Biology and Medicine

Goutam et al., Biol Med (Aligarh) 2016, 8:7

DOI: 10.4172/0974-8369.1000349

Research Article

OMICS International

In vitro Potential of Endophytic Fungus Aspergillus terrus (JAS-2)

Associated with Achyranthus aspera and Study on its Culture Conditions
Jyoti Goutam1*, Shweta Singh1, Ravindra Nath Kharwar1 and Vijayakumar Ramarai2
1Laborotary

of Mycopathology, Department of Botany, Centre of advance study in Botany, Banaras Hindu University, Varanasi, Uttar Pradesh 221005, India

2Department

of Microbiology, Sri Ramachandra Medical College & Research Institute, Porur, Chennai, Tamil Nadu 600116, India

*Corresponding

author: Jyoti Goutam, Centre of advance study in botany, Banaras Hindu University, Varanasi, Uttar Pradesh 221005, India, Tel: 919452917944; Email: jyoti23biotech@gmail.com

Received date: August 30, 2016; Accepted date: October 10, 2016; Published date: October 17, 2016
Copyright: 2016 Goutam J, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution and reproduction in any medium, provided the original author and source are credited.

Abstract
The present study explores the efficacy of an endophytic fungus from symbiotic plant Achyranthus aspera, an
herb of medicinal importance. Considering limitation of secreted fungal metabolites in terms of biologically active
molecules; an endeavour was made to increase the amount of secreted active product. Enhanced secretion of
active compound was observed by optimizing different parameter of culture conditions. The fungal culture was
isolated from stem of Achyranthus aspera and taxonomically identified as Aspergillus terreus. While analysing its
different in vitro potential, culture metabolites showed antibacterial, antifungal and anti-oxidant activity. In order to
increase the yield of compound, culture was optimized for different parameters such as carbon and nitrogen sources
and extracting solvent. All the optimization was performed based on % inhibition of bacterial growth when
challenged with 10 g/l metabolite concentration. Among different media used, potato dextrose broth (PDB) and
sabouraud's dextrose broth (SDB) proven to be better media for growth of fungus as well as metabolites production.
1% yeast extract and 4% dextrose resulted in higher cell inhibition. Ethyl acetate served as good extracting solvent.

Keywords: Endophytes; Achyranthus aspera; Antibacterial activity;

Antifungal activity; Antioxidant activity

Introduction
Endophytes are the cryptic factories of diversified metabolites inside
plant, synthesising significant compounds of unknown or known
medicinal importance. As they are endowed with multitude diversity
structure, their physiological functions [1] are being continuously
chased for bioactive principles. Study on this endophytic mycoflora
revealed that endophytes could be alternative form of drug and by
optimizing the culture conditions; activity of bioactive compounds
could be enhanced. Endophytically derived compounds such as taxol,
cryptocin and isopestacin, justified their significance with respect to
their application in human being [2]. But the increased incidence of
diseases caused by various bacterial and fungal pathogens forces us to
continue our search for newer biometabolites. Apart from identifying
newer metabolites, it is important to unveil the structure of already
identified biometabolite such as taxol, [3] which revealed the
production of host metabolite (host mimetic) from its endophytes. This
led to the discovery of different chemical structures such as piperine
[4] etc. Since the active molecules represent only 0.0001% or 1 ppm of
total biomass [5], the primary step should be increasing the quantity of
bioactive molecules present in metabolites. There are number of
reports are available in direction of conditioning cultural parameters
by altering the fermentation conditions such as media, pH and
temperature.
In the present study we have demonstrated the optimum culture
conditions required for maximum secretion of antibacterial
compounds by the endophytic fungal genus JAS-2, which was isolated
from Achyranthus aspera, commonly known as latzeera, chichida,
apamarga. Considering multiple medicinal properties of this plant

Biol Med (Aligarh), an open access journal

ISSN: 0974-8369

such as cytotoxity [6] and wound healing [7], the plant was chosen for
isolating biologically active endophytic fungi. Since crude metabolite
consists only a meagre amount of bioactive product, it is extremely
important to increase the amount of active compound. This could be
achieved successfully by optimizing the culture conditions. Globally, it
is required to work on diverse fungal metabolomics and its different
biotechnological aspects, which would aid in improving the quality
and quantity of bioactive molecules.

Materials and Methods

Isolation and characterization of endophytic Fungi from medicinal
plant Achyranthus aspera.

Sampling
Healthy samples (leaf, stem and root) were collected in sterile
polythene bags from botanical garden, at Department of Botany,
Banaras Hindu University, Varanasi (25.5 N 82.9E, elevation 279 ft/85
m) India and brought to the laboratory in an icebox. Samples were
stored at 4C after collection until use.

Surface sterilisation healthy tissues and isolation of

endophytic fungi
In order to screen out only fungal endophytes a modified
methodology of Dobranic [8] and Schulz [9] was adapted. Briefly,
samples were immersed sequentially in 75% ethanol for 4-5 min
followed by aqueous 2% sodium hypochlorite for 10 min and then
rinsed in 75% ethanol for 2min or as per requirement of stem/root/leaf
tissue. The exposed time length with sterilent could be decided based
on tissues strength. Tissues were washed in double distilled sterile
water to remove chemical and excess moisture and then blotted on

Volume 8 Issue 7 1000349

Citation:

Goutam J, Singh S, Kharwar RN, Ramarai V (2016) In vitro Potential of Endophytic Fungus Aspergillus terrus (JAS-2) Associated with
Achyranthus aspera and Study on its Culture Conditions. Biol Med (Aligarh) 8: 349. doi:10.4172/0974-8369.1000349

Page 2 of 7
sterilized tissue paper. Samples were cut in the dimensions of 0.5 cm
0.5 cm. Six to seven segments of plant tissues were placed on potato
dextrose agar (PDA) plate with streptomycin (100 g/ml), and
incubated in a BOD incubator for 21 days at 26 2C. In order to
ensure proper surface sterilization, the sterilization protocol was
validated using leaf imprint method [9]. The plotted segments were
monitored on every alternate day.

Identification of endophyte Aspergillus terreus (JAS-2)

Isolated endophytic fungus JAS-2 was maintained in potato
dextrose agar (PDA) and incubated in BOD incubator. On the basis of
macroscopic and microscopical characteristics, the fungus was
identified with the help of standard taxonomic manuals [10,11]. The
microscopic identification was performed with the help of picture was
observed by aniline blue stained slide.

Solvent optimization
Aspergillus terreus JAS-2 was grown at 26C for 21days to complete
its fermentation period. Equal volume of culture broth was extracted
individually with three different organic solvents such as hexane,
chloroform and ethyl acetate. Concentrated crude extract was checked
against different bacterial and fungal pathogens to observe its ability to
inhibit the growth by MTT assay.

Evaluation of basal media against bacterial pathogens

For selecting the best suitable growth medium quantitatively and
qualitatively, four different culture media, i.e. potato dextrose broth
(PDB), sabourauds dextrose broth (SDB), malt extract broth (MEB)
and czapek dox broth (CZB) were used. The potential of bioactive
compound extracted from Aspergillus terreus was assessed by
measuring activity of crude extract against targeted bacterial
pathogens. Tools to measure inhibitory effect of bacterial pathogen
were accessed by MTT assay. The accumulated biomass and bioactive
metabolites were measured separately after drying at 70C until a
constant weight was obtained.

Effect of carbon and nitrogen sources on biomass and

bioactive compound
To enhance the activity of present compound in crude extract,
carbon and nitrogen contents of sabourauds dextrose broth (SDB) had
been replaced with different carbon (starch, sucrose, dextrose and
maltose) and nitrogen sources (yeast extract, peptone, beef extract,
urea extract and casein) 4 gm/100 ml and 1 gm/100 ml respectively.
Each 250 ml flask containing 100 ml of SDB supplemented with 4%
carbon and 1% nitrogen sources and SDB without any modification
used as control.

Cultivation of culture and extraction of metabolites

The fresh mycelium grown on PDA plates were inoculated in
Erlenmeyer flask of 1000 ml containing 500 ml of PDB medium and
incubated for 21 days at 26C in BOD. After 21 days fermented broth
of endophyte was filtered and extracted with ethyl acetate. Extracted
compounds from solvent were further condensed in vacuum by using
rota evaporator.

Staphylococcus aureus (IMS/GN7), Aeromonas hydrophila (IMS/

GN11), Salmonella typhi (MTCC 3216), Shigella flexneri (IMS/GN1),
Escherichia coli (ATCC 25922) and Enterococcus faecalis (ATCC
25923). The fungal pathogens used were Fusarium oxysporum,
Bipolaris soronkinana, Aspergillus flavus, Aspergillus niger and
Alternaria alternata. All bacterial pathogens were procured from
Institute of Medical Science (IMS), and fungal pathogens from
Institute of Agricultural Sciences (IAS), Banaras Hindu University, and
Varanasi respectively.
In vitro antibacterial bioassay by disk diffusion method: The crude
extract from Aspergillus terreus JAS-2 was screened for antibacterial
activity by disc diffusion method [12]. Bacterial suspension was made
in autoclaved distilled water and bacterial lawn culture was prepared
on Mueller Hinton agar plate with sterile cotton swab. Sterile disc
impregnated with crude extract at the concentration of 1.0 mg/disc,
were placed on the lawn culture and observed for the zone of
inhibition after 48 hours. Zone of inhibition was measured using ruler.
Ciprofloxacin (5.0 g/disc) and methanol were used as positive and
negative control respectively.
In vitro antifungal activity: Crude metabolites evaluated for
antifungal activity, by modified method by Ranware et al. [13]. Briefly,
crude metabolite was dissolved in 1% DMSO to get a concentration of
1.0 mg/ml and added to 5 ml of sterile potato dextrose broth (PDB) in
test tube. Each pathogenic fungal strain having 5 mm spores cake were
inoculated in a test tube and incubated. A separate control was
prepared, inoculated with pathogenic fungi without any metabolite. All
cultures were incubated for 7 to 15 days at temperature of 26C. After
15 days, grown biomass was dried at constant temperature at 70C.
Inhibitory effect of metabolite grown under the influence of
metabolites was calculated using formula.
Fungalgrowth

C Fungalgrowthaftertreatment100=%Reductioninfungalgrowth
Fungalgrowth c Control

In vitro antioxidant assay by DPPH free radical scavenging

activity

Crude metabolites of Aspergillus terreus (JAS-2) was evaluated for

antioxidant assay by using DPPH (2, 2-diphyenyl 2 picryl hydrazyl)
free radical scavenging method [14]. Since the free radical scavenging
activity of any compound is considered to reduce the stable DPPH
radical, in order to measure anti-oxidant activity a stock solution of
DPPH was prepared (100 mg/ml) with methanol. Decrease in
absorbance of DPPH while increasing the concentration of crude
extract was recorded. IC-50 values were calculated by taking DPPH as
negative control. Percentage inhibition activity was calculated by the
given formula;
[(A0A1)/A0] 100,
Where A0 is the absorbance of the control, and A1 is the absorbance
of the extract and DPPH. The inhibition curves were prepared and
IC50 value calculated. The free radical scavenging activity of the
extract, which is based on the scavenging activity of the stable DPPH
free radical, was determined by free radical scavenging method. Crude
extract (0.1 ml) was added to 3 ml of a 0.004% methanol solution of
DPPH. Absorbance at 517 nm was determined after 30 min of
incubation period.

Determination of antimicrobial activity of Aspergillus terreus JAS-2:

Test strains: Different bacterial and fungal pathogens were used as test
organisms for antimicrobial assay. The bacterial cultures used were,

Biol Med (Aligarh), an open access journal

ISSN: 0974-8369

Volume 8 Issue 7 1000349

Citation:

Goutam J, Singh S, Kharwar RN, Ramarai V (2016) In vitro Potential of Endophytic Fungus Aspergillus terrus (JAS-2) Associated with
Achyranthus aspera and Study on its Culture Conditions. Biol Med (Aligarh) 8: 349. doi:10.4172/0974-8369.1000349

Page 3 of 7

Percentage cell inhibition through MTT assay in each culture

conditions
MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium
Bromide) assay was introduced by Mossman [13] as a quantitative
measurement of mammalian cell survival and their proliferation, and
was subsequently used to detect the viability of bacteria under adverse
condition. MTT is a yellow tetrazolium salt that is converted into blue
formazone by dehydrogenase of live cell. The principle of present assay
is the amount of formazone produced is directly proportional to
number of live cell. The amount of formazan production, which is
detected by measuring OD at 570 nm, was directly proportional to the
initial bacterial concentration and the duration of incubation at 37C.

screening of antibacterial test by disk diffusion revealed that S. aureus,

A. hydrophila and E. faecalis were sensitive to crude metabolite of
JAS-2 (Figure 2). Apart from bacteria, fungal isolates such as
Alternaria alternata, Bipolaris scrokmiana (MTCC 11857), Fusarium
oxysporum, Phytopthora drchlerii var cajai and Aspergillus were also
found sensitive for JAS-2 crude metabolites which is tabulated (Table
1). The occurrence of Aspergillus and its different species as
endophytes have been reported earlier as an antibacterial, antifungal
compounds [17,18]. Apart from Aspergillus species, other genera of
endophytic fungi have also exhibited themselves as antibacterial,
antifungal and anti-oxidant secreting molecules. It proves that plants
are the platform in which endophytes may flourish with its unique
biological properties beneficial to human being and plants.

The equal amount (10 g) of crude extract from different culture

conditions, were dissolved in DMSO (10 g/ml) and added to the
sterile water glass tubes (4 ml). 2 l of test bacterial culture was
inoculated in 200 l of sterile Mueller Hinton Broth (MHB). A
negative control was prepared by using test bacterium in MuellerHinton broth without any crude extract. An antibiotic 10 g/l was
used as positive control. After incubation period, 60 l MTT solution
prepared in phosphate buffer saline (PBS) was added to each tube and
incubated for 24 hour. Culture was centrifuged at 10,000 rpm for 5
minutes and absorbance was observed at 570 nm.
Calculation of % cell inhibition=
ODofBacterialGrowth C ODofBacterialgrowthwithtestsample100=%Cellviability
ODofBacterialgrowth c Control

Statistical analysis

Data reported is means of triplicate values activity for basal media;

optimum solvent, and each carbon and nitrogen sources were studied.
Value of data was expressed as mean S.D. from triplicate value. The
ANOVA test for unpaired observations between controls and
experimental samples for multiple comparisons was conducted to
evaluate statistical differences; p values of 0.05 or less were considered
statistically significant. The ANOVA test was performed using SPSS
(software package used for statistical analysis) version 16 software.

100 = %

Figure 1: Emergence of endophytic fungus (b, c) (JAS-2) from stem

of Achyranthus aspera (a) and its microscopic appearance (d).

Pathogenic Fungi

Inhibition (%)

Alternaria alternata

53.3

Fusarium oxysporum

30.3

Characterization of endophytic fungus

Bipolaris soronkiniana

47.7

Endophytic fungus A. terreus was isolated from stem of medicinal

plant Achyranthus aspera (Figure 1a) uprooted from botanical garden
BHU Varanasi in the month of June 2014. Initially, the fungal growth
was observed as white mycelia colonies on potato dextrose agar (PDA)
which turned to brownish yellow after few days (Figures 1b, c). On
microscopical observation, conidiophores apex change into globose
vesicles on which bi-seriate conidial heads showed the densely
attached conidia in chains (Figure 1d). Since it is a thermo tolerant
fungus, and its maximum growth reported at 45C-48C [15], isolation
was easy in the month of June.

Aspergilus flavus

27.3

Phytopthora sp

20.7

Clinical human pathogens

ZOI

S. aureus

12

S. typhi

Nil

E. faecalis

10

A. hydrophila

10

Screening for antibacterial anti fungal and antioxidant

activity

S. flexenei

Nil

E. coli

Nil

P. aureogenosa

Nil

Results and Discussions

With the passage of time Endophytic fungal group have been

proved themselves as one of the major source of antibacterial
compounds. Among different groups of endophytic fungi Aspergillus
sp and its telomorph contain a large number of species, which have
been exploited for various interesting bioactive products [16]. In-vitro

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Table 1: Antifungal Inhibition (%).

Volume 8 Issue 7 1000349

Citation:

Goutam J, Singh S, Kharwar RN, Ramarai V (2016) In vitro Potential of Endophytic Fungus Aspergillus terrus (JAS-2) Associated with
Achyranthus aspera and Study on its Culture Conditions. Biol Med (Aligarh) 8: 349. doi:10.4172/0974-8369.1000349

Page 4 of 7
Many similar results were reported earlier which correlates with our
present work. Endophytic fungus Emericella qaudrilineata [20] found
it IC-50 value of 2, DPPH scavenging at 400 g/ml.

Selection for organic solvent

Figure 2: Primary screening of antibacterial compound by disk

diffusion assay.

Determination of antioxidant activity

Free radical scavenging activity of crude metabolite was performed
as described by Marwah [12]. In this activity, a varying concentration
of crude metabolite dissolved in methanol ranges from 10-100 g/ml,
was used. Along with that, ascorbic acid (10 g/ml) and blank
methanol were taken as positive and negative control respectively.
There was gradual change of colour in increasing order of metabolite
concentration 10-100 g/ml (Figure 3a). Absorbance is significantly
correlated with the concentration of crude extract set in range of
concentrations, and graphical interpolation found IC-50 value at 31.4
g/ml concentration (Figure 3b). Although crude metabolites showed
lower antioxidant activity at (30 g/ml) as compared to positive control
ascorbic acid (5 g/ml). Extremely higher IC-50 value i.e. 953 g/ml
was reported from endophytes Phoma sp. [19] as compared to the
present study. This proves that, antioxidant nature of endophytic fungal
metabolites varies among different group of fungi.

Since, selection of suitable solvent system highly depends on the

specific nature of the bioactive compound being targeted. Different
solvents have ability to extract the bioactive compound from culture
broth. Ethyl acetate was recognized as best organic solvent which could
be used to extract the bioactive metabolites [21]. Among the three
organic solvent used, crude metabolite of JAS-2 extracted using ethyl
acetate showed maximum cell inhibition against A. hydrophila
(41.2%), S. aureus (45.4%), E. faecalis (27.3%) followed by chloroform
and hexane respectively (Figure 4 and Table 2). Hexane extract of
culture resulted in negligible activity against any pathogenic bacteria,
no colour change was observed in hexane with reference to negative
control (Deep purple) (Figure 4).

Figure 4: Assessment of coloured formazan produced during

solvent optimization.

Solvents

Figure 3a: Primary observation with the change in colour of DPPH

(Purple colour).

A.
A.
A.
hydrophila hydrophila hydrophila

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%
Inhibition

Ethyl
Acetate

0.498

0.501

0.511

0.503 0.00

41.2

Chloroform

0.614

0.605

0.601

0.607 0.00

11.6

Hexane

0.722

0.671

0.723

0.705 0.02

Positive
Control

0.298

0.299

0.245

0.281 0.02

61

Control

0.775

0.679

0.711

0.722 0.03

S. aureus

Figure 3b: Calculation of EC-50 value with graphical interpretation.

Mean SD

S. aureus

S. aureus

Mean SD

Ethyl
Acetate

0.361

0.328

0.351

0.347 0.01

45.4

Chloroform

0.556

0.558

0.555

0.556 0.00

12.5

Hexane

0.623

0.651

0.603

0.626 0.01

Positive
Control

0.311

0.315

0.281

0.302 0.01

52.3

Control

0.617

0.591

0.701

0.636 0.04

E. faecalis

E. faecalis

E. faecalis

Mean SD

Volume 8 Issue 7 1000349

Citation:

Goutam J, Singh S, Kharwar RN, Ramarai V (2016) In vitro Potential of Endophytic Fungus Aspergillus terrus (JAS-2) Associated with
Achyranthus aspera and Study on its Culture Conditions. Biol Med (Aligarh) 8: 349. doi:10.4172/0974-8369.1000349

Page 5 of 7
Ethyl
acetate

0.487

0.488

0.432

0.469 0.02

27.73

Chloroform

0.616

0.605

0.585

0.602 0.01

7.2

Hexane

0.678

0.611

0.648

0.646 0.02

0.4

Positive
Control

0.342

0.341

0.312

0.332 0.01

49

Control

0.617

0.681

0.651

0.650 0.02

Table 2: Inhibition (%) of bacterial growth by different crude

metabolite secreted by JAS-2 extracted with different organic solvent.
Data of % inhibition of bacterial pathogens were statistically analysed
by one way Anova were found statistically significant at 5% (P<0.05).

Impact of media in isolation of bioactive crude compounds

Medium optimization is considered as integral part of
biopharmaceutical industries with an approach to highest possible
product of interest. With respect to fungal physiology, it is important
for growth, building of primary metabolites and secondary
metabolites. Endophytic fungus Aspergillus terreus was grown in
different medium to find the best supporting medium. Best supporting
medium was calculated with the help of MTT dye measuring viability
of bacterial cells. This work deals with media conditioning
optimization, antibacterial activity by MTT assay so that false positive
and negative results related to antibacterial could be deleted. All four
basal media tested for the production of bioactive metabolites showed
significant level of cell inhibition. But SDB and PDB both revealed
significant level of cell inhibition against target bacterial pathogens.
Sabourauds dextrose broth (SDB) isolated compounds had shown
inhibition 76.5% against Staphylococcus aureus, 49.8% against
Aeromonas hydrophila and 42.2% against Enterococcus faecalis
(Figure 5 and Table 3).

maximum amount of Palmarumycin C13 when grown in a modified

SDB medium [22].
Media

S. aureus

S. aureus

S. aureus

Mean SD

%
Inhibition

PDB

0.199

0.187

0.198

0.195 0.00 78.1

SDB

0.217

0.211

0.203

0.210 0.00 76.5

MEB

0.232

0.275

0.3

0.269 0.02 69.87

CZB

0.249

0.234

0.269

0.251 0.01 70.01

0.192

0.177

0.189

0.186 0.00 79.17

0.898

0.901

0.881

0.893 0.00

A.
hydrophila

A.
hydrophila

A.
hydrophila

Mean SD

PDB

0.212

0.214

0.22

0.215 0.00 48.6

SDB

0.221

0.201

0.208

0.210 0.00 49.8

MEB

0.216

0.236

0.231

0.228 0.00 41.9

CZB

0.235

0.243

0.231

0.236 0.00 43.6

Positive
Control

0.142

0.111

0.131

0.128 0.00 69.45

Control

0.412

0.394

0.452

0.419 0.02

Media

Media

E. faecalis

E. faecalis

E. faecalis

Mean SD

%
Inhibition

%
Inhibition

PDB

0.219

0.231

0.222

0.224 0.00 41.9

SDB

0.218

0.201

0.241

0.220 0.01 42.2

MEB

0.221

0.237

0.301

0.253 0.03 34.4

CZB

0.272

0.251

0.245

0.256 0.01 33.6

Positive
Control

0.115

0.107

0.125

0.116 0.00

Control

0.398

0.381

0.378

0.386 0.00

70.1

Table 3: Inhibition (%) of bacterial growth by different crude

metabolite secreted by JAS-2 in different media. Data of %inhibition of
bacterial pathogens were statistically analysed by one way Anova were
found statistically significant at 5% (P<0.05).

Supplement of Carbon and Nitrogen sources in SDB

Figure 5: Assessment of coloured formazan produced during basal
media selection.
Whereas PDB isolated compounds exhibited little higher inhibition
78.1% against S. aureus and lower inhibition or approximately similar
inhibition against A. hydrophila 48.6% and E. faecalis 41.9% compared
to SDB and other media. P-value of OD (optical density) of reduced
MTT in tube was found statistically significant at 5% (Table 3). All
above results indicates SDB to be the best medium growth of
endophytic fungus (JAS-2) as well as production of bioactive
metabolite. Earlier studies have been reported in the direction of
optimizing basal media, endophytic fungus (Dzf-12) secreted

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It is known that carbon and nitrogen sources are channelled into

metabolic machinery of microorganisms so that interesting
biotechnological product could be attained. SDB was found to be a best
medium to culture JAS-2. Sabourauds dextrose broth (SDB) is well
known defined media which was supplemented with different sources
of carbon 4% and nitrogen 1%. All the media (altered carbon and
nitrogen source) have shown efficient antibacterial activity against S.
aureus, A. hydrophila and E. faecalis illustrated in Table 4 and Figure 6.
Medium supplemented with Dextrose and yeast extract as carbon and
nitrogen sources, resulted in higher cell inhibitory activity by MTT
assay. The inhibitory activity of the crude extract is 54.3% against
Staphylococcus aureus, 51.5% against Aeromonas hydrophila and
56.06% against Enterococcus faecalis (Table 4).

Volume 8 Issue 7 1000349

Citation:

Goutam J, Singh S, Kharwar RN, Ramarai V (2016) In vitro Potential of Endophytic Fungus Aspergillus terrus (JAS-2) Associated with
Achyranthus aspera and Study on its Culture Conditions. Biol Med (Aligarh) 8: 349. doi:10.4172/0974-8369.1000349

Page 6 of 7
C/N SOU

%
Inhibition

Dextrose

0.502

0.501

0.471

0.491
0.01

41.3

Sucrose

0.519

0.513

0.515

0.516
0.00

28.18

Maltose

0.512

0.631

0.556

0.566
0.04

Starch

0.532

0.601

0.556

0.563
0.02

Peptone

0.615

0.604

0.564

0.594
0.02

Beef extract

0.574

0.513

0.489

0.525
0.03

Yeast
extract

0.325

0.299

0.314

0.313
0.01

Urea

0.478

0.617

0.493

0.529
0.06

Casein

0.502

0.615

0.401

0.506
0.08

Positive
control

0.142

0.131

0.111

0.128
0.01

Control

0.718

0.721

0.719

0.719
0.00

S. aureus

S. aureus

S. aureus

Mean SD

Dextrose

0.414

0.378

0.394

0.395
0.01

Sucrose

0.415

0.511

0.527

0.484
0.04

Maltose

0.444

0.501

0.511

0.485 002 28.18

Starch

0.414

0.511

0.412

0.446
0.04

0.452
0.06

0.474
0.02

0.308
0.00

0.356
0.06

0.441
0.01

0.096
0.00

0.674
0.01

Peptone

Beef extract
Yeast
extract
Urea

Casein

0.514

0.486

0.311

0.379

0.459

0.478

0.491

0.311

0.271

0.421

0.364

0.445

0.302

0.418

0.443

Positive
control

0.099

Control

0.676

0.691

0.656

A.
hydrophil
a

A.
hydrophil
a

A.
hydrophil
a

Mean SD

Dextrose

0.518

0.514

0.449

0.494
0.03

Sucrose

0.615

0.709

0.617

0.647
0.04

Maltose

0.723

0.708

0.711

0.714
0.06

Starch

0.637

0.581

0.645

0.621
0.02

Peptone

0.512

0.632

0.621

0.588
0.05

Beef extract

0.525

0.521

0.524

0.523
0.00

Yeast
extract

0.401

0.441

0.451

0.431
0.02

Urea

0.565

0.601

0.642

0.603
0.03

Casein

0.632

0.607

0.612

0.617
0.01

Positive
control

0.181

0.141

0.101

0.141
0.03

Control

0.849

0.858

0.961

0.889
0.00

C/N SOU

C/N SOU

0.101

0.089

E. faecalis E. faecalis E. faecalis

Biol Med (Aligarh), an open access journal

ISSN: 0974-8369

Mean SD

33.97

32.93

29.67

54.3

47.18

34.56

85.75

%
Inhibition

44.54

31.7

28.37

21.37

21.69

17.38

27.26

56.06

26.42

29.62

82.19

Table 4: Inhibition (%) of bacterial growth by different crude

metabolite secreted by JAS-2 in different carbon and nitrogen source
replaced in Sabourauds Dextrose Broth (SDB). Data of %inhibition of
bacterial pathogens were statistically analysed by one way Anova were
found statistically significant at 5% (P<0.05).

27.21

19.68

30.14

33.85

41.16

51.51

32.28

Figure 6: Assessment of coloured formazan produced in carbon and

nitrogen sources optimization.

30.59

84.17

%
Inhibition

In 1971 Gupta and chaudhary [23] studied the effect of 20 nitrogen

sources in antibiotic production followed by Basak and Majumdar
[24], who studied the role of carbon and nitrogen sources metabolism
in fungal growth. Dextrose is thought to be readily absorbed by
microorganisms.With reference to our result a number of
microorganisms have elevated their secondary metabolites, when
supplemented with yeast extract. In earlier studies on two major
metabolites, Tanshinones [25] and Spirobisnaphthalenes [26], yeast
extract was found to work as biotic elicitors.

Volume 8 Issue 7 1000349

Citation:

Goutam J, Singh S, Kharwar RN, Ramarai V (2016) In vitro Potential of Endophytic Fungus Aspergillus terrus (JAS-2) Associated with
Achyranthus aspera and Study on its Culture Conditions. Biol Med (Aligarh) 8: 349. doi:10.4172/0974-8369.1000349

Page 7 of 7
Considering major experiment of optimization discussed above
revealed that endophytes could be a better subject for isolating new bio
molecules. The level of these bio molecules could be elevated thereby
fulfilling the requirement of pharmaceutically important compound.
Medically important compounds could be increase using advance
fermentation methods.

Conclusion
Endophytes have been a great source of bioactive compounds and
their effective applications in the field of agriculture, medicine and
other industries parts significant importance. Earlier studies had
influenced us to show interest on this topic with hope to find a novel
bioactive compound. This work has been associated with the study of
fungal endophyte JAS-2 that includes isolation of microorganism,
optimization of different broth media, impact of carbon and nitrogen
sources, in vitro potentials (antibacterial, antifungal and antioxidant)
of metabolites and search of leading molecules.

10.
11.
12.
13.
14.
15.
16.
17.

Acknowledgments

18.

Our research program on fungal endophytes is being supported by

University Grants Commission (RGNF), India. Authors are thanking
to Prof. Gopal Nath (IMS, BHU) and Prof. R. Chand (IAS, BHU)
Varanasi for their support and help in antibacterial and antifungal
assays.

19.

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