drought, heat or insect

damage during fungus growth usually increases aflatoxin levels. Aflatoxin contamination

will reduce feeding value and hinder sales. Because it is extremely poisonous to warm-

blooded animals even at relatively low levels, grain handling facilities often check for the

presence of the toxin before purchasing corn (Sumner, n.d.)

Table 1 shows the optimum conditions for Aspergillus growth and aflatoxin

development. When temperatures are below 65 degrees F and the moisture of the corn is

below 12 to 13 percent, development of the fungus usually stops (Lee, n.d.)

Table 1. Conditions Favoring Aspergillus

flavus Development

Factor Optimum Range

Temperature 86°F 80-110°F

Relative Humidity 85% 62-99%

Kernel Moisture 18% 13-20%

Corn

Corn kernels are the seeds of maize. Though technically a grain, maize kernels are

used in cooking as a vegetable or starch. They are used as pelletized fuel for pellet stoves and

furnaces. Corn kernels are a natural pellet, which gives them an economic advantage over

other man-made biomass pellets and wood pellets.The use of corn and other grains as a

renewable biofuel may have environmental and cost benefits, compared to other energy

source, and may create additional forms of revenue for farmers and other economic

industries (Benson, 2002).

One reference lists over 500 different uses for corn. Corn is a component of canned

corn, baby food, hominy, mush, puddings, tamales, and many more human foods. Some

industrial uses of corn include filler for plastics, packing materials, insulating materials,

adhesives, chemicals, explosives, paint, paste, abrasives, dyes, insecticides, pharmaceuticals,

organic acids, solvents, rayon, antifreeze, soaps, and many more.Corn also is used as the

major study plant for many academic disciplines such as genetics, physiology, soil fertility

and biochemistry. It is doubtful that any other plant has been studied as extensively as has

the corn plant (Gibson, 2002).

II. Related Studies

G.L. Windhan, W.P. Williams, and F.M. Davis. “Effects of the Southwestern
Corn Borer on Aspergillus flavus Kernel Infection and Aflatoxin Accumulation in

Maize hybrids”. Field studies were conducted in 1995 to 1997 to determine the effect of the

southwestern corn borer (SWCB) on Aspergillus Flavus kernel infection and aflatoxin

accumulation in maize hybrids. In 1995, when A. flavus conidia were applies to silks in a

spray and SWCB neonate larvae in maize cob grits were placed in the leaf axil at the top ear

of commercial hybrids, aflatoxin contamination and A. flavus kernel infection were highest

in plants treated with both the fungus and the insect. In 1996, using the same inculcation and

infestation techniques, aflatoxin levels and kernel infection were much lower than in 1995

and SWCB had no effect on aflatoxin contamination on kernel infection.

Kazemi. “Effect of Carum copticum essential oil on growth and aflatoxin

formation by Aspergillus strains”. The objective of this study were to determine the

antiaflotoxin B1 activity in vitro of the essential oil (EO) extracted from the seeds of Carum

copticum and to evaluate its antifungal activity in vivo as a potential food preservative. The

results indicated the percentage of infected fruits is significantly (p<0.01) reduced by the EO

at 16 degrees Celsius for 30 days. In this case, the oil at 100 mL concentration showed the

highest inhibition of fungal infection with a value of 80.45% compared with the control.

Thus, the EO of dill could be used to control food spolage and as a potential source of food

preservative.

Seno G.M.M. , “Antibacterial and Antifungal Activity of Lato(Caulerpa

Racemosa)”. Ethanolic and lipid soluble extracts from a marine algal species (C. racemosa)

from the coast of Tacloban City, Philippines were evaluated for antibacterial and antifungal
activity against pathogenic microbes (1 gram-positive, 2 gram-negative, 4 fungi). This

species with antibacterial activity was active against the gram-positive bacteria

(Staphylococcus aureus), and the gram-negative bacteria (Pseudomonas aeruginosa and

Escherichia coli). In addition, this species with antifungal activity was active against fungi.

Results generally indicate that the extract of C. racemosa has the highest antibacterial

effectivity against P. aeruginosa and the highest antifungal effectivity against Aspergillus

flavus. 75 µL of the chloroform-methanol or lipid soluble extracts of C. racemosa exhibited

the highest activity against the species test.

S-H Brorson, 2007. “Grapefruit Seed Extract is a Powerful in Vitro Agent

against Motile and Cystic Forms of Borrelia Burgdorferi Sensu Lato”. Lyme borreliosis

, caused by Borrelia burgdorferi sensulato, may lead to long-term tissue infection, which

may be difficult to cure. The outcome of Lyme borreliosis is highly dependent on the

antibiotic treatment .The observation of the ability of B. burgdorferi sensu lato to convert

(and reconvert) to cystic forms may explain why the infection sometimes is persistent and

reactivating. Therefore, it might be important to eradicate all germative forms (not only the

motile form) of the bacterium to obtain a proper treatment for Lyme borreliosis. Grapefruit-

seed extract (GSE) contains bioactive flavenoids (e.g., hesperitin, resveratrol, and

naringenin) and has been shown to possess anti-microbiological effect against bacteria and

fungus. Many

studies indicate that GSE is a substance whose therapeutic effect ranks equal to or better than

other known anti-bacterial agents. Positive effects of GSE are decreased levels of TNF-α,

Nuclear factor Kb, NO, protection of the gastrointestinal tract against mechanical stress, and

has anti-allergic and other antioxidative properties . Naringenin, hesperidin and other citrus

flavones have been found in plasma and tissue after ingestion . Lactobacillus and
bifidobacteria in the gut seems to be insignificantly affected by GSE , and no severe side

effects have been observed. B. burgdorferi sensu lato has a gene for efflux mechanism which

may be responsible for antibiotic resistance . GSE is an efflux inhibitor, which can be used to

enhance the activity of antibacterial agents.

Chapter III

I. Methodology

Research experiments undergo different procedures or methods in order to collect the

data of the results of the study.

Preparation of Lato Extract

One hundred grams of lato were set aside, washed, and refrigerated until processing. The

parts of the algal sample (ramuli, stipe, and holdfast) were separated by hand, and each was

placed in a prelabeled 100- mL beaker. Twenty grams of each part was prepared.

For the extraction, the separated parts were each pounded using a sterilized mortar

and pestle. Each part was extracted using 20 mL of 80 percent ethanol and homogenized for

6 to 12 hours. The extracts were then heated at 76 degrees Celsius (°C) to 80°C to evaporate

the ethanol.

And set aside for 30 minutes. The extracts were each filtered using Whatman 110

filter paper. The clear less dense liquid formed was decanted. The remaining lipid liquid

extract was stored in a 60 mL reagent bottle until further bacterial sensitivity testing.

Culture condition

Aflatoxin B1 producing strain of A. Flavus will be isolated from a corn and will be

obtained from a laboratory. The fungal strain cultures will be reconstituted in sterile water

and inoculated in a corn meal agar for 10 days at 25 deg. Celsius. After the period, the

cultures will be washed with sterile solution.

Test of antifungal activity

Disk diffusion assay: filter paper disc (6mm diameter) containing 5.0 micro litres of

essential oils and will be applied on the surface of the corn meal agar plates previously

inoculated with A.flavus. The inoculated plates will be incubated at 25 deg. Celsius for 5
days. At the end of the period, antifungal activity will be evaluated by measuring the zone

inhibition (mm) against the test fungus.

Test in grains: the fungal growth was also evaluated in grains, using the same

methods above, but the paper disc will be replaced by grains of corn.

Commercial fungicide will be used as positive control. All treatments consisted of

three replicates and repeated once more. The averages of the experimental results will be the

determined.

Evaluation of aflatoxin production in corn impregnated with essential oils.

Samples with 60 g of each grain of corn will be irradiated with a dose of 20 kGy, to

eliminate the fungal contamination. For evaluation of aflatoxin B1 production in grains, the

samples were placed in Erlenmeyer flasks; the water activity will be adjusted to 0.94, the

samples will be treated with 200, 100, 50, and 10 micro litres of lato essential oil. Untreated

samples will be used as controls.

Suspension of A. Flavus that will contain a certain amount of spores will be

inoculated in the grains treated and untreated with essential oil. Four replicates will be

performed and the experiment will be repeated twice.

The cultures will be inoculated at 25 deg cesius for 10 days for aflatoxin production,

and then grain samples will be grind. 50 g of each samples will be used for extraction of

aflatoxin. Aflatoxin B1 will be extracted with chloroform and solvent evaporated until

1.0mL in a volumetric flask. An aliquot will bw spotted on silica gel-G thin layer plate and
will be developed with chloroform and acetone as solvent system. The concentration of

aflatoxin B1 will be determined comparing the area of the spot samples with aflatoxin B1.

Test of Lato

Determination of lesion of plasma membrane

A spore suspension of A. flavus was obtained from its 3-day-old cultures,

which were harvested by adding 5 ml PBS with 2% (w/v) D-glucose (PBS-2%G) to each

petri dish and gently scraping the mycelial surface three times with a sterile L-shaped

spreader to free the spores. The spore suspension of A. flavus containing 4×106 spores/ml

adjusted by a hemocytometer was then added into each glass tube. A requisite amount of the

dill oil was added in the tubes to obtain 0.25, 0.5, 1.0, 1.5, and 2.0 ìl/ml concentrations.

Samples without any oil treatment were considered as controls. The mixtures were then

incubated for 12 h at 28±2°C in an incubator shaker. The cells were washed and resuspended

in 0.5 ml PBS, and stained with a final concentration of 1 µg/ml PI solution in PBS for 30

min at room temperature. All the incubations were carried out in the dark. Unstained cell

suspensions were always included as autofluorescence controls. For each sample, a

Scattergram analysis was performed to evaluate morphological changes, and the percentage

of PI-positive cells was determined using flow cytometer (Epics Altra, Beckman Coulter,

Miami, USA) at PMT4 channel (620 nm).

Preparations of mitochondria

The mitochondria of A. flavus were isolated using the previously described

method with some modifications. An amount of 100 µl containing 107 spores/ml of A.

flavus spore suspension was inoculated in PDB medium containing 0, 0.25, 0.5, 0.75, and
1.0 µl/ml of dill oil (the failure of mycelia generation at 2.0 µl/ml) for four days at 28±2°C.

After incubation, mycelia was harvested and washed twice with distilled water. The net wet

weight of the cell pellet was determined. Each approximately 2 g wet weight of mycelium

was resuspended in 10 volumes of isolation buffer (10 mM Tris-HC1, 1 mM EDTA, 0.5 M

mannitol, pH 7.0), and then disrupted in a high-speed homogenizer (XHF-D, Xinzhi

Scientific Biotechnology Co., Ltd., Ningbo, China) for 45 s twice. The homogenate was

centrifuged at 2000×g for 10 min at 4°C to remove mycelial fragments and conidia. The

supernatant was carefully removed from the loose pellet and centrifuged at 12000×g for 40

min at 4°C. The pellet containing the mitochondria was washed in an isolation buffer and re-

centrifuged using the same rotor for 20 min. The final mitochondrial pellet was resuspended

in 1 ml isolation buffer and maintained at 4°C until use.

Determination of mitochondrial ATPase activity

The mitochondrial ATPase activity in dill oil-treated fungal cells was detected

using a Micro-ATPase Assay Kit obtained from the Institute of Biological Engineering of

Nanjing Jianchen (Nanjing, China) following the manufacturer's protocol. Protein content

was determined as described by Bradford [20], and bovine serum albumin was used as a

standard. The optical density (OD) value was determined at 636 nm after 5 min. Next, 20 nM

inorganic phosphate was used as a control. One unit of ATPase activity was defined as 1

µmol inorganic phosphorus catalyzed by this enzyme in 1 mg protein for 1 h

(µmolPi/mgpro/h).

Determination of mitochondrial dehydrogenases activity

The change of mitochondrial dehydrogenases in A. flavus after treatment with

dill oil was measured using the XTT method. Briefly, 200 µl of 2×106 spore/ml was added

to 96-well flat-bottom microplates (Corning, Corning Incorporated, New York, USA) and

incubated with different concentrations of dill oil (0.0313, 0.0625, 0.125, 0.25, 0.5, 1.0, 2.0,

and 4.0 µl/ml). Samples without any oil treatment were considered as controls. After 24 h of

incubation at 28°C, 50 µl aliquots stock XTT with menadione was added to the wells in

order to obtain a final concentration of 50 µg/ml XTT and 25 µM menadione. The optical

densities at 450 nm (OD450) were determined directly using a 96-well scanner (KHB ST-

360, Experimental System Co., Ltd. Shanghai, China) after 2 h of exposure to XTT. The

XTT assay was performed in triplicate.

Determination of endogenous ROS production

Endogenous ROS levels of A. flavus were detected by a fluorometric assay using

the fluorescent dye DCFH-DA as a ROS indicator as previously reported with some

modifications [23]. Fungal cell suspension obtained was adjusted to 4×106 spore/ml and

exposed to dill oil with final concentrations of 0.25, 0.5, 1.0, and 2.0 µl/ml for 12 h. Samples

without any oil treatment were considered as controls. At the end of the treatments, DCFH-

DA was added into the mixture with a final concentration of 10 µM for 4 h at 28°C. After

incubation, the fungal cells were centrifuged at 5000×g for 5 min and washed twice with

PBS; the pellet was resuspended in 0.5 ml PBS. Fluorescence intensities of the resuspended

cells were quantified via FACScan flow cytometer (Epics Altra, Beckman Coulter, Miami,

USA) using excitation and emission wavelengths of 485 and 535 nm, respectively.

Additionally, these experiments were performed with the presence of the antioxidant Cys at

concentrations of 80 mM. Results were corrected by subtracting the fluorescence value of

lato oil ± antioxidant in the corresponding concentration without cells but with DCFH-DA.
All tests were performed in triplic

Morphology of Aspergillus Flavus

         Aseptically place the sterile filter paper in thepetri plate, and then place the sterile u-

shaped rod inside the petri plate. Moist it with sterile distilled water. Sterilized the scalpel

blade, then cut the prepared agar and transfer it to the slide. Aseptically place the cover slip.

Flame the inoculating loop. Take the small portion of the fungal culture to be examined and

inoculate the four sides of the agar square with mycelial fragment of the fungus. Cover the

petri dish and incubate for 48 hours at room temperature. After 48 hours, examine the slide

with mycelia growth and spore production spreading over the cover slips.

Antifungal investigation by poison method

          2 ml of extract was added to 15 ml of potato dextrose agar media which was

inovulated with 5mm agar disc and incubated in 27 degrees Celsius. Positive and negative

control was also taken. PDA plates with clotrimazole were used as positive control. Below is

the formula to get the percent of inhibition of fungal pathogen:

        

 Antifungal activity (%) =DC-DSDCx 100

         Where:

                 DC = diameter of growth in control plate

               

  DS = diameter of growth in the plate containing tested antifungal agent

Experimental Design

Legend: A B C

A: Oregano C A B

B: + Control B C A

C: - Contro

H. Data Collection

         After 48 hours of incubation, the plates will be taken out and the zone of inhibition will

be measured with a caliper that will be placed below the plate. It will be measured by

millimeters. To get the zone of inhibition:

Zone of inhibition =  x+y2

Where,

x = vertical diameter

y=  horizontal diameter.

Bibliography

 Mediterranean Journal of Chemistry 2015, 3(6), 1093-1099 of Alexandra Wilson

 Public Affairs Specialist, NIFA

 Susan Jongeneel, University of Illinois

 September 2011, Annual Review of Phytopathology, Vol. 49: 107-133

 Seno, G.M.M.

 S-H Brorson, 2007

 Gibson, 2002

 Plant resources of South-East Asia No. 15(1).

 Machida, 2010

 Ramirez, 2012

 Agrios, 2005

 Amaike. 2005

 Anon, 1989

 Sumner, n.d.

 Lee, n.d.

 Benson, 2002
“The Antifungal effect of Essential Oil from Lato (Caulerpa Racemosa) on Aspergillus

Flavus in Corn”

RESEARCH 2

Submitted to:

Mr. Reynaldo Huervana

Submitted by:

Cheveem Grace C. Emnace

Raesl Vien G. Somuelo

Aira Fe E. Ibanez