1

INTRODUCTION

Background of the Study

Soil is the soul of infinite life that promotes diverse microflora. Soil

organisms contribute a wide range of essential services to the sustainable function of all

ecosystems. They act as the primary driving agents of nutrient cycling and regulating the

dynamics of organic matter (Hesammi et al., 2014). Earthworms are popularly known as

the “farmer’s friend” or “nature’s plowman”. Earthworm influences microbial

community, physical and chemical properties of soil. They breakdown large soil

particles and leaf litter and thereby increase the availability of organic matter for

microbial degradation and transforms organic wastes into valuable vermicomposts by

grinding and digesting them with the help of aerobic and anaerobic microbes (Maboeta &

Van Rensburg, 2003). Earthworms activity is found to enhance the beneficial microflora

and suppress harmful pathogenic microbes. Soil worm casts are rich source of micro and

macro-nutrients, and microbial enzymes (Lavelle & Martin, 1992). Vermicomposting is

the term given to the process of conversion of biodegradable matter by earthworms into

vermicast (Abbasi & Ramasamy, 2001).

Vermicast has an active amounts of beneficial bacteria, enzymes and nutrient

as well as remnants of plant material that were not digested by the earthworm (Holmer,

2008). Vermicast is believed to contain hormones and enzymes which it acquires during

the passage of the organic matter through the earthworm gut and a very good soil

conditioner (Gajalakshmi & Abassi, 2003).

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Fungi are eukaryotic microorganisms that play fundamental ecological roles

as decomposers, mutualists, or pathogens of plants and animals; they drive carbon

cycling in forest soils, mediate mineral nutrition of plants, and alleviate carbon

limitations of other soil organisms (Blackwell, 2011). They are an important component

of the soil microbiota typically constituting more of the soil biomass than bacteria,

depending on soil depth and nutrient conditions. Many important plant pathogens (smuts

and rusts) and plant growth promoting microorganisms (e.g., ecto and endo mycorrhizae)

are fungi. The saprobic fungi represent the largest proportional of fungal species in soil

and they perform a crucial role in the decomposition of plant structural polymers such as

cellulose, hemi-cellulose and lignin thus contributing to the growth on inexpensive

substrates (Hawksworth et al., 1996). Thus, the present study was undertaken to screen

the mycochemical constituents and biological activities mainly the antibacterial and

antioxidant activity of fungi (Aspergillus niger and Rhizomucor pusillus) associated with

vermicast in their ethanolic extract and fungal spent. Staphylococcus aureus and

Escherichia coli were used as the test organisms in the antibacterial assay.

Objectives of the Study

This study generally aimed to screen the phytochemical constituents and to

determine the biological activities of fungi (A. niger and R. pusillus) associated with

vermicat in their ethanolic extract and fungal spent.

Specifically, it intended to:

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1. determine the presence of mycochemical constituents of the different fungi associated

with vermicast in their ethanolic extract and fungal spent which includes alkaloids,

cardiac glycosides, flavonoids, saponins, steroids, tannins, and terpenoids;

2. evaluate the antibacterial activity as protectant and eradicant of the fungal extracts

and fungal spent against S. aureus and E. coli at 12 and 24 hours of incubation; and

3. determine the antioxidant property as radical scavenging activity of fungal extract and

fungal spent of the different fungi isolated from vermicast.

Significance of the Study

Although a considerable number of studies have been carried out on

vermicompost which has a great impact on the soil and plant growth (Gajalakshmi et al.,

2001, 2002; Singh & Sharma, 2002; Gajalakshmi & Abbasi, 2003, 2004;

Padmavathiamma et al., 2008), there is still a lack of knowledge on the vermicast and the

microorganisms associated with it and its biological activities. Hence, this study gave

further information regarding on the mycochemical constituents and biological activities

of the fungi A. niger and R. pusillus associated with vermicast. Investigating these

properties is of great importance with respect to understanding the biological potential of

A. niger and R. pusillus that can lead to the improvement and discovery of new

therapeutic and pharmacological medicine.

Scope and Limitations of the Study

This study was divided into three sub-studies. Sub-study 1 focused mainly on

the screening of the mycochemical constituents of A. niger and R. pusillus in their

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ethanolic extract and fungal spent which includes alkaloids, cardiac glycosides,

flavonoids, saponins, tannins, steroids and terpenoids following the standard method

described in the Laboratory Manual for UNESCO (1986). Sub-study 2 evaluated the

antibacterial activity as protectant and eradicant against S. aureus and E. coli using disc

diffusion method. Sub-study 3 elucidated the antioxidant property as radical scavenging

activity or the ability of the extracts to inhibit or delays cellular damage. Pure cultures of

fungi (A. niger and R. pusillus) associated with vermicast were obtained from the

collection of Mary Jhane G. Valentino, M.Sc., and these were identified by Philippine

Center for Post-Harvest Development and Mechanization, Science City of Munoz, Nueva

Ecija.

Time and Place of the Study

Extraction of fungi, antibacterial assay and screening of phytochemical

constituents were done at the Bamboo Tissue Culture Laboratory, Department of

Biological Sciences, College of Arts and Sciences, Central Luzon State University,

Science City of Munoz, Nueva Ecija, Philippines. Whereas, rotary evaporation of the

samples was conducted at Araullo University, Cabanatuan City, Nueva Ecija.

Determination of the antioxidant property as radical scavenging activity was done at

Saint Mary’s University, Nueva Vizcaya, Philippines. This study was undertaken from

December-April, 2018.
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REVIEW OF RELATED LITERATURE

Vermicast

Vermicast has an active amounts of beneficial bacteria, enzymes and nutrient

as well remnants of plant material that were not digested by the earthworm (PUVEP,

2008). Microbial activity of beneficial microorganisms in worm castings is ten to twenty

times higher than that of in the soil and other organic matter. Among beneficial soil

microbes stimulated by earth worms are “nitrogen-fixing & phosphate solubilizing

bacteria”, the “actinomycetes” & “mycorrhizal fungi”. Studies found that the total

bacterial count was more than 10/gm of vermin- compost. It included Actinomycetes,

Azotobacter, Rhizo- bium, Nitrobacter & Phosphate solubilizing bacteria ranges from

102-106 per gm of vermicompost (Edwards, 1995).

The vermicast contained higher amounts of nitrate nitrogen and possessed a

greater nitrifying power than the corresponding soils. Application of vermicompost or

vermicast improves the soil structure by increasing porosity and reducing the bulk

density. It improvises soil aeration, water-holding capacity, buffer capacity, and cation

exchange capacity of soil (Nada et al., 2011). It is also reported to contain biologically

active substances such as plant growth regulators and offers a promise as alternate to
 6

inorganic fertilizer as source of plant nutrients (Brady, 1974). Microfungi in the casts of

earthworms, Perionyx millardi, Eudrilus eugeniae, Lampito mauritii and certain other

earthworm species are available. But, the scientific knowledge available on the fungal

flora in the casts of the earthworm, Perionyx ceylanensis Mich., Aspergillus fumigatus,

Mucor circinelloides, F. circinelloides and Penicillium expansum were dominant in the

intestine of earthworms (Vaclav & Novakova, 2003).

Vermicompost

Vermicompost is a nutrient-rich, microbiologically-active organic amendment

that results from the interactions between earthworms and microorganisms during the

breakdown of organic matter. It is a stabilized, finely divided peat-like material with a

low C:N ratio, high porosity and high water-holding capacity, in which most nutrients are

present in forms that are readily taken up by plants (Dominguez, 2004). Unlike compost,

vermicompost is produced under mesophilic conditions, and although microorganisms

degrade the organic matter biochemically, earthworms are the crucial drivers of the

process, as they aerate, condition and fragment the substrate, thus drastically altering the

microbial activity. Earthworms act as mechanical blenders, and by fragmenting the

organic matter they modify its physical and chemical status by gradually reducing the

ratio of C:N and increasing the surface area exposed to microorganisms - thus making it

much more favorable for microbial activity and further decomposition (Dominguez et al.,

2010).

Mycochemical Constituents of Fungi

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Yadav et al. (2014), studied the phytochemical constituents of different

endophytic fungi isolated from Eugenia jambolana Lam. Results showed that Fusarium

sp., Coprinopsis cinerea, Penicillium spinilosum, Aspergillus flavus, Isaria tenuipes,

Aspergillus sp., Aspergillus niger, Aspergillus peyronelii, Aspergillus tubingensis,

Curvularia lunata, Alternaria alternata, Syncephalastrum racemosum, Choanephora sp.,

Chaetomium sp., Trichoderma longibrachiatum, Aspergillus japonicas, Aspergillus

terreus, Paecilomyces formosus, A. niger strain and Aspergillus fumigatus contained

alkaloids, phenols, flavonoids, saponins and terpenes.

In addition, Ramesha and Srinivas, (2013) revealed that ethyl acetate extracts

of C. gloeosporioides contains alkaloids and steroids; whereas F. oxysporum extract

contains flavonoids, phenol and phenolic compounds. Whereas, Agrawal et al. (2015)

found out that Alternaria alternata, Geotrichium albida, Penicillium, Frequentans and

Thielaviopsis basicola contains flavonoids, alkaloids, phenols, saponins, steroids, tannins,

terpenoids in endophytic fungi.

Similarly, Ladoh et al. (2015), proved that Aspergillus species has flavonoids,

anthroquinones, tannins, phenols, steroids, coumarins and terpenoids and absence of

alkaloids and saponins. Thus their potential as new bioactive compounds, can be used in

the fields of health and agriculture.

Biological Activities of Fungi

Antibacterial Activity

Nowadays endophytic fungi serve as a source of antimicrobial compounds. In

the study of Hema et al. (2015), a total of 21 endophytic fungi were isolated from Basella
 8

rubra L. a medicinal plant and screened for their antibacterial potential and for the

presence phytochemical constituents and these resulted to inhibition of all the test

pathogens with a maximum zone of inhibition of 20 mm and a minimum of 11 mm. The

need for new antimicrobial agents, in general, comes from the increasing rates of

resistance to existing antibiotics. This problem extends beyond the clinical application of

antimicrobial drugs such as agricultural microorganisms are also known to have acquired

resistance to commonly used antimicrobial chemicals (Agrawal et al., 2015)

Antioxidant Activity and Total Phenolic Compound

Yadav et al. (2014), studied In-vitro antioxidant and phenolic content of

different endophytic fungi isolated from Eugenia jambolana Lam. Results revealed

Aspergillus niger strain showed the highest antioxidant activity ranging from 50% to 80%

having 58 mg/g to 60 mg/g GAE total phenolics. Ascorbic acid used as a standard

showed 90% reducing potential.

Similarly, Nitya et al. (2015), evaluated the antioxidant potential of the

methanolic extracts of the fungi Aspergillus and Mucor species and found out that the

two fungi have significant antioxidant potential and the antioxidant nature of the extracts

were dependent on the concentration. There was a positive correlation between the

phenolic content and the antioxidant capacity of the endophyte extracts. These studies

confirm the medicinal values of the endophytic fungi.

Fungi Associated with Vermicast

Aspergillus niger
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A. niger is a filamentous ascomycete fungus that is ubiquitous in the

environment and has been implicated in opportunistic infections of humans (Perfect et

al., 2001). A. niger is most widely known for its role as a citric acid producer (Magnuson

& Lasure, 2004). With production of citric acid at over one million metric tons annually,

A. niger citric acid production serves as a model fungal fermentation process. As a

common member of the microbial communities found in soils, A. niger plays a

significant role in the global carbon cycle. This organism is a soil saprobe with a wide

array of hydrolytic and oxidative enzymes involved in the breakdown of plant

lignocellulose. A variety of these enzymes from A. niger are important in the

biotechnology industry. A. niger is also an important model organism for several

important research areas including the study of eukaryotic protein secretion in general,

the effects of various environmental factors on suppressing or triggering the export of

various biomass degrading enzymes, molecular mechanisms critical to fermentation

process development, and mechanisms involved in the control of fungal morphology.

Rhizomucor pusillus

In 1978, the genus Rhizomucor was established for Mucor like fungi forming

stolons and rudimentary rhizoids and expressing thermophilism (Ribes et al., 2002).

Rhizomucor is commonly found contaminating air, soil and organic matter. Various

species of Rhizomucor are known. R. pusillus, R. miehei and R. variabilis with two

subspecies (R. variabilis var. variabilis and R. variabilis var. regularior) can cause

mucormycosis in humans (Gomes et al., 2011). R. pusillus is the most common species

seen and has been detected in a variety of food items, including grains, seeds, nuts and
 10

beans. It is a thermophilic saprobic mucormycete with a wide distribution but it is not

commonly associated with human disease (Hernanz et al., 1983). Its mode of

transmission is by inhalation of spores and percutaneous introduction of spores into a

susceptible host (Lu et al., 2009). Various studies in the literature have shown this

species to be mostly associated with patients who are severely immunocompromised,

especially those undergoing therapy for leukemia and those who have uncontrolled

diabetes mellitus (Germain et al., 1993).

Rhizopus stolonifer

Rhizopus stolonifer, a Zygomycete has a filamentous growth habit. Its

filaments are coenocytic, that is, they are non-septate. It is the only fungus yet known to

produce rhizoids which penetrate the substratum in order to obtain nutrients. The rhizoids

also serve as support. Opposite the rhizoids, a sporangiophore juts into the atmosphere

and this terminates in a club-shaped collumelum enclosed within the sporangial wall.

Between the collumelum and wall are numerous asexual reproductive structures known

as sporangiospores. This organism is also characterized by the presence of stolons, which

connect rhizoid joints. Other members of the Zygomycetes, especially species of the

genus Mucor, have been shown to undergo fungal dimorphism (McIntyre et al., 2002;

Lubberhusen et al., 2003). In such phase transitions, sporangiospores convert to yeast like

cells, with a central globose mother cell which multilaterally produces daughter buds by

blastic action. This occurs under CO2 tension or high hexose concentration.

Aspergillus fumigatus
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Aspergillus species are opportunistic filament forming moulds comprising

over 180 different species, of which A. fumigatus causes the majority of human

Aspergillus infections (Hope et al., 2005). A. fumigatus is now the second most common

fungal infection found in hospitalized patients, after Candida albicans. A. fumigatus is

ubiquitous, with a worldwide distribution due to the production of small spores called

conidia that have an average size of 2, 3.5 mm, resulting in the conidia dispersing in the

air and remaining in the atmosphere for prolonged periods (Rivera et al., 2006).
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MATERIALS AND METHODS

This study was divided into three sub-studies. Sub-study 1 focused on the

screening of the mycochemical constituents; sub-study 2 determined the antibacterial

asctivity as protectant and eradicant against S. aureus and E. coli; and sub-study 3

elucidated the antioxidant property as radical scavenging activity of fungi (A. niger and

R. pusillus) associated with vermicast in their ethanolic extract and fungal spent.

Source of Fungal Inoculum

Pure cultures of fungi associated with vermicast were obtained from the

collection of Mary Jhane G. Valentino, M.Sc. and these were identified by Philippine

Center for Post-Harvest Development and Mechanization, Science City of Munoz, Nueva

Ecija.

Preparation of the Sub Culture

Potato Dextrose Agar (PDA) was used in the sub culture. Thirty-nine grams of

powdered PDA was dissolved in a liter of distilled water. After sterilization, media was

pour-plated and mycelia was aseptically inoculated into sterile PDA plates and incubated

at room temperature for 7 days.

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Preparation of Liquid Media

Potato Dextrose Broth (PDB) was used in the mycelial production of the

fungi. Twenty-four grams of powdered PDB was dissolved in a liter of distilled water.

Approximately 50 ml of liquid media was dispensed into each bottle covered with

polypropelyne bag and sealed with clean paper and rubber band. All the prepared liquid

media were sterilized using an autoclave at 121°C, 15psi for 20 minutes

.
Preparation of Mycelial Production

The previously sterilized liquid media was inoculated with 7 day old mycelial

discs prepared using flame sterile 10-mm-diameter cork-borer and it was incubated at

room temperature for ten (10) days to allow mycelial growth. The mycelia were

harvested and it was air-dried for seven days.

Preparation of Ethanol Extracts

Twenty-five grams (25g) of powdered fungi was used. The weighed samples

were mixed with 100 ml of 95% ethanol in a sterile flask for 48 hours. The extracts were

filtered with Whatman No. 1 filter paper prior to rotary evaporator. Then, it was placed in

amber bottles and sealed with aluminum foil to avoid exposure to light and dust (adapted

from Onyeagba et al. (2004) with some modification).

Preparation of Fungal Spent

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The liquid media used in the mycelial production was kept after the mycelia

were harvested. Mycelial spent was filtered using Whatman No. 1 filter paper then it was

placed in an amber bottle and stored in a refrigerator until needed.

Sub-study 1. Screening of Mycochemical Constituents of Fungi (A. niger and R.

pusillus) Associated with Vermicast

Screening of mycochemical constituents of fungi (A. niger and R. pusillus)

was carried out by following the standard methods described in Laboratory Manual for

the UNESCO (1986). The various mycochemical constituents evaluated were alkaloids,

cardiac glycosides, flavonoids, saponins, steroids, tannins, and terpenoids

Test for Alkaloids

Five milliliters of the different extracts were prepared in a beaker and 200 mL

of 10% HCH3CO2 in C2H5OH was added. The mixture was filtered and the extracts was

allowed to become concentrated in water bath until it reached one fourth of the original

volume then concentrated NH4OH was added. Formation of the white precipitate or

turbidity was observed for the presence of alkaloids (Trease & Evans, 1983).

Test for Cardiac glycosides

One milliliter of concentrated sulfuric acid (H 2SO4) was prepared in the test

tube. Five milliliters of different fungi extracts were mixed with 2 mL of glacial
 15

HCH3CO2 containing one drop of FECl3. The mixture was added carefully to 1 mL of

concentrated H2SO4 so that the concentrated H2SO4 was underneath the mixture. The

appearance of brown ring was observed for the presence of cardiac glycosides.

Test for Flavonoids

In 5 ml different extracts of fungi few drops of 1% ammonium (NH 3) solution

was added in the test tube. Yellow coloration was observed for the presence of

flavonoids.

Test for Saponins

A volume of 0.5 mL of different extracts were added to 10 ml distilled water

and were shaken vigorously to obtain a stable persistent froth. The persistent frothing was

observed for the presence of saponins.

Test for Steroids

Two milliliters of acetic anhydride were added to a 5 mL extracts of different

fungi sample with 2 mL of H2SO4.Violet to blue or green precipitate was observed for the

presence of steroids.

Test for Tannins

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An aliquot of 0.5 mL extracts of different fungi were added to 10 ml of

distilled water on the test tube and filtered. Two ml of 5% FeCl was added to the filtered

sample. The formation of brownish green to blue-black coloration was observed for the

presence of tannins.

Test for Terpenoids

Five milliliters extracts of different fungi samples were added with 2 mL

CHCl3 in a test tube. Then, 3 mL of H2SO4 was added carefully to the mixture to form a

layer. The formation of the reddish brown of the interface was observed for the presence

of terpenoids.

Sub-study 2. Determination of Antibacterial Activity as Protectant and Eradicant of

Fungi (A. niger and R. pusillus) Associated with Vermicast

Preparation of inoculum

Antibacterial activities (as protectant and eradicant) of the fungal extracts

were evaluated using disc diffusion method at 12 and 24 hours of incubation. Pure

cultures of S. aureus (Gram positive bacteria) and E. coli (Gram negative bacteria) were

obtained from the Department of Biological Sciences in CLSU, Science City of Muñoz,

Nueva Ecija. The bacteria were grown in Nutrient Agar for 24 hours and transferred to

Nutrient Broth to standardize to 1.5 X 108 cells/ml using McFarland standards.

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Preparation of Mueller Hinton Agar

Thirty-eight grams (38g) of Mueller Hinton agar II powder was dissolved in

1000 ml of distilled water prior to sterilization using an autoclave at 121◦C (15psi) for 30

minutes.

Preparation of the assay plates

After sterilization, the agar was distributed in the assay plates with

approximately 20 ml each. Treatments used are presented in Table 1.

Table 1. The different treatments to be used in the antibacterial activity

TREATMENTS FUNGAL EXTRACTS
T1 Aspergillus niger ethanolic extract
T2 Rhizomucor pusillus ethanolic extract
T3 Aspergillus niger spent
T4 Rhizomucor pusillus spent
T5 (Negative control) Distilled Water
T6 (Positive control) Streptomycin sulfate

Preparation of the paper disc

Whatman filter paper No. 1 was used as the paper disc with the use of paper

puncher. The paper discs were sterilized in autoclave at 121◦C (15psi) for 15 minutes.

Protectant test

The paper discs were soaked in the bacterial suspension. Then the sterile plate

with MH agar was swabbed with 0.1 ml of the different extracts. The paper discs were

seeded equidistantly in the plates together with the control. The plates were stored at
 18

room temperature and zones of bacterial colonization were measured using a Vernier

caliper after 12 and 24 hours of incubation.

Eradicant test

The eradicant test was adapted from the work of Valentino et al. (2015). The

paper discs were soaked in the different extracts. Then, the sterile plates with MH agar

was poured and spread with 0.1 mL of the bacterial suspension using a T-rod. The discs

were soaked in each treatment and were seeded equidistantly in the plates. The plates

were incubated within 24 hours at room temperature. The zones of inhibition were

measured using Vernier caliper at 12 and 24 hours of incubation.

Sub-study 3. Determination of Antioxidant as Radical Scavenging Activity of Fungi (A.

niger and R. pusillus) Associated with Vermicast

The DPPH radical scavenging activity of ethanolic extracts and fungal spent

of fungi A. niger and R. pusillus were sent and analyzed at the Chemistry Laboratory of

Center for Natural Sciences at St. Mary’s University, Bayombong, Nueva Vizcaya

following the procedure described by Kolak et al. (2006).

DPPH radical scavenging assay

The extracts were dissolved in methanol to a final concentration of 500 ppm.

A 0.1 mM DDPH in methanol was freshly prepared by diluting 1 mL DPPH stock

solution (3.49 mg DPPH in 10 ml methanol) to 100 mL methanol. Then, 1 ml of the

extracts and 4 mL of DPPH solution was mixed and incubated in the dark at 37 o C for 30

minutes. Triplicate tests were done in each extracts. The absorbance reading was
 19

monitored at 517 nm using UV- Vis spectrophotometer (APEL-100) and the ability to

scavenge the DPPH radical will be calculated using the equation below:

% DPPH scavenging effect = [ (Acontrol-Asample)/ Acontrol] x l00

Where Acontrol is the absorbance of the control, which is the DPPH solution

without the extract, the Asample is the absorbance of the test sample containing the mixture

of DPPH and the ethanol and culture spent extract. The synthetic antioxidant cathechin

was used as positive control.

Data Gathered

The following data gathered were:

1. The presence of phytochemicals in ethanolic and fungal spent of fungi (A. niger and

R. pusillus) associated with vermicast which includes alkaloids, cardiac glycosides,

flavonoids, saponins, steroids, tannins, and terpenoids.

2. The zone of colonization in the antibacterial assay as protectant of A. niger and R.

pusillus in their ethanolic extract and fungal spent against S. aureus and E. coli at 12

and 24 hours of incubation.

3. The zone of inhibition in the antibacterial assay as eradicant of A. niger and R.

pusillus in their ethanolic extract and fungal spent against S. aureus and E. coli at 12

and 24 hours of incubation.

4. The DPPH radical scavenging activity of the antioxidant activity of ethanolic and

fungal spent of fungi (A. niger and R. pusillus) associated with vermicast.

Statistical Analysis
 20

This study used Completely Randomized Design (CRD) and data were

analyzed using Analysis of Variance (ANOVA) and means were compared using

Multiple Duncan Range Test (DMRT) at 5% level of significance.

RESULTS AND DISCUSSION

This study was conducted to determine the mycochemical constituents of

Aspergillus niger and Rhizomucor pusillus ethanolic extracts and fungal spent.

Antibacterial activity as protectant and eradicant against Staphylococcus aureus and

Escherichia coli and their ability to scavenge free radicals or antioxidant property were

also evaluated.

Sub Study 1. Screening of Mycochemical Constituents of Fungi (A. niger and R.

pusillus) Associated with Vermicast

Fungi are a promising source of novel bioactive compounds as lead structures for

medicine and plant protection (Duarte et al., 2012). These compounds are responsible for

different medicinal properties of the extracts. Mycochemical screening of A. niger and R.

pusillus ethanolic extracts and fungal spent revealed the presence of flavonoids through

the presence of yellow coloration (Appendix Figure 5A and 5C for A. niger ethanolic

extract and fungal spent, respectively and Appendix Figure 5B and 5D for R. pusillus

ethanolic extract and fungal spent, respectively) and terpenoids indicated by the
 21

formation of reddish brown in the interface (Appendix Figure 5E and 5G for A. niger

ethanolic extract and fungal spent, respectively and Appendix Figure 5F and 5H for R.

pusillus ethanolic extract and fungal spent, respectively). Presence of tannins was only

detected in the A. niger ethanolic extract (Appendix Figure 5I) as the formation of blue-

black coloration was observed. In addition, formation of the white precipitate or turbidity

was observed only in R. pusillus ethanolic extract for the presence of alkaloids (Appendix

Figure 5J). All these are shown in Table 2.

Table 2. Bioactive components of the ethanolic extracts and fungal spent of fungi (A. niger
and R. pusillus) associated with vermicast
FUNGAL EXTRACTS
BIOACTIVE A. niger R. pusillus
COMPONENTS ETHANOLI FUNGAL ETHANOLIC FUNGAL
C SPENT EXTRACT SPENT
EXTRACT
Alkaloids - - + -
Cardiac glycosides - - - -
Flavonoids + + + +
Saponins - - - -
Steroids - - - +
Tannins + - - -
Terpenoids + + + +
*(+) determines the presence of bioactive compounds; (-) absence of bioactive compounds

This coincides with the studies of Madhusudan and Mishra, (2017); Murthy et al.

(2011), wherein the presence of terpenoids was detected in A. niger and flavonoids in

Mucor sp., respectively. Similarly, findings of Yadav et al. (2014), showed the

phytochemical constituents of different endophytic fungi A. niger, and A. flavus

contained flavonoids and terpenes. Also, Ladoh et al. (2015), revealed that fungi of

genus Aspergillus, Penicillium, Trichoderma and Fusarium contains flavonoids,

anthroquinones, tannins, phenols, coumarins and terpenoids.

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Alkaloids are large and structurally diverse group of compounds that have

served as scaffolds for important antibacterial drugs (Cushnie et al., 2014). Alkaloids

have been reported as powerful poison and many alkaloids derived from medicinal

plants show biological activities like anti-inflammatory (Augusto et al., 2011),

antimicrobial (Benbott et al., 2012), cytotoxicity and pharmacological effects (Thite et

al., 2013). Alkaloids show bioactivity against Gram-positive bacteria and cytotoxicity

against leukemia (Omar et al., 1992).

Flavonoid are a group of natural compounds with variable phenolic structures and

are found in plants and are known to be synthesized by plants in response to microbial

infection (Du et al., 2011). It has anti-oxidative, free radical scavenging capacity,

coronary heart disease prevention and anti-cancer activity (Yao et al., 2004). Their

activity is probably due to their ability to complex with extracellular and soluble proteins

and to complex with bacterial cell wall (Marjorie, 1996).

Terpenoids accounts for the major class of secondary metabolites produced by

plants (Prakash, 2017). Several terpenoids are biologically active and are exploited in the

fight against cancer, malaria, inflammation, and a variety of infectious diseases.

Nonetheless, some compounds of this group showed toxic effects causing gastrointestinal

problems or central nervous system manifestations among others (Mbaveng et al., 2014). 

Also, terpenes are reported to be the main chemical constituents responsible for reducing

lipid peroxidation and hence act as primary and secondary antioxidants (Gulcin, 2006;

Hajdu et al., 2007).

Tannins (polyphenols with widely varying chemistry) are one of the major

phytochemicals found in many higher plants. They are useful as an anti-inflammatory

 23

agent and in the treatment of burns and other wounds based on their anti-hemorrhagic and

antiseptic potentials. In particular, tannins-rich remedies are used as antihelmintics

(Ketzis et al., 2006), antioxidants (Koleckar et al., 2008).

Sub-study 2. Determination of Antibacterial Activity as Protectant and Eradicant of

Fungi (A. niger and R. pusillus) Associated with Vermicast

Protectant

At 12 and 24 hours of incubation, the least zone of colonization of S. aureus and

E. coli were recorded in plates treated with A. niger ethanolic extract (9.64 mm and 12.35

mm for S. aureus; 9.60 mm and 12.37 mm for E. coli) and R. pusillus ethanolic extracts

(0.00 mm and 13.03 mm for S. aureus: 19.35 mm and 32.44 mm for E. coli) (Figure 1A,

B, D and E). In addition, plates treated with A. niger spent shows minimum colonization

(10.18 mm for S. aureus and 12.75 mm for E. coli) but colonized the whole plate after 24

hours of incubation for both test organisms (Figure 1C and F) as presented in Table 3.

Statistical analysis revealed that R. pusillus ethanolic extract was comparable with

that of the Streptomycin sulfate (positive control) for S. aureus after 12 hours of

incubation. Meanwhile, fungal ethanolic extracts were also significantly lower as

compared to the spent and distilled water, therefore the inhibitory activity of A. niger and

R. pusillus ethanolic extracts against E. coli and S. aureus indicates potential as protectant

agent.
 24

Table 3. Antibacterial activity as protectant of fungal extracts against S. aureus and E.

coli after 12 and 24 hours of incubation
TREATMENTS S. aureus (ZONE OF E coli (ZONE OF
COLONIZATION) COLONIZATION)
12HOURS 24 HOURS 12 HOURS 24HOURS
A. niger ethanolic extract 9.64b 12.35b 9.60c 12.37c
c b b
R. pusillus ethanolic extract .00 13.03 19.35 32.44b
A. niger spent 10.18b 32.42a 12.75c 32.42a
a a a
R. pusillus spent 32.42 32.42 32.42 32.42a
Distilled water 32.42a 32.42a 32.42a 32.42a
c c d
Streptomycin sulfate 0.00 0.00 0.00 0.00d
*Treatments with the same letter are not significantly different with each other at 5% level of
significance
 25

C D

A B C

D
D E
Figure 1. Antibacterial activity as protectant of fungal extracts treated
F with (A) A. niger
ethanolic extract; (B) R. pusillus ethanolic extract; (C) A. niger fungal spent
against S. aureus; (D) A. niger ethanolic extract; R. pusillus ethanolic extract;
and (F) A. niger fungal spent against E. coli

Eradicant

Table 4 presents the antibacterial activity as eradicant which revealed the absence

of zone of inhibition discs with A. niger and R. pusillus spent both for S. aureus and E.

coli. Whereas, zone of inhibition at 12 and 24 hours of incubation against the bacterial

pathogens were recorded in ethanolic extract of A. niger (9.43 mm and 9.28 mm in S.

aureus) and R. pusillus (11.94 mm and 11.75 mm in S. aureus; 9.40 mm and 9.08 mm E.

coli) (Figure 2A and B).

Table 4. Antibacterial activity as eradicant of fungal extracts against S. aureus and E.

coli after 12 and 24 hours of incubation
 26

TREATMENTS S. aureus (ZONE OF E coli (ZONE OF

INHIBITION) INHIBITION)
12HOURS 24 HOURS 12 HOURS 24HOURS
A. niger ethanolic extract 9.43b 9.28b 0.00c 0.00c
b
R. pusillus ethanolic extract 11.94 11.75b 9.40 b
9.08b
A. niger spent 0.00c 0.00c 0.00c 0.00c
c
R. pusillus spent 0.00 0.00c 0.00 c
0.00c
Distilled water 0.00c 0.00c 0.00c 0.00c
a
Streptomycin sulfate 23.99 23.86a 26.34 a
25.88a
*Treatments with the same letter are not significantly different with each other at 5% level of
significance

Statistical analysis revealed that the zone of inhibition produced by A. niger and

R. pusillus spent were comparable to the negative control which indicates the lack of

antibacterial activity in 12 and 24 hours of incubation against S. aureus and E. coli.

Whereas, ethanolic extracts of A. niger and R. pusillus were significantly higher than

negative control, which signifies their antibacterial activity against S. aureus and E. coli,

however it is still incomparable with the positive control.

 27

+
1 2 +

2 1
2 1
3

A B

Figure 2. Antibacterial activity as eradicant (A) against S. aureus; and (B) against E.
coli: (1) A. niger ethanolic extract; (B) R. pusillus ethanolic extract; (+)
Streptomycin sulfate

This coincides with the study of Madsuhudan and Mishra (2017), that A. niger

crude extract, contains significant pathogen inhibiting compounds in both Gram positive

and Gram negative bacterial strains. In addition, according to Durairaj et al. (2015), A.

niger exhibited excellent antimicrobial activity against both bacterial species and fungi

thus their potential for external used as antibacterial agent in surface coatings on various
 28

substrates to prevent microorganisms from attaching, colonizing, spreading and forming

biofilms in indwelling medical devices. The same results were obtained by Kalyani and

Hemalatha (2017).

The ability of both fungal extracts to prevent bacteria from colonizing and their

inhibitory activity might be due to the bioactive compounds present in the extracts like

alkaloids and flavonoids. Results link with some previous findings including Mishra et al.

(2013) in which flavonoids are known to be synthesized by plants in response to

microbial infection; thus it should not be surprising that they have been found in vitro to

be effective antimicrobial substances against a wide array of microorganisms. Flavonoid

rich plant extracts from different species have been reported to possess antibacterial

activity. Several flavonoids including apigenin, galangin, flavone and

flavonolglycosides, isoflavones, flavanones and chalcones have been shown to possess

potent antibacterial (Cushnie & Lamb, 2005). Similar findings from the study of Cushnie

et al. (2014), stated that alkaloids have a power truck record as drug and scaffold

substructures in modern antibacterial therapy.

Sub-study 3. Determination of Antioxidant as Radical Scavenging Activity of Fungi

(A. niger and R. pusillus) Associated with Vermicast

The field of free radical chemistry is gaining more attention these days. Free

radicals are reactive oxygen and nitrogen species which are generated by various

physiological processes in the body. Uncontrolled generation of free radicals leads to

attack on membrane lipids, proteins, enzymes and DNA causing oxidative stress and
 29

ultimately cell death (Gulcin, 2007). Free radical scavenging property of antioxidants

delays or inhibits cellular damage (Halliwell, 1995). Table 5 presents the radical

scavenging activity (RSA) of the ethanol extracts of A. niger, R. pusillus and their fungal

spent.

Among the four extracts, A. niger ethanolic extract was recorded as the treatment

having the highest ability to scavenge free radicals with 73.03% followed by R. pusillus

ethanolic extract with 71.86% and A. niger spent and lastly R. pusillus spent with the

percentage of 42.50% and 29.96%, respectively. In the previous study, it has been

reported by Yadav et al. (2014), that Aspergillus niger strain showed the highest

antioxidant activity ranging from 50% to 80%. In addition, findings of Hameed et al.

(2017), showed that Mucor strains proved to be rich sources of antioxidants and

secondary metabolites, which could be used in the development of nutraceutical and

natural antioxidants.

Table 5. Free radical scavenging activity (%) of fungi A. niger and R. pusillus in their
ethanolic extract and fungal spent
TREATMENTS % Radical Scavenging Activity
Aspergillus niger ethanolic extract 73.03%
Rhizomucor pusillus ethanolic extract 71.86%
A. niger spent 45.20%
R. pusillus spent 29.96%
Cathecin (Positive control) Ethanol/Spent 72.86% / 76.06%
*Abs DPPH-1.874; Wavelength 517 nm using Spectrum lab 752s UV Vis spectrophotometer;
Concentration: 1000 nm
Also, from the study of Nitya et al. (2011), the antioxidant activity of endophytic

fungi isolated from Lobelia nicotianifolia, all the fractions showed significantly higher

inhibition percentage (stronger hydrogen – donating ability). Aspergillus extract showed

highest total antioxidant activity just equivalent value to the positive control being used
 30

which is the ascorbic acid. Meanwhile, from the study of Yen and Lee (1996), reported

that Aspergillus sp. possessed a high antioxidant up to 78% just like Penicillium.

Ishikawa (1995), reported that some fungi, especially Penicillium and Aspergillus, might

produce antioxidants. This was confirmed by the results of this study.

Potential of the fungal extracts to scavenge free radicals are in correlation with

their secondary metabolites present like terpenoids, tannins and flavonoids. Recent

studies have shown that these compounds have anti-oxidative property. This coincides

with the findings of Gulcin, (2007) and Hajdu et al. (2007), that terpenes are reported to

be the main chemical constituents responsible for reducing lipid peroxidation and hence

act as primary and secondary antioxidants. Flavonoids is also a compound which exhibits

anti-oxidative, free radical scavenging (Yao et al., 2004). In addition, tannins also have

antioxidants property as reported by Koleckar et al. (2008).

SUMMARY, CONCLUSION AND RECOMMENDATION

Summary
 31

This study aimed to determine the mycochemical constituents and biological

activities of fungi associated with vermicast; Aspergillus niger, Rhizomucor pusillus and

their fungal spent.

Mycochemical screening of A. niger and R. pusillus ethanolic extract and fungal

spent revealed the presence of flavonoids and terpenoids Other bioactive compounds like

tannins and alkaloids were only detected in A. niger and R. pusillus ethanolic extracts,

respectively.

For the antibacterial activity as protectant, at 12 hours and 24 hours of incubation,

the least zone of colonization of Staphylococcys aureus and Escherichia coli were

recorded in plates treated with A. niger ethanolic extract and R. pusillus ethanolic

extracts. On the other hand, antibacterial activity as eradicant revealed the absence of

zone of inhibition disks with A. niger and R. pusillus spent both for S. aureus and E. coli.

Whereas, zone of inhibition at 12 and 24 hours of incubation against the bacterial

pathogens were recorded in ethanolic extract of A. niger and R. pusillus.

For the DPPH radical scavenging activity, among the four extracts, A. niger

ethanolic extract was recorded as the treatment having the highest ability to scavenge free

radicals.

Conclusion

Based on the results of the study the following are being concluded:
 32

1. A. niger and R. pusillus contains several bioactive compounds like alkaloids,

terpenoids, flavonoids and tannins.

2. R. pusillus ethanolic extract and A. niger ethanolic extract possess the least zone of

colonization which means that they can be a good source of antibacterial as

protectant agent. Moreover, aforementioned treatments have the potential to be an

antibacterial as eradicant agent with the highest zone of inhibition after 12 and 24

hours of incubation.

3. A. niger and R. pusillus exhibit high DPPH radical scavenging activity.

Recommendations

Based on the results of the study, the following recommendations are suggested:

1. To estimate the total phenolic content of the fungi associated with vermicast and

their fungal spent.

2. Further studies should be conducted in elucidating their antifungal property and

antibacterial property with the use of other pathogenic bacteria and fungi.

3. The use of other extraction in evaluating their phytochemical constituents and

their biological activities.

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 39

APPENDICES
 40

APPENDIX A

Preparation of 0.5 McFarland Standard (Archarya, 2016)

1. Prepare a 0.05 ml of 1% solution of anhydrous barium chloride (BaCl2).

2. Prepare a 9.95 ml of 1% solution of sulfuric acid (H4SO4).

3. Combine and completely mix the barium chloride and sulfuric acid solutions

to form a turbid suspension and BaSO4.

4. Place the resulting mixture in a foil-covered screw-cap tube.

5. Store the McFarland standard at room temperature (25̊ C) when not in use.

McFarland standard density solution will precipitate and clump over time, and

it needs vigorous vortexing before each use. Mark the tube to indicate the

level of fluid, and check before use to be sure that evaporation has not

occurred.

6. Prepare a fresh standard solution every six months.

 41

APPENDIX B

Statistical Analysis

Appendix Table 1. One way analysis of variance of the antibacterial activity as

protectant of the different extracts of fungi associated with
vermicast against S. aureus after 12 hours of incubation
SUM OF DF MEAN F SIGNIFICANCE
SQUARES SQUARE
SAPRO1 Between 34369.568 5 34385.021 5337. 920 .000
Groups
2
Within 15.453 12 1.288
Groups
Total 34385.021 17 6873.914 5337.920 .000

Appendix Table 2. One way analysis of variance of the antibacterial activity as

protectant of the different extracts of fungi associated with
vermicast against S. aureus after 24 hours of incubation
SUM OF DF MEAN F SIGNIFICANCE
SQUARES SQUARE
SAPRO24 Between 35799.646 5 7159.929 421.976 .000
Groups
Within 203.612 12 16.968
Groups
Total 36003.257 17

Appendix Table 3. One way analysis of variance of the antibacterial activity as

protectant of the different extracts of fungi associated with
vermicast against E. coli after 12 hours of incubation
SUM OF DF MEAN F SIGNIFICANCE
SQUARES SQUARE
ECPRO12 Between 30737.688 5 6147.538 639.825 .000
Groups
Within 115.298 12 9.608
Groups
Total 30852.985 17
 42

Appendix Table 4. One way analysis of variance of the antibacterial activity as

protectant of the different extracts of fungi associated with
vermicast against E. coli after 24 hours of incubation
SUM OF DF MEAN F SIGNIFICANCE
SQUARES SQUARE
ECPRO24 Between 32095.800 5 6419.160 191.911 .000
Groups
Within 401.385 12 33.449
Groups
Total 32497.185 17

Appendix Table 5. One way analysis of variance of the antibacterial activity as eradicant
of the different extracts of fungi associated with vermicast against S.
aureus after 12 hours of incubation
SUM OF DF MEAN F SIGNIFICANCE
SQUARES SQUARE
SAERA12 Between 1393.120 5 278.624 48.283 .000
Groups
Within 69.248 12 5.771
Groups
Total 1462.933 17

Appendix Table 6. One way analysis of variance of the antibacterial activity as eradicant
of the different extracts of fungi associated with vermicast against S.
aureus after 24 hours of incubation
SUM OF DF MEAN F SIGNIFICANCE
SQUARES SQUARE
SAERA24 Between 1372.728 5 274.546 46.269 .000
Groups
Within 71.205 12 5.934
Groups
Total 1443.933 17

Appendix Table 7. One way analysis of variance of the antibacterial activity as eradicant
of the different extracts of fungi associated with vermicast against E.
coli after 12 hours of incubation
SUM OF DF MEAN F SIGNIFICANCE
SQUARES SQUARE
ECERA12 Between 1707.385 5 341.477 209.644 .000
Groups
Within 19.546 12 1.629
Groups
Total 1726.931 17
 43

Appendix Table 8. One way analysis of variance of the antibacterial activity as eradicant
of the different extracts of fungi associated with vermicast against E.
coli after 24 hours of incubation
SUM OF DF MEAN F SIGNIFICANCE
SQUARES SQUARE
ECERA24 Between 1645.995 5 329.199 306.439 .000
Groups
Within 12.891 12 1.074
Groups
Total 1658.887 17 .000
 44

APPENDIX C

Laboratory results of the fungal identification

Appendix Figure 1. Identification of the fungal isolates from

vermicast
 45

Appendix D

Laboratory result of the re-identification of one fungal isolate

Appendix Figure 2. Re-identification of one fungal isolate

 46

Appendix E

Laboratory Result of the DPPH Radical Scavenging Activity of the fungal ethanolic
extracts

Appendix Figure 4. DPPH RSA of the fungal

ethanolic extracts
 47

Appendix F

Laboratory result of the DPPH Radical Scavenging Activity

Appendix Figure 4. DPPH RSA of the fungal spent

 48

Appendix G

Results of the phytochemical screening of ethanolic extract and fungal spent of fungi
associated with vermicast
 49

A B C

D E F
 50

H I
G

J
Appendix Figure 5. Positive result of flavonoids in (A) A. niger ethanolic extract; (B) R. pusillus
ethanolic extract; (C) A. niger fungal spent; (D) R. pusillus fungal spent;
terpenoids in (E) A. niger ethanolic extract; (F) R. pusillus fungal spent; (G)
A. niger fungal spent; (H) R. pusillus fungal spent; tannins in (I) A. niger
ethanolic extract; and alkaloids in (J) R. pusillus ethanolic extract

Appendix H

Antibacterial activity as protectant against S. aureus and E. coli

 51

A B C

D E F

Figure 6. Antibacterial activity as protectant treated with (A) R. pusillus spent; (B) distilled
water; (C) Streptomycin sulfate against S. aureus; (D) R. pusillus spent; distilled
water; and (F) Streptomycin sulfate against E. coli

Appendix I
 52

Antibacterial activity as eradicant against S. aureus and E. coli

5 3 4 3
1 4

1 2 6
5 3 2

A B C

6 4 3
6 5

1 4
2 1
5 6

D E F

5
1 5 2 6
3 2

3
2 6 4 4
1
H I
G

Appendix Figure 11. Antibacterial activity as eradicant against S. sureus (A, B, C, D, and E)
and E. coli (F, G, H, I, and J) of discs treated with (1) A. niger
ethanolic extract; (2) R. pusillus ethanolic extract; (3) A. niger fungal
spent; (4) R. pusillus fungal spent; (5) distilled water; and (6)
Streptomycin sulfate