ph.d. thesis harpreet kaur...
TRANSCRIPT
Results
58
4.0 RESULTS
The present study was carried out to investigate the antimicrobial potential of
fungi isolated from soil of different areas of Punjab, India. On the basis of colony
characteristics and microscopic morphology of the 113 fungal isolates obtained, 48.6 %
belonged to Aspergillus group while 28.3 % were from Penicillium, 6 % belonged to
Rhizopus and rest 13.2 % belonged to different systematic groups. Out of 113 isolates,
only 58 (51.3 %) showed antimicrobial activity against at least one or more of the tested
microorganisms. Four isolates viz. Penicillium sp. close to P. expansum (HT-28),
Penicillium citrinum (HT-46), Aspergillus sp. close to Aspergillus wentii (HT-113),
Aspergillus terreus (HT-66) were selected for further study. The antimicrobial potential
of the selected strains was tested against ten reference strains of bacteria and two yeasts
obtained from Microbial Type Culture Collection (MTCC), Institute of Microbial
Technology (IMTECH), Chandigarh, India and the clinical isolate, Methicillin resistant
Staphylococcus aureus (MRSA) was obtained from Post Graduate Institute of Medical
Education and Research, (PGIMER), Chandigarh, India. Reference strains included
Gram positive bacteria: Enterococcus faecalis (MTCC 439), Staphylococcus aureus
(MTCC 740), Staphylococcus epidermidis (MTCC 435), Gram negative bacteria:
Escherichia coli (MTCC 119), Klebsiella pneumoniae 1 (MTCC 109), Klebsiella
pneumoniae 2 (MTCC 530) Pseudomonas aeruginosa (MTCC 741), Salmonella
Typhimurium 1 (MTCC 98), Salmonella Typhimurium 2 (MTCC 1251), Shigella
flexneri (MTCC 1457) and two yeast strains viz. Candida albicans (MTCC 227),
Candida tropicalis (MTCC 230). Optimization of physiochemical parameters by one –
factor-at-a time classical method and statistical methods such as response surface
methodology by Box-Behnken design were applied to standardize the media in order to
enhance the antimicrobial activity. Extraction of extracellular broth of these selected
fungi with different solvents (butanol, hexane, chloroform, ethyl acetate and diethyl
ether) was done to find the best solvent to elute the antimicrobial compounds for further
studies. Minimum inhibitory concentration (MIC), viable cell count and post antibiotic
effect of the solvent extracted compounds of all the selected fungi were carried out.
Results
59
Purification and characterization of antimicrobial compounds from all these selected
fungi was done by various analytical and spectroscopic techniques (NMR, IR and Mass
spectrometry). The purified compounds isolated from these selected fungi were also
tested for their bioactivity studies as above. Comparison of solvent extract of these
selected fungi and the purified compounds with standard antibiotics revealed these to be
equally effective or better than some standard antibiotics. Probable mechanism of
antimicrobial action was investigated by ethidium bromide uptake. To demonstrate the
biosafety of the purified compounds these were checked for their mutagenicity and
cytotoxicity and Sulforhodamine B assay. Further the purified compounds were
checked for cytotoxicity against some human cancer cell lines.
4.1 Isolation of fungi from soil samples collected from neighboring areas of
Amritsar, Punjab
Sixty five soil samples were collected from neighboring areas of Amritsar,
Punjab (30˚ 4’ N 75˚ 5’ E), closed tightly and stored at 4˚C. These soil samples were
processed to isolate 113 fungal isolates which were further screened for their
antimicrobial potential by agar well diffusion assay. Among the 113 fungal isolates,
48.6% belonged to Aspergillus group while 28.3% were from Penicillium, 6% belonged
to Rhizopus and rest 13.2% belonged to different systematic groups (Table 4.1.1)
(Figure 4.1.1.).
Results
60
Table 4.1.1: Collection and isolation of fungi from soil.
Fungal
isolate
Collection
Site
Vegetation Colony characteristics Microscopic
Examination
HT-1 GT road,
Khasa,
Amritsar
Ploughed field Colonies are fast growing and cover an
agar surface with a dense cottony growth.
Rhizopus sp.
HT-2 Attari road,
Amritsar
Soil and leaf
litter
Black on the surface and white or yellow
underneath.
Aspergillus
sp.
HT-3 Wagah
border,
Amritsar
Ploughed field Light yellow surface becoming dark green Aspergillus
sp.
HT-4 Wagah
border,
Amritsar
Compost Brownish in color and gets darker as it
ages on culture media
Aspergillus
sp.
HT-5 Attari Nursery
(Mango
garden)
White color of the colony with dark
purple from reverse.
Unidentified
HT-6 Attari Railway station White color of the colony with dark
purple from reverse.
Unidentified
HT-7 Attari bye
pass
Pear garden Light yellow surface become light green Aspergillus
sp.
HT-8 Ram Tirath
road
Rice field wet
soil
Light yellow to dark greenish colony Penicillium
sp.
HT-9 Ajnala Road side
popular tress
Light yellow to dark green colony Penicillium
sp.
HT-10 Ajnala Maize field Black sporulating fungus, white margins Aspergillus
sp.
HT-11 Airport road,
Amritsar
Ploughed field Black sporulating fungus with white
margins
Aspergillus
sp.
HT-12 Airport road,
Amritsar
Maize field Black sporulating fungus with white
margins
Aspergillus
sp.
HT-13 Amritsar-
Jalandhar GT
road
Poplar trees Dark green, powdery, back side of the
colony was yellowish cream
Penicillium
sp.
HT-14 Amritsar-
Jalandhar GT
road
Oat/barley field Light yellow surface become light green Aspergillus
sp.
HT-15 Amritsar-
Jalandhar GT
road
Compost Light greenish powdery colony Aspergillus
sp.
HT-16 Jandiala-
Khadoor sahib
Road, rice field Fast growing Rhizopus sp.
HT-17 Jandiala -
Khadoor sahib
Road, compost Green colored colony with yellow
pigmentation on reverse side of the plate
Penicillium
sp.
Ht-18 Chogawan Barren land Light greenish powdery colony Aspergillus
sp.
HT-19 Bhullar Soil and leaf
litter
Green colored colony with yellow
pigmentation on reverse side of the plate
Penicillium
sp.
HT 20 Hussainpur Dullowal,
Sugarcane field
Black sporulating fungus with off white
edges
Aspergillus
sp.
HT 21 Hussainpur Rice field Green colored colony with yellow
pigmentation
Penicillium
sp.
HT 22 Hussainpur Decomposing
leaf litter
Black on the surface and white on
margins
Aspergillus
sp.
Results
61
Fungal
isolate
Collection
Site
Vegetation Colony characteristics Microscopic
Examination
HT 23 Talwandi
Chaudhrian
Seesham trees Black on the surface and white underneath Aspergillus
sp.
HT 24 Talwandi
Chaudhrian
Maize field Black on the surface and white underneath Aspergillus
sp.
HT 25 Talwandi
Chaudhrian
Compost White colony and purple underneath Unidentified
HT 26 Mewa Singh
village
Bitter gourd Black on the surface and white on
margins
Aspergillus
sp.
HT 27 Mewa Singh
village
Decomposing
leaf litter
White colony Unidentified
HT 28 Sultanpur
lodhi
Barley field Green colored colony with yellow
pigmentation on the reverse side
Penicillium
sp.
HT 29 Sultanpur
lodhi
Decomposing
paddy husk
Greenish powdery Aspergillus
sp.
HT 30 Sultanpur
lodhi
Eucalyptus Black on the surface and white on
margins
Aspergillus
sp.
HT 31 Mirapur
village
Chilly field Green powdery Penicillium
sp.
HT 32 Miran Kot
Kalan
Decomposing
leaf litter
Black on the surface and white on the
margins
Aspergillus
sp.
HT 33 Tajpur Chilly field Brown in color and its getting darker as
its ages
Aspergillus
sp.
HT 34 Tajpur Banana tress Black on the surface and white on the
margins
Aspergillus
sp.
HT 35 Tajpur Barley field Dark green coloured colony Penicillium
sp.
HT 36 Mothanwal Ploughed field Powder green colony Aspergillus
sp.
HT 37 Mothanwal Brinjal Green colored with white margins Aspergillus
sp.
HT 38 Mothanwal Road side soil Black on the surface and white on the
margins
Aspergillus
sp.
HT 39 Mahabalipur Brinjal field Green colored colony with white margins Penicillium
sp.
HT 40 Mahabalipur Eucalyptus Green colored colony with white margins Penicillium
sp.
HT 41 Mahabalipur Ploughed field Green colored colony with reverse yellow
pigmentation
Penicillium
sp.
HT 42 Khatkar Kalan Grape wine Green colored colony powdery Penicillium
sp.
HT 43 Khatkar Kalan Compost Black on the surface with margins Aspergillus
sp.
HT 44 Khatkar Kalan Mango trees White cottony fast growing colony Rhizopus sp.
HT 45 Kapurthala Road side soil
sample
Brownish colony Aspergillus
sp.
HT 46 Kapurthala Mango trees Green colored colony with yellow
pigmentation on the reverse side
Penicillium
sp.
HT 47 Kapurthala Field Banyan
tree
Black on the surface with white margins Aspergillus
sp.
HT 48 Kapurthala Road side soil Cottony white fast growing colony Rhizopus sp.
HT 49 Kapurthala-
Nakodar Road
Compost Green colored colony with white margins Penicillium
sp.
Results
62
Fungal
isolate
Collection
Site
Vegetation Colony characteristics Microscopic
Examination
HT 50 Kapurthala-
Nakodar Road
Ploughed field Black on the surface Aspergillus
sp.
HT 51 Kapurthala-
Nakodar Road
Rice field Green on the surface with white edge Penicillium
sp.
HT 52 Kapurthala-
Nakodar Road
Seesham and
poplar trees
Black on the surface with white margins Aspergillus
sp.
HT 53 Kapurthala-
Nakodar Road
Grape vine Green on the surface, powdery Penicillium
sp.
HT 54 Kapurthala-
Nakodar Road
Sugarcane field Black on the surface with white margins Aspergillus
sp.
HT 55 Talwandi
Salem
Harvested
wheat field
White on the surface with purpulish on
reverse side of the plate
Unidentified
HT 56 Talwandi
Salem
Sugarcane field white coloured cottony colony with
greenish centre
Unidentified
HT 57 Talwandi
Salem
Bitter gourd Black on the surface with white margins Aspergillus
sp.
HT 58 Nakodar
Jalandhar GT
road
Straw compost Green powdery Penicillium
sp.
HT 59 Nakodar
Jalandhar GT
road
Seesham trees Black on the surface with white margins Aspergillus
sp.
HT 60 Nakodar
Jalandhar GT
road
Road side Green powdery Penicillium
sp.
HT 61 Batala Sugarcane field Fast growing cottony covering the surface Rhizopus sp.
HT 62 Batala Wheat field Black on the surface with white margins Aspergillus
sp.
HT 63 Batala Decomposing
leaf litter
White cottony colony Unidentified
HT 64 Qadian Soil and
decaying
matter
Brown in color and its getting darker as
its ages
Aspergillus
sp.
HT 65 Qadian ploughed field
eucalyptus tree
Brown in color and its getting darker as
its ages
Aspergillus
sp.
HT 66 Qadian Straw compost Brown in color and its getting darker as
its ages
Aspergillus
sp.
HT 67 Qadian Sugarcane field White cottony colony Unidentified
HT 68 Tugalwal Ploughed field Green powdery Penicillium
sp.
HT 69 Tugalwal Harvested
wheat field
Green colored with white margins Penicillium
sp.
HT 70 Darapur Kalan fodder field Brown in color and its getting darker as
its ages
Aspergillus
sp.
HT 71 Tugalwal Peach garden Black on the surface with white margins Aspergillus
sp.
HT 72 Darapur Kalan Wheat field Black on the surface with white margins Aspergillus
sp.
HT 73 Darapur Kalan Mango garden White colored colony Unidentified
HT 74 Darapur Kalan Road side Green Powdery Penicillium
sp.
Results
63
Fungal
isolate
Collection
Site
Vegetation Colony characteristics Microscopic
Examination
HT 75 Saidowal
Kalan
Compost Brown colony with cream margins Aspergillus
sp.
HT 76 Saidowal
Kalan
Ploughed field White colony Unidentified
HT 77 Saidowal
Kalan
Sugarcane field Black on the surface with white margins Aspergillus
sp.
HT 78 Saidowal
Kalan
Grape vine Brown in color and its getting darker as
its ages
Aspergillus
sp.
HT 79 Saidowal
Kalan
Grassland Powdery green Penicillium
sp.
HT 80 Mukerian Seesham,
Acacia
White colored colony with white and
purple from centre
Unidentified
HT 81 Mukerian Paddy field White cottony colony Unidentified
HT 82 Mukerian Arhar Field Powdery green Penicillium
sp.
HT 83 Mukerian Oat/Barley
field
White colony Unidentified
HT 84 Fatehgarh
churian
Compost Green powdery Penicillium
sp.
HT 85 Fatehgarh
churian
Seesham White cottony colony Unidentified
HT 86 Fatehgarh
churian
Ploughed field Green colored colony with yellow
pigmentation on the reverse side
Penicillium
sp.
HT 87 Nagoke Arhar field Green colored powdery Aspergillus
sp.
HT 88 Nagoke Wheat field Black on the surface with white margins Aspergillus
sp.
HT 89 Nagoke road side Brown colony Unidentified
HT 90 Tugalwal Ploughed field Brown colony with white edge Aspergillus
sp.
HT 91 Tugalwal Brinjal field Powdery green colored colony Penicillium
sp.
HT 92 Tugalwal soil and leaf
litter
Brown in color and its getting darker as
its ages
Aspergillus
sp.
HT 93 Gurdaspur
Road
Wheat field Green colored colony with white margins Penicillium
sp.
HT 94 Gurdaspur
Road
Compost Brown colony with white edge Aspergillus
sp.
HT 95 Gurdaspur
Road
Rice field Black on the surface with white margins Aspergillus
sp.
HT 96 Bhikhiwind Rice field White cottony colony Rhizopus sp.
HT 97 Bhikhiwind ploughed field Black on the surface with white margins Aspergillus
sp.
HT 98 Talwara Eucalyptus Powdery green Penicillium
sp.
HT 99 Talwara Barren land Black on the surface with white margins Aspergillus
sp.
HT
100
Talwara rice field White colored colony Unidentified
HT
101
Hajipur Mango garden Brown colony with white edge Aspergillus
sp.
Results
64
Fungal
isolate
Collection
Site
Vegetation Colony characteristics Microscopic
Examination
HT
102
Hajipur Road side
shrubs
Green colored colony Penicillium
sp.
HT
103
Hajipur Wheat field Light green colored with white edge Penicillium
sp.
HT
104
Gurdaspur
road
Wheat field White colony Unidentified
HT
105
Gurdaspur
road
Sugarcane field Green colored colony with white edge Penicillium
sp.
HT
106
Gurdaspur
raod
Eucalyptus Powdery green colony Penicillium
sp.
HT
107
Gurdaspur
road
Ploughed field Brown colony with white edge Aspergillus
sp.
HT
108
Dhadha
Daulatpur
Ploughed field Black on the surface with white margins Aspergillus
sp.
HT
109
Dhadha
Daulatpur
Maize field Powdery green Penicillium
sp.
HT
110
Dhadha
Daulatpur
Soil and leaf
litter
Green colored with white margins Penicillium
sp.
HT
111
Gharinda Barren land Brown colony with white edge Aspergillus
sp.
HT
112
Gharinda Compost Black on the surface with white margins Aspergillus
sp.
HT
113
Gharinda Rice field White colony Aspergillus
sp.
Figure 4.1.1 Fungal isolates from neighboring areas of Amritsar, Punjab
65
113
48.6%
28.3%
6%13.2%
0
20
40
60
80
100
120
Soil samples Total fungal
isolates
Aspergillus Penicillium Rhizopus others
systematic gr
Results
65
4.2 Screening of fungal isolates for their antimicrobial potential
All the 113 fungal isolates were screened against 12 microorganisms viz. E.
faecalis, S. aureus, S. epidermidis, E. coli, K. pneumoniae 1, K. pneumoniae 2, Sh.
flexneri, Salm. Typhimiurium1, Salm. Typhimiurium2, C. albicans, C. tropicalis for
their antimicrobial potential by agar well diffusion assay. Out of 113 isolates screened
only 50 (44.2 %) showed antimicrobial activity against at least one or more of the tested
microorganisms. The growth of Pseudomonas aeruginosa was inhibited by Aspergillus
spp. (HT-4, HT-64, HT-65, HT-66, HT-70, HT-75 and HT-78), whereas C. tropicalis
was found to be totally resistant to all the fungal extracts. From the entire range of
Penicillium spp., HT-28, HT-41, HT 46, HT-86 and HT-110 demonstrated better
antimicrobial activity with inhibition zone ranging from 15 mm to 37 mm against four
microorganism viz. S. aureus, S. epidermidis, K. pneumoniae 1 and C. albicans.
However, the extracellular culture broth of these Penicillium spp. did not showed any
activity against rest of the microorganism and the best two; Penicillium expansum (HT
28) and Penicillium citrinum (HT 46) were selected for further studies. Similarly, from
all the Aspergillus spp., HT-4, HT-64, HT-65, HT-66, HT-70, HT-75 and HT-78
showed potent antimicrobial activity against almost all tested microbial strains with
zone of inhibition ranging from 15 mm to 18 mm. HT-66 was selected from this group
as it showed broad antimicrobial spectrum. Further, HT-113 another Aspergillus spp,
with a better zone of inhibition effective against a few organisms such as S. aureus, S.
epidermidis, K. pneumoniae 1 and C. albicans with zone of inhibition 26 mm, 26 mm,
16 mm and 23 mm, respectively. Thus, in total four fungal isolates i.e. HT 28, HT 46,
HT 66 and HT 113 were selected for further studies (Table 4.2.1).
Res
ult
s
66
Tab
le 4
.2.1
: S
cree
nin
g o
f var
ious
fungal
iso
late
s fo
r th
eir
Anti
mic
rob
ial
acti
vit
y b
y A
gar
wel
l dif
fusi
on m
ethod
.
E.f
aeca
lis
S.a
ure
us
S.e
pid
erm
idis
E
.co
li
K.p
en
um
on
iae 1
K
.pen
um
on
iae 2
P
.aeru
gin
osa
S
h. fl
exneri
S
alm
.T y
phim
uri
um
2
Sa
lm.
Typ
him
uri
um
1
C.a
lbic
an
s C
.tro
pic
ali
s
HT
2
0
15
.5±
0.7
0
0
0
0
0
0
0
0
1
4.5
±0
.7
0
HT
4
0
17
±1.4
1
8.5
±0
.7
16
.5±
0.7
1
8±
1.4
0
1
6.5
±0
.7
15
±0
15
±1.4
0
1
7.5
±0
.7
0
HT
5
0
0
0
0
14
.5±
0.7
0
0
0
0
0
1
4.5
±2
.1
0
HT
6
0
0
0
0
14
±1.4
0
0
0
0
0
1
6±
2.1
0
HT
7
0
16
.5±
0.7
0
0
0
0
0
0
0
0
0
0
HT
8
0
0
0
0
17
.5±
2.1
0
0
2
1.5
±0
.7
0
0
15
.5±
2.1
0
HT
9
0
0
18
±1.4
0
0
0
0
0
0
0
1
5±
1.4
0
HT
10
0
0
0
0
1
7.5
±0
.7
0
0
0
0
0
14
.5±
0.7
0
HT
11
12
.5±
0.7
0
0
0
1
6.5
±0
.7
0
0
0
0
0
16
±0
0
HT
12
0
0
0
0
1
4±
0.7
0
0
0
0
0
1
5.5
±2
.1
0
HT
17
0
21
.5±
2.1
2
5.5
±2
.1
0
25
.5±
0.7
0
0
0
0
0
2
0.5
±0
.7
0
HT
19
0
18
.5±
0.7
0
0
0
0
0
0
0
0
2
0.5
±2
.1
0
HT
21
0
30
.5±
2.1
3
5.5
±0
.7
0
19
±1.4
0
0
0
0
0
2
5±
1.4
0
HT
23
0
0
0
0
0
0
0
0
0
0
18
±1.4
0
HT
24
0
0
0
0
0
0
0
0
0
0
18
±0
0
HT
25
0
0
0
0
0
0
0
0
0
0
1
6±
0
0
HT
27
0
0
0
0
0
0
0
0
0
0
1
7.5
±0
.7
0
HT
28
0
3
4±
1.4
3
7.5
±0
.7
0
20
.5±
0.7
0
0
0
0
0
1
6±
1.4
0
HT
29
0
0
0
0
0
0
0
0
0
0
1
6±
1.4
0
HT
32
0
0
0
0
0
0
0
0
0
0
16
±0
0
HT
33
0
1
5.5
±2
.1
15
.5±
0.7
0
1
6.5
±0
.7
0
0
0
0
0
15
±0
0
HT
40
0
3
2±
1.4
3
4±
1.4
0
1
6.5
±0
.7
0
0
0
0
0
16
.5±
2.1
0
HT
41
0
3
5.5
±2
.1
31
.5±
0.7
0
1
5.5
±0
.7
0
0
0
0
0
15
.5±
2.1
0
HT
46
0
3
4.5
±0
.7
35
±0.7
0
1
8±
1.4
0
0
0
0
0
1
7±
1.4
0
HT
47
0
0
0
0
1
6±
0
0
0
0
0
0
15
.5±
0.7
0
HT
48
0
0
0
0
1
5.5
±2
.1
0
0
0
0
0
15
.5±
0.7
0
HT
51
0
0
0
0
1
6.5
±0
.7
0
0
0
0
0
16
±1.4
0
HT
52
0
0
1
5.5
±0
.7
0
15
±1.4
0
0
0
0
0
1
5.5
±0
.7
0
HT
56
0
0
0
0
0
0
0
0
0
1
5±
1.4
0
0
HT
61
0
0
0
0
0
0
0
0
0
0
1
6.5
±2
.1
0
HT
62
0
0
0
0
1
5.5
±2
.1
0
0
0
0
0
15
.5±
0.7
0
Res
ult
s
67
E.f
aeca
lis
S.a
ure
us
S.e
pid
erm
idis
E
.co
li
K.p
en
um
on
iae 1
K
.pen
um
on
iae 2
P
.aeru
gin
osa
S
h. fl
exneri
S
alm
.T y
phim
uri
um
2
Sa
lm.
Typ
him
uri
um
1
C.a
lbic
an
s C
.tro
pic
ali
s
HT
63
0
0
0
0
0
0
0
0
0
0
1
4.5
±0
.7
0
HT
64
0
1
6±
1.4
1
6±
1.4
1
5.5
±0
.7
16
.5±
2.1
0
1
5.5
±0
.7
15
.5±
0.7
1
5.5
±0
.7
15
.5±
0.7
1
5.5
±0
.7
0
HT
65
0
1
5.5
±2
.1
15
.5±
0.7
1
5.5
±0
.7
16
.5±
2.1
0
1
5.5
±0
.7
16
.5±
0.7
1
5.5
±2
.1
16
.5±
0.7
1
6.5
±2
.1
0
HT
66
1
6±
1.4
1
6±
1.4
1
7±
1.4
1
6±
1.4
1
8±
0
0
17
±1.4
1
7±
1.4
1
6.5
±2
.1
16
.5±
2.1
1
8±
1.4
0
HT
69
0
2
0±
1.4
0
0
0
0
0
0
0
0
1
6.5
±0
.7
0
HT
70
0
1
7.5
±2
.1
15
.5±
0.7
1
6.5
±0
.7
16
.5±
0.7
0
1
6±
1.4
1
5.5
±0
.7
16
.5±
2.1
1
4.5
±2
.1
16
.5±
0.7
0
HT
71
0
0
0
0
0
0
0
0
0
0
1
6±
1.4
0
HT
72
0
0
0
0
1
7±
1.4
0
0
0
0
0
1
6.5
±2
.1
0
HT
74
0
0
0
0
1
6.5
±2
.1
0
0
0
0
0
15
.5±
2.1
0
HT
75
0
1
5.5
±0
.7
15
.5±
2.1
1
5.5
±0
.7
15
±1.4
0
1
6.5
±0
.7
15
±1.4
1
5±
1.4
1
5.5
±2
.1
16
.5±
0.7
0
HT
76
0
0
0
0
1
5±
1.4
0
0
0
0
0
1
5±
1.4
0
HT
78
0
1
7.5
±2
.1
16
.5±
2.1
1
6.5
±2
.1
17
±1.4
0
1
4.5
±2
.1
15
.5±
0.7
1
5.5
±0
.7
15
±1.4
1
6.5
±0
.7
0
HT
79
0
0
0
0
1
5±
0.7
0
0
0
0
0
0
0
HT
80
0
0
0
0
1
4.5
±2
.1
0
0
0
0
0
0
0
HT
81
0
0
0
0
0
0
0
0
0
0
1
6.5
±2
.1
0
HT
83
0
0
0
0
0
0
0
0
0
1
6.5
±0
.7
0
0
HT
85
0
0
0
0
0
0
0
0
0
0
1
5.5
±0
.7
0
HT
86
0
2
5±
1.4
2
9±
1.4
0
1
8.5
±2
.1
0
0
0
0
0
17
.5±
0.7
0
HT
87
0
0
0
0
0
0
0
0
0
1
5±
1.4
0
0
HT
90
0
0
0
0
0
0
0
0
0
0
1
4±
1.4
0
HT
91
0
0
0
0
0
0
0
0
0
0
0
0
HT
92
0
1
5.5
±2
.1
15
.5±
0.7
1
6.5
±2
.1
15
.5±
0.7
0
0
1
5.5
±2
.1
15
.5±
2.1
0
1
5.5
±0
.7
0
HT
93
0
1
6.5
±2
.1
17
±1.4
0
1
6±
1.4
0
0
0
0
0
1
6.5
±2
.1
0
HT
94
0
0
0
0
0
0
0
0
0
0
1
5±
1.4
0
HT
95
0
1
9.5
±0
.7
0
0
0
0
0
0
0
0
0
0
HT
11
0
0
21
.5±
0.7
2
0.5
±0
.7
0
15
.5±
2.1
0
0
0
0
0
0
0
HT
11
3
0
26
±1.4
2
6.5
±0
.7
0
16
.5±
0.7
0
0
0
0
0
2
3.5
±2
.1
0
Val
ues
are
exp
ress
ed i
n t
erm
s of
mea
n ±
Sta
ndar
d d
evia
tion
Results
68
4.3 Identification of Fungal Isolates
All the 113 fungal isolates screened were identified on the basis of morphology
of the fungal culture. The identification of selected four strains was confirmed by
National Fungal Culture Collection of India (NFCCI), Agharkar Research Institute,
Pune, India and cultures were deposited in NFCCI. Identification remarks provided by
National Fungal Culture Collection of India, Agharkar Research Institute, Pune, India
are presented in Table 4.3.1.
Table 4.3.1: Identification remarks provided by National Fungal Culture Collection.
Culture No. Identification Remarks Accession No.
HT 28 Penicillium sp. close to P. expansum Link. NFCCI* 2554
HT 46 Penicillium citrinum NFCCI* 2555
HT 113 Aspergillus sp. close to A. wentii gr. NFCCI* 2565
HT 66 Aspergillus terreus gr. NFCCI* 2556
*National Fungal Culture Collection of India
4.4 Antimicrobial potential of fungal isolates as assayed under different physio-
chemical conditions
Different physiochemical parameters were optimized for selected four fungal
isolates to enhance their antimicrobial potential. Various basal media were screened to
find out their suitability for the fungal growth and best expression of antimicrobial
potential. The experiments were carried out to see the effect of shaking at different
RPM and compared with the results of static culture. The antimicrobial potential was
measured at different, incubation periods, and pH values by agar well diffusion assay.
4.4.1 Effect of different growth media on antimicrobial activity of Penicillium
spp.
Various basal media viz. Czapek dox’s medium (CZ), Potato dextrose medium
(PDM), Malt extract medium (ME), Yeast extract medium (YE) and Yeast Peptone
dextrose (YPDS) medium were screened to find out their suitability for the growth and
best expression of the antimicrobial activity. All the selected fungi showed the highest
Results
69
antimicrobial activity in YPDS medium followed by YE, PDM. Both the Penicillium
spp. showed better antimicrobial activity, when grown on YPDS medium with zone of
inhibition ranging from (20 - 37 mm) in Penicillium expansum (Fig. 4.4.1.1 ) and (16 -
33 mm) in Penicillium citrinum (Fig. 4.4.1.2) followed by yeast extract medium with
zone of inhibition ranging from (15- 30 mm) and (15 -32 mm) for respective fungi.
While the activity decreased when both the fungi were grown in PDM and ME and the
antimicrobial activity decreased to its lowest levels when grown on CZ medium and
showed activity against S. aureus and S. epidermidis with 16 and 15 mm zone of
inhibition, respectively.
CZ- Czapek dox’s medium, ME- Malt extract medium, PDM- Potato dextrose medium,
YE- Yeast extract medium, YPD- Yeast peptone dextrose medium.
Figure 4.4.1.1 Effect of different growth media on antimicrobial activity of Penicillium
expansum
0
5
10
15
20
25
30
35
40
CZ ME PDM YE YPDS
Zo
ne
of
inh
ibit
ion
(m
m)
Different basal media
E. faecalis
S. aureus
S.epidermidis
E.coli
K.pneumoniae 1
K.pneumoniae 2
P.aeruginosa
Sh. flexneri
Salm.Typhimurium 1
Salm.Typhimurium 2
C.albicans
C.tropicalis
MRSA
Biomass
Results
70
CZ- Czapek dox’s medium, ME- Malt extract medium, PDM- Potato dextrose medium,
YE- Yeast extract medium, YPD- Yeast peptone dextrose medium.
Figure 4.4.1.2 Effect of different media on antimicrobial activity of Penicillium
citrinum
4.4.2 Effect of different growth media on antimicrobial activity of Aspergillus
spp.
With YPDS medium, Aspergillus terreus (HT66) (Figure. 4.4.2.1) showed
maximum zone of inhibition, ranging from (15 - 19 mm) followed by yeast extract (13 -
16 mm), potato dextrose medium (13 -14mm), malt extract medium showed
antimicrobial activity against S. aureus and C. albicans with zone of inhibition 16 and
14 mm, respectively. Czapek dox’s medium supported the least antimicrobial activity
against S. aureus and C. albicans with zone of inhibition 15 and 12 mm, respectively.
Similarly, YPDS was found to support antimicrobial activity by Aspergillus wentii
(Fig. 4.4.2.1) with inhibition zone ranging from 24 -26 mm, followed by yeast extract
medium (zone size 16 mm to 22 mm) followed by potato dextrose medium and malt
extract. The least activity was found when Czapek dox’s was used as a basal medium.
From the above observations, YPDS medium was found to be best to support maximum
antimicrobial activity by all the four fungi.
0
5
10
15
20
25
30
35
CZ ME PDM YE YPDS
Zo
ne
of
inh
ibit
ion
(m
m)
Different basal media
E. faecalis
S . aureus
S .epidermidis
E.coli
K.pneumoniae 1
P.aeruginosa
Sh. flexneri
Salm.Typhimurium 1
Salm.Typhimurium 2
C.albicans
MRSA
Biomass
Results
71
CZ- Czapek dox’s medium, ME- Malt extract medium, PDM- Potato dextrose medium,
YE- Yeast extract medium, YPD- Yeast peptone dextrose medium.
Figure 4.4.2.1 Effect of different media on antimicrobial activity of Aspergillus terreus
CZ- Czapek dox’s medium, ME- Malt extract medium, PDM- Potato dextrose medium,
YE- Yeast extract medium, YPD- Yeast peptone dextrose medium.
Figure 4.4.2.2 Effect of different media on antimicrobial activity of Aspergillus wentii
0
2
4
6
8
10
12
14
16
18
20
CZ ME PDM YE YPDS
zon
e o
f in
hib
itio
n (
mm
)
Different basal media
E. faecalis
S . aureus
S .epidermidis
E.coli
K.pneumoniae 1
K.pneumoniae 2
P.aeruginosa
Sh. flexneri
Salm.Typhimurium 1
Salm.Typhimurium 2
C.albicans
C.tropicalis
MRSA
Biomass
0
5
10
15
20
25
30
CZ ME PDM YE YPDS
Zo
ne
of
inh
ibit
ion
(m
m)
Different basal media
E. faecalis
S . aureus
S .epidermidis
E.coli
K.pneumoniae 1
P.aeruginosa
Sh.flexneri
Salm.Typhimurium 1
Salm.Typhimurium 2
C.albicans
MRSA
Biomass
Results
72
4.4.3 Effect of incubation period on the antimicrobial activity of Penicillium spp
The culture broth obtained from Penicillium expansum and Penicillium citrinum
during different incubation periods demonstrated the antimicrobial activity against five
organisms viz. S. aureus, S. epidermidis K. pneumoniae 1, MRSA and C. albicans. Rest
of the tested bacterial cultures (Salm. Typhimurium1, Salm. Typhimurium2, E. coli, Sh.
flexneri, E. faecalis, K. pneumoniae1, P. aeruginosa) did not show any sensitivity
during the entire period of incubation. The antimicrobial activity of both the fungi
reached their maxima on 7th
day, remained static or showed a slight decline on 15day
onwards. Staphylococcus aureus was the most sensitive organism to Penicillium
expansum, with a maximum inhibition zone of 42 mm, followed by S. epidermidis (36
mm), Klebsiella pneumoniae (28 mm), followed by MRSA and C. albicans, showed
inhibition zone of 17mm. Staphylococcus epidermidis was the most sensitive to
Penicillium citrinum with a zone size of 36 mm closely followed by Staphylococcus
aureus (34 mm), Klebsiella pneumoniae (17 mm), C. albicans (18mm) and MRSA with
17mm zone of inhibition. Maximum biomass of both the organisms was obtained on
day 5th (Figure 4.4.3.1, 4.4.3.2). Thus further experiments involving Penicillium spp
were carried out for 7 days as optimal incubation period.
Figure 4.4.3.1: Effect of incubation period on antimicrobial activity of Penicillium
expansum.
0
2
4
6
8
10
12
14
0
5
10
15
20
25
30
35
40
45
5 6 7 8 9 10 15 20 25 30
bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Days of incubation
S.aureus
S.epidermidis
K.pneumoniae 1
MRSA
C.albicans
Biomass
Results
73
Figure 4.4.3.2: Effect of incubation period on antimicrobial activity of Penicillium
citrinum
4.4.4. Effect of incubation period on the antimicrobial activity of Aspergillus spp.
Of the two Aspergillus spp., Aspergillus terreus was found to be much effective
as it inhibited all the tested organisms viz. S. aureus, S. epidermidis, K. pneumoniae 1,
K. pneumoniae 2, Sh. flexneri, Salm. Typhimurium 1, Salm. Typhimurium 2, E. faecalis,
P. aeruginosa, MRSA, C. albicans except C. tropicalis (Fig. 4.4.4.1, 4.4.4.2). The
maximum antimicrobial activity was observed on 5th
day of incubation for both the
Aspergillus cultures though A. terreus gave the activity only under shaking conditions.
It remained stable upto day 10 and then it declined. Though Aspergillus terreus was
effective against a wide range of organisms but a better zone size was observed for
Aspergillus wentii where it ranged from 11-28 mm. C. albicans was most sensitive
organism to extracellular culture broth of Aspergillus terreus and Aspergillus wentii
with maximum zone of inhibition of 20 and 25 mm respectively. Aspergillus wentii
showed maximum biomass (8.34 mg/ml) at 5th
day of incubation which then declined
slightly whereas biomass was increased steadily in case of Aspergillus terreus and was
maximum at 30th day of incubation. Maximum antimicrobial activity was observed at
day 5 which remain more or less stable till 9th
day of incubation and then declined.
0
2
4
6
8
10
12
0
5
10
15
20
25
30
35
40
5 6 7 8 9 10 15 20 25 30
bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Days of incbation
S.aureus
S.epidermidis
K.pneumoniae1
MRSA
C.albicans
Biomass
Results
74
Figure 4.4.4.1 Effect of incubation period on the antimicrobial activity of Aspergillus
terreus
Figure 4.4.4.2 Effect of incubation period on the antimicrobial activity of Aspergillus
wentii
0
5
10
15
20
25
30
35
40
0
5
10
15
20
25
4 5 6 7 8 9 10 15 20 25 30
Bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Days of incubation
E. faecalis
S. aureus
S.epidermidis
E.coli
K.pneumoniae 1
P.aeruginosa
Sh. flexneri
Salm.Typhimurium 1
Salm.Typhimurium 2
MRSA
C.albicans
Biomass
0
1
2
3
4
5
6
7
8
9
0
5
10
15
20
25
30
4 5 6 7 8 9 10 15 20 25 30
bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Days of incubation
S.aureus
S.epidermidis
k.pneumoniae1
MRSA
C albicans
Biomass
Results
75
4.4.5 Effect of shaking conditions on antimicrobial activity of Penicillium spp.
To study the best expression of antimicrobial activity of all the selected fungi,
these were grown under shaking conditions at different rpm. (Fig. 4.4.5.1; 4.4.5.2).
With increase in RPM, both the fungi showed a steady decline in their biomass and
antimicrobial activity against tested organisms and at 250 RPM, least or no
antimicrobial activity was observed. Maximum biomass of 13.06 mg/ml and
10.23mg/ml and antimicrobial activity was observed at 100 RPM against S. aureus, S.
epidermidis, K. pneumoniae 1, C. albicans and MRSA in Penicillium expansum and
Penicillium citrinum respectively. As maximum antimicrobial activity was observed at
stationary conditions, so further experimentations for both the Penicillium spp. was
carried out under stationary conditions.
Figure 4.4.5.1 Effect of shaking on antimicrobial activity of Penicillium expansum.
0
2
4
6
8
10
12
14
0
5
10
15
20
25
30
35
100 150 200 250
bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
on
(m
m)
RPM
S.aureus
S.epidermidis
K.pneumoniae 1
MRSA
C.albicans
Biomass
Results
76
Figure 4.4.5.2 Effect of shaking conditions on antimicrobial potential of Penicillium
citrinum.
4.4.6 Effect of shaking conditions on antimicrobial activity of Aspergillus spp.
The antimicrobial activity of Aspergillus terreus was better expressed under
shaking conditions as there was no activity in static conditions. Of the thirteen
microorganism tested , Aspergillus terreus showed antimicrobial activity against eleven
microorganism as K. pneumoniae 2 and C. tropicalis was found to be resistant (Fig.
4.4.6.1). No activity was observed at 100 rpm, and the activity increased with the
increase in rpm. Best antimicrobial activity was observed at 200 rpm with an inhibition
zone ranging from 15 mm to 20 mm and the activity remained same till 250 rpm.
Biomass of the fungus also increased with the increase in the RPM and found to be
maximum (20.35 mg/ml) at 250 rpm. The antimicrobial activity of Aspergillus terreus
was better expressed under shaking conditions as there was no activity in static
conditions whereas the antimicrobial activity of Aspergillus wentii was better expressed
under stationary conditions as compared to shake flask cultures. Of all the thirteen
organisms tested, Aspergillus wentii showed antimicrobial activity against S. aureus, S.
epidermidis, K. pneumoniae 1, MRSA and C. albicans. The antimicrobial activity
decreases with increase in RPM and showed no activity at 200 and 250 RPM.
0
2
4
6
8
10
12
0
5
10
15
20
25
30
100 150 200 250
bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
RPM
S.aureus
S.epidermidis
K.pneumoniae 1
MRSA
C.albicans
Biomass
Results
77
Maximum biomass of 8.06mg/ml was found at 100 RPM (Fig. 4.4.6.2) which also
declined with the increase in RPM. Thus, further experimentation for Aspergillus wentii
was carried out under stationary conditions while for Aspergillus terreus the
experimentation was carried out at 200 RPM.
Figure 4.4.6.1: Effect of shaking on antimicrobial activity of Aspergillus terreus
Figure 4.4.6.2: Effect of shaking conditions on antimicrobial activity of Aspergillus
wentii.
0
5
10
15
20
25
0
5
10
15
20
25
100 150 200 250
Bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
RPM
E. faecalis
S. aureus
S.epidermidis
E.coli
K.pneumoniae 1
P.aeruginosa
Sh. flexneri
Salm.Typhimurium 1
Salm.Typhimurium 2
MRSA
C.albicans
Biomass
0
1
2
3
4
5
6
7
8
9
0
5
10
15
20
25
100 150 200 250
bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibti
tio
n (
mm
)
RPM
S.aureus
S.epidermidis
K.pneumoniae 1
C.albicans
MRSA
Biomass
Results
78
4.4.7 Effect of pH on antimicrobial Activity of Penicillium spp.
Both the Penicillium spp. were grown at different pH ranging from pH 3-9 and
the resulting extracellular culture broth were tested for their antimicrobial activity. The
antimicrobial activity increased from pH 3 to 5 and then remained more or less stable
till pH 9 in both the Penicillium spp. In both the Penicillium spp. biomass was
maximum at pH 3 with 11.4 and 11.24mg/ml in Penicillium expansum and Penicillium
citrinum respectively which then declined. The pH optima for antimicrobial activity of
both the Penicillium spp. was in the range of pH 6-9. (Fig. 4.4.7.1, 4.4.7.2)
Figure 4.4.7.1: Effect of different pH on antimicrobial activity of Penicillium
expansum.
0
2
4
6
8
10
12
0
5
10
15
20
25
30
35
40
45
3 4 5 6 7 8 9
bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
pH
S.aureus
S.epidermidis
K.pneumoniae
MRSA
C.albicans
Biomass
Results
79
Figure 4.4.7.2: Effect of different pH on antimicrobial activity of Penicillium citrinum
4.4.8 Effect of pH on Antimicrobial Activity of Aspergillus spp.
Aspergillus terreus when grown at different pH showed no antimicrobial activity
at pH 3-4 and the activity increased from pH 5 which remained more or less stable upto
pH 7 and then declined upto pH 10, so the pH optima lies between pH 5-pH 7. There
was no significant difference in the antimicrobial activity from pH 5 to pH 7. Biomass
of the fungus increased from pH 3-7 and remain stable till pH 9. (Fig. 4.4.8.1).
Similarly, Aspergillus wentii, when grown at different pH showed relatively low
antimicrobial activity at pH 3- 4 and the activity increased from pH 5 which remained
more or less stable upto pH 9 and then declined , so the pH optima lies between pH 5-
pH 7. Maximum biomass of 9.54 mg/ml was observed at pH 3 which declined till pH 5
and again increased upto pH 7 (Fig. 4.4.8.2).
0
2
4
6
8
10
12
0
5
10
15
20
25
30
35
40
3 4 5 6 7 8 9
Bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
pH
S.aureus
S.epidermidis
K.pneumoniae
MRSA
C.albicans
Biomass
Results
80
Figure 4.4.8.1: Effect of different pH on antimicrobial activity of Aspergillus terreus
Figure 4.4.8.2: Effect of different pH on antimicrobial activity of Aspergillus wentii
0
1
2
3
4
5
6
7
8
9
0
5
10
15
20
25
3 4 5 6 7 8 9 10
Bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
pH
E. faecalis
S. aureus
S.epidermidis
E.coli
K.pneumoniae 1
P.aeruginosa
Sh. flexneri
Salm.Typhimurium 1
Salm.typhimurium 2
MRSA
C.albicans
Biomass
7
7.5
8
8.5
9
9.5
10
0
5
10
15
20
25
30
3 4 5 6 7 8 9 10
bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
pH
S.aureus
S.epidermidis
K.pneumoniae1
MRSA
C.albicans
Biomass
Results
81
4.4.9 Effect of different temperature on antimicrobial activity of Penicillium spp.
Both the Penicillium spp. when grown at different temperature, their resulting
extracellular culture broth were tested for antimicrobial activity. Maximum
antimicrobial activity was found at 25°C. Both the fungi showed a good growth upto
30°C and none of these could grow at 35°C. The temperature optima for the maximum
biomass and antimicrobial activity were best observed in the culture broth of both the
fungi at 25-30°C. However in case of Penicillium citrinum the activity was more or less
stable from 15-30°C. Biomass of both the fungi increased as the temperature increased
upto 25°C and then declined (Fig. 4.4.9.1; 4.4.9.2).
Figure 4.4.9.1: Effect of different temperature on antimicrobial activity of Penicillium
expansum.
0
2
4
6
8
10
12
14
0
5
10
15
20
25
30
35
40
45
15 20 25 30 35 40 45
bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Temperature (˚C)
S.aureus
S.epidermidis
K.pneumoniae 1
MRSA
C.albicans
Biomass
Results
82
Figure 4.4.9.2 Effect of different temperature on antimicrobial activity of Penicillium
citrinum.
4.4.10 Effect of different temperature on antimicrobial activity of Aspergillus spp.
Maximum biomass of 8.8 mg/ml and antimicrobial activity of Aspergillus wentii
was observed at 25°C which further decreased with the increase in temperature. The
antimicrobial activity was more or less stable upto 30°C and the fungus were not able to
grow after 30°C. Similarly Aspergillus terreus showed no growth at 15°C but showed
less biomass at 20°C which increase with increase in temperature at reaches its
maximum at 30°C. However the antimicrobial activity was found to be similar at 25°C
and 30°C which decreased slightly at 35°C and further the fungus was not able to grow
above 35°C (Fig. 4.4.10.2; 4.4.10.2).
0
1
2
3
4
5
6
7
8
9
10
0
5
10
15
20
25
30
35
40
15 20 25 30 35
bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Temperature (°C)
S.aureus
S.epidermidis
K.pneumoniae 1
MRSA
C.albicans
Biomass
Results
83
Figure 4.4.10.1 Effect of different temperature on antimicrobial activity of Aspergillus
wentii
Figure 4.4.10.2: Effect of different temperature on antimicrobial activity of Aspergillus
terreus.
0
1
2
3
4
5
6
7
8
9
10
0
5
10
15
20
25
30
15 20 25 30 35 40 45
bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Temperature in °C
S.aureus
S.epidermidis
K.pneumoniae1
MRSA
C.albicans
Biomass
0
1
2
3
4
5
6
7
8
9
0
5
10
15
20
25
15 20 25 30 35 40 45
Bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Temperature C
E. faecalis
S. aureus
S.epidermidis
E.coli
K.pneumoniae 1
P.aeruginosa
Sh. flexneri
Salm.Typhimurium 1
Salm.Typhimurium 2
MRSA
C.albicans
Biomass
Results
84
4.4.11 Effect of media components and their concentration on Antimicrobial
activity of Penicillium spp.
To find out the best concentration of different media components, each
component was added at variable concentrations without altering the remaining
composition.
In the experimental setup, YPDS I containing different concentration (1-10%) of
dextrose, biomass of both the Penicillium spp. showed a consistent increase upto 10%.
The antimicrobial activity for both the Penicillium spp. declined with the increase in
concentration and no activity was found at 8 and 10 %. The maximum antimicrobial
activity was observed at 1% dextrose in both the Penicillium spp. (Fig. 4.4.11.1;
4.4.11.2)
Similarly in setup, YPDS II containing different concentration of starch,
biomass of both the Penicillium spp. increased with increase in concentration and was
found to be maximum at 10% of starch while the antimicrobial activity in both the
species was found to be maximum at 1 % of starch which decreased with the increase in
concentration of starch. (Fig. 4.4.11.3; 4.4.11.4)
In the experimental setup YPDS-III and YPDS-IV, where different
concentration (1-10%) of peptone and yeast extract, respectively were tested, 2 %
concentration of peptone and yeast extract was found to be the best for antimicrobial
activity. Biomass of both the fungi viz. Penicillium expansum and Penicillium citrinum
increased with increase in concentration of nitrogen sources. Thus, for obtaining the
best antimicrobial activity, carbon and nitrogen source, were used at 1 and 2 %,
respectively, in the subsequent experiments. (Fig. 4.4.11.5; 4.4.11.6; 4.4.11.7; 4.4.11.8)
Results
85
Figure 4.4.11.1. Effect of different concentration of dextrose on antimicrobial activity
of Penicillium expansum
Figure 4.4.11.2 Effect of different concentration of dextrose on antimicrobial activity of
Penicillium citrinum
0
5
10
15
20
25
0
5
10
15
20
25
30
35
40
1% 2% 4% 6% 8% 10%
bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Concentration of dextrose
S.aureus
S.epidermidis
K.pneumoniae 1
MRSA
C.albicans
Biomass
0
5
10
15
20
25
0
5
10
15
20
25
30
35
1% 2% 4% 6% 8% 10%
bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Concentration of dextrose
S.aureus
S.epidermidis
K.pneumoniae
MRSA
C.albicans
Biomass
Results
86
Figure 4.4.11.3 Effect of different concentration of starch on antimicrobial activity of
Penicillium expansum
Figure 4.4.11.4 Effect of different concentration of starch on antimicrobial activity of
Penicillium citrinum
0
5
10
15
20
25
30
35
40
45
0
5
10
15
20
25
30
35
40
1% 2% 4% 6% 8% 10%
bio
ma
ss (
mg
/ml)
Zo
ne
f i
nh
ibit
ion
(m
m)
Concentration of starch
S.aureus
S.epidermidis
K.pneumoniae 1
MRSA
C.albicans
Biomass
0
5
10
15
20
25
30
35
40
0
5
10
15
20
25
30
35
40
1% 2% 4% 6% 8% 10%
bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Concentration of starch
S.aureus
S.epidermidis
K.pneumoniae 1
MRSA
C.albicans
Biomass
Results
87
Figure 4.4.11.5 Effect of different concentration of peptone on antimicrobial activity of
Penicillium expansum.
Figure 4.4.11.6 Effect of different concentration of peptone on antimicrobial activity of
Penicillium citrinum
0
5
10
15
20
25
0
5
10
15
20
25
30
35
1% 2% 4% 6% 8% 10%
bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Concentration of peptone
S.aureus
S.epidermidis
K.pneumoniae 1
MRSA
C.albicans
Biomass
0
2
4
6
8
10
12
14
0
5
10
15
20
25
30
1% 2% 4% 6% 8% 10%
bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Concentration of peptone
S. aureus
S. epidermidis
K. pneumoniae1
MRSA
C. albicans
Biomass
Results
88
Figure 4.4.11.7 Effect of different concentration of yeast extract on antimicrobial
activity of Penicillium expansum
Figure 4.4.11.8 Effect of different concentration of yeast extract on antimicrobial
activity of Penicillium citrinum
0
5
10
15
20
25
0
5
10
15
20
25
30
35
40
1% 2% 4% 6% 8% 10%
bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Concentration of yeast extract
S.aureus
S.epidermidis
K.pneumoniae 1
MRSA
C.albicans
Biomass
0
2
4
6
8
10
12
14
16
18
0
5
10
15
20
25
30
35
40
1% 2% 4% 6% 8% 10%
bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibti
on
(m
m)
Concentration of yeast extract
S.aureus
S.epidermidis
K.pneumoniae 1
MRSA
C.albicans
Biomass
Results
89
4.4.12 Effect of media components and their concentration on Antimicrobial
activity of Aspergillus spp.
Aspergillus wentii when grown in experimental set up I, the maximum
antimicrobial activity was observed at 1 % dextrose which declined appreciably at 4%
and showed no activity at all at 6% against any bacteria except the yeast strain which
showed only a moderate decline up to 10% dextrose. Similarly, in set up II, maximum
biomass was obtained at the highest concentration of starch i.e. 10% while the best
antimicrobial activity was observed at 1% starch which declined with further increase in
the concentration. In the third set of experiment, 1% peptone gave the best antimicrobial
activity which was more or less stable upto 2%. Biomass of the fungus increased with
increase in concentration of nitrogen sources. Thus, to work out the combined effect of
carbon and nitrogen sources, these were respectively tested at 1% concentration (Figure
4.4.12.1-4.4.12.4). Similarly in case of Aspergillus terreus maximum activity was
observed at 1% dextrose which remain more or less stable upto 6% and then declined.
In experimental setup II 1% starch give maximum antimicrobial activity which remain
more or less stable to 6% and then declined. Further in experimental setup III and IV,
1% peptone and 1% yeast extract showed maximum antimicrobial activity which
remain more or less stable to 4% and then declined. Biomass of both the fungi
increased with increase in concentration of nitrogen sources. Biomass of both the fungi
increased with increase in concentration of nitrogen sources. Thus, to work out the
combined effect of carbon and nitrogen sources, these were respectively tested at 1%
concentration.
Results
90
Figure 4.4.12.1 Effect of different concentration of dextrose on antimicrobial activity of
Aspergillus wenti
Figure 4.4.12.2 Effect of different concentration of dextrose on antimicrobial activity of
Aspergillus terreus
0
2
4
6
8
10
12
14
0
5
10
15
20
25
30
1% 2% 4% 6% 8% 10%
bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Concentration of dextrose
S.aureus
S.epidermidis
K.pneumoniae1
MRSA
C.albicans
Biomass
0
2
4
6
8
10
12
14
16
18
20
0
5
10
15
20
25
0.50% 1% 2% 4% 6% 8% 10%
Bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Concentration of dextrose
E.faecalis
S. aureus
S.epidermidis
E.coli
K.pneumoniae 1
P.aeruginosa
Sh. flexneri
Salm.Typhimurium 1
Salm.Typhimurium 2
MRSA
C.albicans
Biomass
Results
91
Figure 4.4.12.3 Effect of different concentration of starch on antimicrobial activity of
Aspergillus wentii
Figure 4.4.12.4 Effect of different concentration of starch on antimicrobial activity of
Aspergillus terreus
0
5
10
15
20
25
0
5
10
15
20
25
30
1% 2% 4% 6% 8% 10%
Bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Concentration of starch
S.aureus
S.epidermidis
K.pneumoniae
MRSA
Calbicans
Biomass
0
10
20
30
40
50
60
0
5
10
15
20
25
0.50% 1% 2% 4% 6% 8% 10%
Bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Concentration of starch
E. faecalis
S. aureus
S.epidermidis
E.coli
K.pneumoniae 1
P.aeruginosa
Sh. flexneri
Salm.Typhimurium 1
Salm.Typhimurium 2
MRSA
C.albicans
Biomass
Results
92
Figure 4.4.12.5 Effect of different concentration of peptone on antimicrobial activity of
Aspergillus wentii
Figure 4.4.12.6 Effect of different concentration of peptone on antimicrobial activity of
Aspergillus terreus
0
2
4
6
8
10
12
14
0
2
4
6
8
10
12
14
16
18
20
1% 2% 4% 6% 8% 10%
bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Concentration of peptone
S.aureus
S.epidermidis
K.pneumoniae1
MRSA
C. albicans
Biomass
0
5
10
15
20
25
30
35
40
0
5
10
15
20
25
0.50% 1% 2% 4% 6% 8% 10%
Bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Concentration of peptone
E. faecalis
S. aureus
S.epidermidis
E.coli
K.pneumoniae 1
P.aeruginosa
Sh. flexneri
Salm.Typhimurium 1
Salm.Typhimurium 2
MRSA
C.albicans
Biomass
Results
93
Figure 4.4.12.7 Effect of different concentration of yeast extract on antimicrobial
activity of Aspergillus wentii
Figure 4.4.12.8 Effect of different concentration of yeast extract on antimicrobial
activity of Aspergillus terreus
0
2
4
6
8
10
12
14
16
0
5
10
15
20
25
30
1% 2% 4% 6% 8% 10%
bio
ma
ss(m
g/m
l)
Zo
ne
of
inh
ibit
ion
(m
m)
Concentration of yeast extract
S.aureus
S.epidermidis
K.pneumoniae1
MRSA
C.albicans
Biomass
0
5
10
15
20
25
30
35
40
45
0
5
10
15
20
25
0.50% 1% 2% 4% 6% 8% 10%
Bio
ma
ss (
mg
/ml)
Zo
ne
of
inh
ibit
ion
(m
m)
Concentration of yeast extract
E. faecalis
S. aureus
S.epidermidis
E.coli
K.pneumoniae 1
P.aeruginosa
Sh. flexneri
Salm.Typhimurium 1
Salm.Typhimurium 2
MRSA
C.albicans
Biomass
Results
94
4.4.13 Effect of different carbon sources on antimicrobial activity of Penicillium spp.
To find out the best carbon source all the selected fungi were grown in different
media replaced with different carbon sources. Starch was found to the best carbon
source for both the Penicillium spp. with an inhibition zone ranging from 18-40 mm in
Penicillium expansum and 17-35 mm in Penicillium citrinum. The order of
antimicrobial activity under different carbon sources was as follows Starch > Dextrose
> Glycerol > Lactose > Maltose > Sucrose (Table 4.4.13.1). Maximum biomass of
Penicillium expansum was recorded in the presence of glycerol while starch was best
for Penicillium citrinum (Table 4.4.13.2).
Table 4.4.13.1 Effect of different carbon sources on antimicrobial activity Penicillium
expansum
Carbon Source Starch Dextrose Lactose Maltose Sucrose Glycerol
Biomass (mg/ml) 13.7 13.04 8.78 13.4 13.5 14.9
Microorganisms Zone of inhibition (mm)
S. aureus 39.5±0.7 37.5±2.12 33±2.82 31±1.41 25±0 35±0
S. epidermidis 40±0 38±1.41 38±0 38±2.12 26±0 40±0
K. pneumoniae 27±0 23±2.8 22±0 20±0 19±1.41 25±0
MRSA 18.5±0.7 17±1.41 17.5±0.7 16±2.12 16±0 18.5±0.7
C. albicans 18±1.41 18±0 17.5±0.7 17.5±0.7 17±2.12 18±2.12
Values are expressed as Mean ± Standard Deviation
Table 4.4.13.2 Effect of different carbon sources on antimicrobial activity of
Penicillium citrinum
Carbon Source Starch Dextrose Lactose Maltose Sucrose Glycerol
Biomass (mg/ml) 9.7 6.92 4.94 8.16 8.04 8.54
Microorganisms Zone of inhibition (mm)
S. aureus 35±0 31.5±2.1 30±0 27±1.41 20.5±0.7 28±0
S. epidermidis 35±2.82 32±0 29±1.41 27.5±3.5 23.5±2.1 29.5±2.1
K. pneumoniae 18±2.8 16±0 20±0 12±0 11±0 15±0
MRSA 17±2.12 17±0 0 0 0 0
C.albicans 17±0 17±1.41 15±0.7 15±0.7 15±0.7 15±0
Values are expressed as Mean ± Standard Deviation
Results
95
4.4.14 Effect of different carbon sources on antimicrobial activity of Aspergillus spp.
Starch was found to the best carbon source for both the fungi and the order of
antimicrobial activity expressed by Aspergillus wentii in different carbon sources was as
follows Starch > Dextrose > Lactose > Maltose > Sucrose> Glycerol (Table 4.4.14.1).
Maximum biomass of Aspergillus wentii was recorded in the presence of maltose.
Aspergillus terreus showed maximum antimicrobial activity when starch was used as a
carbon source with a zone of inhibition ranging from 16 mm to 20mm and order of
antimicrobial activity in different carbon sources was as follows Starch> Dextrose >
Sucrose > Lactose > Glycerol >Maltose. No antimicrobial activity was found when
maltose was used as a carbon source. Maximum biomass was found when starch was
used as a carbon source (Table 4.4.14.2).
Table 4.4.14.1 Effect of different carbon sources on antimicrobial activity of
Aspergillus wentii
Carbon Source Starch Dextrose Lactose Maltose Sucrose Glycerol
Biomass(mg/ml) 14 13 8 15 13 12
Microorganisms Zone of inhibition (mm)
S. aureus 23±0.57 20±0 18±2.12 14±1 14±0 12±0.57
S. epidermidis 24±0.57 23±1.5 17±0.57 15±1.5 15±0.57 14±0.57
K. pneumoniae1 15±1 14±0.57 12±0.57 0 0 12±0.57
MRSA 26±0.57 22±0.57 15±0 16±0 15±0.57 12±1
C. albicans 25±0 20±0 14±0 13±0.57 14±0.57 0
Values are expressed as Mean ± Standard Deviation
Results
96
Table 4.4.14.2 Effect of different carbon sources on antimicrobial activity of
Aspergillus terreus
Dextrose Starch Sucrose Lactose Glycerol Maltose
Biomass (mg/ml) 9.4 11.78 10.04 3.34 5.56 4.5
Microorganisms Zone of inhibition (mm)
E. faecalis 16±0 16±1.4 15±0 0 0 0
S. aureus 15.5±0.7 15±1.4 15±1.4 15±1.4 15.5±0.7 0
S. epidermidis 16±0.7 16±1.4 16.5±0.7 16±1.4 16.5±0.7 0
E. coli 16±0 16.5±0.7 15.5±0.7 0 0 0
K. pneumoniae 1 17.5±0.7 18±0 17.5±0.7 15±1.4 15±1.4 0
P. aeruginosa 16±1.4 16±1.4 15±0 0 0 0
Sh. flexneri 16±1.4 16.5±0.7 16.5±0.7 0 0 0
Salm. Typhimurium 2 17.5±0.7 17.5±0.7 16±0 0 0 0
MRSA 17±0 18±0 16±1.4 0 0 0
C. albicans 20±0 21.5±0.7 19±0 15±0 14±1.4 0
Values are expressed as Mean ± Standard Deviation
4.4.15 Effect of different nitrogen sources on antimicrobial activity of Penicillium spp.
Similarly, to work out best nitrogen source yeast extract was the best among
organic and inorganic nitrogen sources. Though, soybean meal and peptone were also
good sources of nitrogen for bioactivity, however biomass of both the fungi was
maximum in soya bean meal based medium. Nevertheless, both the fungi could not
grow in the presence of urea (Table 4.4.15.1). However, other nitrogen sources such as,
ammonium chloride, ammonium sulphate, malt extract, ammonium dihydrogen
phosphate, L-aspargine, and ammonium nitrate showed variable biomass with no
antibacterial activity except in sodium nitrate (Table 4.4.15.2) .
Res
ult
s
97
Tab
le 4
.4.1
5.1
: E
ffec
t of
dif
fere
nt
nit
rogen
sourc
es o
n a
nti
mic
rob
ial
ac
tivit
y o
f P
enic
illi
um
exp
ansu
m (
HT
-28)
Nit
rog
en S
ourc
e P
epto
ne
Yea
st e
xtr
act
So
yab
ean m
eal
Mal
t ex
trac
t C
asei
n
Sod
ium
Nit
rate
A
mm
oniu
m n
itra
te
Pota
ssiu
m n
itra
te
Am
mo
niu
m c
hlo
rid
e U
rea
Bio
mas
s (m
g/m
l)
16
.1
14
.56
25
.6
8.8
5
8.9
3
.74
3.2
5
4.6
2
3.5
0
Mic
roorg
anis
ms
Zon
e of
inhib
itio
n (
mm
)
S.a
ure
us
30
±0
36
±1
.41
36
±0
12
±0
0
11
±0
0
0
0
0
S.e
pid
erm
idis
3
1±
1.4
1
40
±0
37
.5±
0.7
07
0
0
0
0
0
0
0
K.p
neu
mon
iae
16
.5±
0.7
07
23
±0
20
±0
0
0
0
0
0
0
0
MR
SA
1
6±
2.1
2
16
±0
.7
16
±1
.41
0
0
0
0
0
0
0
C.a
lbic
an
s 1
8±
0
18
±1
.41
18
±0
.7
17
±0
.7
0
17
±2
.12
0
0
0
0
Val
ues
are
ex
pre
ssed
as
Mea
n ±
Sta
nd
ard
Dev
iati
on
Tab
le 4
.4.1
5.2
Eff
ect
of
dif
fere
nt
nit
rogen
sourc
es o
n a
nti
mic
rob
ial
acti
vit
y o
f P
enic
illi
um
cit
rinum
(H
T-4
6)
Nit
rog
en
sourc
es
Pep
ton
e Y
east
Ex
trac
t S
oyab
ean m
eal
Mal
t E
xtr
act
Cas
ein
S
od
ium
Nit
rate
A
mm
oniu
m N
itra
te
Pota
ssiu
m n
itra
te
Am
mo
niu
m c
hlo
rid
e U
rea
Bio
mas
s
(mg/m
l)
16
.8
14
.72
21
.4
8.6
5
8.5
5
.56
4.4
5
3.7
5
3.7
5
0
Mic
roorg
anis
ms
Zon
e of
inhib
itio
n (
mm
)
S.a
ure
us
23
±0
33
±0
.70
7
20
±0
0
0
10
±1
.41
0
0
0
0
S.e
pid
erm
idis
2
5±
0
29
.5±
0.7
07
18
±0
0
0
0
0
0
0
0
K.p
neu
mon
iae
12
.5±
0.7
07
19
±0
15
±1
.41
0
0
0
0
0
0
0
MR
SA
1
2±
1.4
1
17
±2
.12
17
±1
.41
0
0
0
0
0
0
0
C.a
lbic
an
s 1
6±
0.7
07
17
±0
.7
18
±1
.41
15
±0
.7
0
15
±2
.1
0
0
0
0
Val
ues
are
ex
pre
ssed
as
Mea
n ±
Sta
nd
ard
Dev
iati
on
Results
98
4.4.16 Effect of different nitrogen sources on antimicrobial activity of Aspergillus spp.
Aspergillus wentii, showed, yeast extract was the best among organic and
inorganic nitrogen sources. Soybean meal and peptone were also good sources of
nitrogen for bioactivity. (Table 4.4.16.1)Maximum biomass of both the fungi viz.
Aspergillus terreus and Aspergillus wentii was recorded in the soya bean meal based
medium and both the fungi was not able to grow in the presence of urea. C. albicans
was found to be most sensitive and showed sensitivity when casein was used as a
nitrogen source in the medium. Aspergillus terreus showed the maximum activity in
yeast extract, soyabean meal and peptone based medium followed by malt extract,
casein and ammonium sulphate. However, other nitrogen sources such as, ammonium
chloride, ammonium dihydrogen phosphate, sodium nitrate and ammonium nitrate
showed variable biomass with no antimicrobial activity. C. albicans was found to be the
most sensitive organism and showed sensitivity to Aspergillus terreus grown in almost
all the nitrogen based medium (Table 4.4.16.2).
Res
ult
s
99
Tab
le 4
.4.1
6.1
Eff
ect
of
dif
fere
nt
nit
rogen
sourc
es o
n a
nti
mic
rob
ial
acti
vit
y o
f A
sper
gil
lus
wen
tii
Pep
ton
e
Yea
st
extr
act
Mal
t
extr
act
So
yab
ean
mea
l
Pota
ssiu
m
nit
rate
C
asei
n
Am
mo
niu
m
sulp
hat
e S
od
ium
nit
rate
Am
mo
niu
m
chlo
rid
e U
rea
Bio
mas
s (m
g/m
l)
14
.34
18
.62
11
.5
18
.56
7.2
5
12
.75
5.5
6
4.2
5
2
0
Mic
roorg
anis
m
Zon
e of
inhib
itio
n (
mm
)
S. a
ure
us
16
±0
.70
7
32
±0
15
±0
.57
23
±0
0
0
0
0
0
0
S.e
pid
erm
idis
1
4±
1.4
1
32
±0
16
±0
.57
20
±0
0
0
0
0
0
0
K.p
neu
mon
iae1
1
1±
0
20
±0
12
±1
.41
14
±0
0
0
0
0
0
0
MR
SA
1
8±
1.4
2
7±
0.5
7
19
±0
.57
23
±0
0
0
0
0
0
0
C. a
lbic
an
s 2
0±
0.5
7
26
±1
.41
19
±0
24
±0
.57
0
0
0
14
±0
.57
0
0
Tab
le 4
.4.1
6.2
Eff
ect
of
dif
fere
nt
nit
rogen
sourc
es o
n a
nti
mic
rob
ial
acti
vit
y o
f A
sper
gil
lus
terr
eus
Pep
ton
e Y
east
ex
trac
t M
alt
extr
act
So
yab
ean m
eal
Pott
assi
um
nit
rate
C
asei
n
Am
mo
niu
m s
ulp
hat
e
Sod
ium
nit
rate
A
mm
oniu
m c
hlo
rid
e U
rea
Bio
mas
s (m
g/m
l)
8.5
1
2
10
.02
29
.5
9.5
2
6
11
.52
3.4
4
0
Zon
e of
inhib
itio
n (
mm
)
E. fa
eca
lis
16
±0
15
±1
.4
12
±0
15
.5±
2.1
0
0
0
0
0
0
0
S. a
ure
us
17
.5±
0.7
1
7±
1.4
1
5.5
±2
.1
17
±1
.4
0
14
±1
.4
12
±1
.4
0
0
0
0
S.e
pid
erm
idis
1
6±
1.4
1
7±
1.4
1
4.5
±2
.1
16
±1
.4
0
14
.5±
0.7
0
0
0
0
0
E.c
oli
1
6.5
±0
.7
17
.5±
0.7
1
3±
0
16
.5±
0.7
0
1
2.5
±2
.1
0
0
0
0
0
K.p
enu
mon
iae
1
17
±1
.4
18
±1
.4
14
±1
.4
18
.5±
0.7
0
1
5.5
±2
.1
13
±0
0
0
0
0
P.a
eru
gin
osa
1
7±
0
16
±1
.4
14
.5±
2.1
1
6±
1.4
0
0
0
0
0
0
0
S f
lexn
eri
16
.5±
0.7
1
6.5
±0
.7
14
±1
.4
17
.5±
0.7
0
0
0
0
0
0
0
S.t
yph
imuri
um
1
16
.5±
0.7
1
7.5
±2
.1
12
.5±
0.7
1
6.5
±2
.1
0
0
0
0
0
0
0
S.t
yph
imuri
um
2
16
±1
.4
18
±1
.4
14
.5±
2.1
1
5.5
±2
.1
0
0
0
0
0
0
0
C.a
lbic
an
s 2
1±
0
21
±1
.4
16
.5±
0.7
2
0±
0
15
.5±
2.1
1
7.5
±0
.7
16
±1
.4
15
.5±
2.1
1
6.5
±0
.7
0
MR
SA
1
7±
1.4
1
7±
0
12
±0
18
±0
0
0
0
0
0
0
0
Results
100
4.5 Box-Behnken design for statistical optimization of carbon and nitrogen
sources
On the basis of above experiments where carbon and nitrogen sources were
optimized qualitatively and quantitatively they were further optimized by Box-Behnken
design for all the selected four fungi. On the basis of results obtained from screening of
different carbon and nitrogen sources through one-factor-at-a-time classical method;
starch, dextrose and yeast extract were taken independent variables for the optimization
by RSM using Box-Behnken design of experiments. Each variable was studied at three
levels (-1, 0, +1); for starch, dextrose and yeast extract at different concentrations. The
experiment for each organism included 17 flasks with five replicates having all the three
variables at their central coded values. The mathematical relationship of response G (for
each parameter) and independent variable X (X1, dextrose; X2, Starch; X3, Yeast
extract) was calculated by the quadratic model equation.
4.5.1 Statistical optimization of carbon and nitrogen sources of Penicillium
expansum
Fitting the model
The data obtained from quadratic model equation was found to be significant. It
was verified by F- value and the analysis of variance (ANOVA) by fitting the data of all
independent observations in response surface quadratic model. The results for model F-
value imply that the model is significant. R2 value for all the responses ranged between
90-94.1 %, which showed suitable fitting of the model in the designed experiments. The
final predictive equations for each response S. aureus (G1), S. epidermidis (G2), K.
pneumoniae 1 (G3), C. albicans (G4) and MRSA (G5) obtained were as follows:
S. aureus (G1)
36-1.25 X1+ 5 X2+ 2.25 X3- 1.6 X12- 3.1 X2
2-0.1 X3
2-1.5 X1X2+ 2 X1X3-0.5 X2X3
Starch significantly affected the antimicrobial activity. Linear effect of starch was
highly significant with P value ≤ 0.005. Linear effect of yeast extract (X3) and squared
effect of starch was found significant at P value ≤ 0.05. The response surface graphs
Results
101
showed the highest activity at 2 % Starch, 1 % Dextrose and 1.2 % Yeast extract (Fig.
4.5.1.1).
S. epidermidis (G2 )
34- 4.37 X1+5.5 X2+2.37 X3-1.62 X12-1.87 X2
2-1.12 X3
2+2.00 X1X2+2.75 X1X3-2.5
X2X3
The seventeen flasks of different combinations of medium demonstrated variable
activity (Table 4.5.1.1). Linear effect of dextrose and starch was found to be highly
significant with P value ≤ 0.005. Dextrose also affects significantly in interactive terms
with yeast extract with P value ≤ 0.05. Linear effect of yeast extract was also significant
with P value ≤ 0.05. The response surface graph showed highest activity at 2 % Starch,
1.5 % Yeast extract and 1.25 % Dextrose (Fig. 4.5.1.2).
K. pneumoniae 1 (G3 )
22.6-5.5 X1+0.625 X2+5.125 X3-0.425 X12-2.175 X2
2+0.325 X3
2+1.00 X1X2+3.00
X1X3+0.25 X2X3
As predicted by the contour plots the maximum antimicrobial activity was supported by
2 % Yeast extract, 0.5% Dextrose and Starch 1.25 % (Fig. 4.5.1.3).. Linear effect of
dextrose and yeast extract was found to be highly significant with P value ≤ 0.05.
Interactive effect of dextrose and yeast extract was also found to be significant with P
value ≤ 0.05.
C. albicans (G4)
20.6-0.5 X1-1.375 X2+4.125 X3-2.8 X12-1.05 X2
2-1.55 X3
2+0.25 X1X2-0.25 X1X3+1.5
X2X3
The results of Box Behnken design are described in Table 4.5.1. Linear effect of yeast
extract was highly significant with P value ≤ 0.005. Dextrose showed squared
significant effect on antimicrobial activity with P value ≤ 0.05. The response surface
graph showed highest activity at 2 % yeast extract, 1-1.5 % starch and 1.25 % dextrose
(Fig. 4.5.1.4).
MRSA (G5)
Results
102
26.4+0.125 X1+3.75 X2+0.875 X3-3.325 X12-1.575 X2
2-1.325 X3
2+0.5 X1X2-5.25 X1X3-
2.5 X2X3
Linear effect of starch significantly affected the antimicrobial activity with P value ≤
0.005. Dextrose was found to be significant in terms of squared effect (P value ≤ 0.05)
and in interactive terms (P value ≤ 0.005). The response surface graph showed highest
activity at 2 % Starch, 1 % Yeast extract and 1.25 % Dextrose (Fig. 4.5.1.5).
4.5.1.1 Validation of results
Thus from the overall assessment, 2 % Starch, 1.25 % Dextrose and 1.5 % Yeast
extract and 1% peptone in YPDS medium may be regarded as the optimized conditions
for antimicrobial activity The F- value and R2 value showed that the model, correlated
well with measured data and was statistically significant. To confirm the adequacy of
the model the verification experiments using optimum medium composition as
described above were carried out in triplicates. The results showed that antimicrobial
activity (Table 4.5.1) was enhanced by, 1.2 folds (C. albicans), 1.8 folds (MRSA), 1.1
folds (K. pneumoniae 1).
Figure 4.5.1.1 Contour Plot of S. aureus
Results
103
Figure 4.5.1.2 Contour Plot of S. epidermidis.
Figure 4.5.1.3 Contour Plot of K. pneumoniae1.
Figure 4.5.1.4 Contour Plot C. albicans.
Results
104
Table 4.5.1: Result of Box –Behnken design experiment for antimicrobial potential of
Penicillium expansum
Run Order
*X1 *X2 *X3 S. aureus S. epidermidis K. pneumoniae C. albicans MRSA
(g/100ml) Zone of inhibition (mm)
1 1.25 0.5 0.4 25 21 15 17 15
2 1.25 2 2 40 36 27 22 27
3 1.25 1.25 1.2 38 35 25 21 27
4 1.25 1.25 1.2 37 32 23 22 26
5 1.25 1.25 1.2 37 37 23 22 27
6 1.25 1.25 1.2 37 34 21 20 25
7 2 1.25 2 36 31 26 21 15
8 1.25 1.25 1.2 32 32 21 18 27
9 0.5 2 1.2 39 37 27 16 24
10 2 1.25 0.4 29 20 11 12 27
11 2 0.5 1.2 27 20 11 17 18
12 0.5 0.5 1.2 25 30 27 20 17
13 0.5 1.25 0.4 37 37 25 11 18
14 1.25 2 0.4 35 37 15 12 27
15 1.25 0.5 2 32 30 26 21 25
16 0.5 1.25 2 36 37 28 21 27
17 2 2 1.2 35 35 15 14 27
*X1 Dextrose,
*X2 Starch,
*X3 Yeast extract
4.5.2 Statistical optimization of carbon and nitrogen sources of Penicillium
citrinum
S. aureus (G1)
32.4+6.87 X1+1.25 X2-2.1 X3-3.0 X12-5.32 X2
2-1.57 X3
2+0.5 X1X2-2.75 X1X3+2.0 X2X3
Results
105
Linear effect of starch and squared effect of dextrose was highly significant with P
value ≤ 0.001. Similarly linear effect of yeast extract and squared effect of starch was
significant with P value ≤ 0.05. R2 value 94.4%. The response surface graph showed the
highest activity at 2% starch, 1-1.5% dextrose, with yeast extract 0.2 %.(Figure 4.5.2.1)
S. epidermidis (G2 )
32.2+7.25 X1+0.5 X2-2.5 X3-1.1 X12-6.6 X2
2-1.6 X3
2+2 X1X2-3.5 X1X3+1.0 X2X3
Linear effect of starch and squared effect of dextrose was highly significant with P
value ≤ 0.001. Similarly linear effect of yeast extract was found to be significant with P
value ≤ 0.05. Interactive effect of starch and yeast extract was also found to be
significant P value ≤ 0.05. R2 value 92.7 % (Figure. 4.5.2.2)
K. pneumoniae 1 (G3 )
16.2+4.1 X1+1.87 X2-1.25 X3+3.15 X12-1.35 X2
2+0.4 X3
2+ 3.0 X1X2-3.25 X1X3-0.25 X2X3
Linear effect of starch was highly significant with P value ≤ 0.001. Linear effect of
dextrose and squared effect of starch was also significant with P value ≤ 0.05.
Interactive effect of starch and yeast extract, starch and dextrose was significant with P
value ≤ 0.05. R2 value 91.3 % (Figure. 4.5.2.3).
MRSA (G5)
16.4+3.25 X1+1.75 X2-1 X3+3.4 X12-0.57 X2
2+0.92 X3
2+1.75 X1X2-3.25 X1X3-0.25 X2X3
Linear effect of starch was highly significant with P value ≤ 0.005. Squared effect of
starch and interactive effect starch and yeast extract was significant with P value ≤ 0.05.
R2 value 87.1% (Figure. 4.5.2.4).
C. albicans
17.4+4.12 X1+2.12 X2-2.0 X3+3.92 X12-0.57 X2
2+0.17 X3
2+3.75 X1X2-4.5 X1X3-0 X2X3
Linear effect of starch was highly significant with P value ≤ 0.005. Linear effect
of dextrose and squared effect of starch was significant with P value ≤ 0.05. Interactive
effect of starch and dextrose; starch and yeast extract was significant with P value ≤
0.05. R2 value 90.8 % (Figure. 4.5.2.5).
Results
106
4.5.2.1 Validation of results
Thus from the overall assessment 2 % Starch, Dextrose 1-1.5 % and Yeast
extract 0.2 % and 1 % peptone in YPDS medium may be regarded as the optimized
conditions for antimicrobial activity. The F value and R2 value showed that the model
correlated well with measured data and was statistically significant. To confirm the
adequacy of the model the verification experiments using optimum medium
composition as described above were carried out in triplicates. The results showed the
antimicrobial activity (Table 4.5.2) was enhanced by 1.1 folds.
Figure 4.5.2.1: Contour Plot of Staphylococcus aureus
Figure 4.5.2.2: Contour Plot of Staphylococcus epidermidis.
Results
107
Figure 4.5.2.3: Contour Plot of Klebsiella pneumoniae 1.
Figure 4.5.2.4: Contour Plot of MRSA.
Figure 4.5.2.5: Contour Plot of C. albicans.
Results
108
Table 4.5.2: Result of Box –Benhken design experiment for antimicrobial activity of
Penicillium citrinum
Std.
Dextrose Starch Yeast extract S. aureus S. epidermidis K. pneumoniae C. albicans MRSA
(g/100 ml) Zone of Inhibition (mm)
1 0.5 0.5 0.5 15 17 14 17 15
2 0.5 2 0.5 27 28 16 17 18
3 2 0.5 0.5 20 17 14 17 17
4 2 2 0.5 34 36 28 32 27
5 1.25 0.5 0.2 20 20 15 16 17
6 1.25 2 0.2 40 41 30 34 30
7 1.25 0.5 0.8 21 25 16 18 18
8 1.25 2 0.8 30 32 18 18 18
9 0.5 1.25 0.2 30 30 14 17 15
10 2 1.25 0.2 25 26 16 18 17
11 0.5 1.25 0.8 22 20 15 16 17
12 2 1.25 0.8 25 20 16 17 18
13 1.25 1.25 0.5 33 34 16 17 16
14 1.25 1.25 0.5 30 28 17 17 17
15 1.25 1.25 0.5 32 33 16 18 17
16 1.25 1.25 0.5 35 33 16 18 16
17 1.25 1.25 0.5 32 33 16 17 16
4.5.3 Statistical optimization of carbon and nitrogen sources of Aspergillus wentii
Fitting the model
The data obtained from quadratic model equation was found to be significant. It
was verified by F value and the analysis of variance (ANOVA) by fitting the data of all
independent observations in response surface quadratic model. The results for model F-
value implies that the model is significant which indicate it to be suitable to represent
adequately the real relationship among the parameters used. R2 value for all the
Results
109
responses ranged between 90 -95.1 %, which showed suitable fitting of the model in the
designed experiments. The final predictive equations for each response S. aureus (G1),
S. epidermidis (G2), K. pneumoniae1 (G3), C. albicans (G4) and MRSA (G5) obtained are
as follows
The optimized values for factors were validated by repeating the experiment in
triplicates:
S. aureus (G1)
25+2.75X1+6.125X2+0.125X3-2X12-1.25X2
2-6.75X3
2-0.25X1X2+0.25X1X3-1X2X3
Linear effect of starch (X2) and squared effect of yeast extract (X3)2 were highly
significant with P value ≤ 0.005 similarly linear (X1) and squared effect (X1)2 of
dextrose was significant with P value ≤ 0.05 and ≤ 0.5, respectively. The response
surface graph showed highest activity at Dextrose 1 %-2 %, Starch 2 % and Yeast
extract 0.4 % (Figure. 4.5.3.1).
S. epidermidis (G2)
26+2.75X1+6.125X2+0.125X3-1.80X12-1.05X2
2-6.55X3
2-0.25X1X2+0.25X1X3-
1X2X3
Linear effect of starch (X2) and squared effect of yeast extract (X3)2 was highly
significant with P value ≤ 0.005. Linear effect of dextrose (X1) and squared effect
of starch (X2)2 was significant with P value ≤ 0.05 and ≤ 0.5, respectively. Highest
activity was found with Dextrose 1 %-2 %, Starch 2 % and Yeast extract 0.4 %
(Figure. 4.5.3.2).
K. pneumoniae1(G3)
16.6+1X1+4.375X2+0.875X3-0.05X12+1.20X2
2-2.80X3
2+0.25X1X2-0.25X1X3-
1.5X2X3
Linear effect of starch (X2) was highly significant with P value ≤ 0.005.Squared
effect of yeast extract (X3)2 and starch (X2)
2 was also significant with P value ≤
0.05 and ≤0.5 respectively. Linear effect of dextrose (X1) and yeast extract (X3)
was significant with P value ≤ 0.5, respectively. The response surface graph showed
Results
110
highest activity at 2 % Starch, 2 % Dextrose and Yeast extract 0.4 % (Figure.
4.5.3.3).
C. albicans (G4)
30+2.75X1+7.25X2-0.25X3-0.75X12-1.75X2
2-7.75X3
2-0.50X1X2-0.0X1X3-0.50X2X3
Linear effect of starch (X2) and squared effect of yeast extract (X3)2 was highly
significant with P value ≤ 0.005. Linear effect of dextrose (X1) and squared effect
of starch (X2)2 was significant with P value ≤ 0.05 and ≤ 0.5, respectively. The
response surface graph of (G4) showed highest activity at Starch 2 %, Dextrose 2 %
and Yeast extract 0.4 % (Figure. 4.5.3.4).
MRSA (G5)
29.2+2.625X1+7.75X2-0.375X3-0.60X12-1.35X2
2-7.1X3
2-0.75X1X2-0.0X1X3-
0.75X2X3
Linear effect of starch (X2) and squared effect of yeast extract (X3)2 was highly
significant with P value ≤ 0.005. Similarly, linear effect of dextrose (X1) and
squared effect of starch (X2)2
was significant with P value ≤ 0.05 and ≤ 0.5,
respectively. Thus the highest activity was expressed at Starch 2 %, Dextrose 2 %
and Yeast extract 0.4 % (Figure. 4.5.3.5).
4.5.3.1 Validation of Results
Thus from the overall assessment 2 % Dextrose, 2 % Starch and 0.4 % Yeast
extract and (1%) peptone in YPDS medium may be regarded as the optimized
conditions for antimicrobial activity The F value and R2 value showed that the model
correlated well with measured data and was statistically significant. To confirm the
adequacy of the model the verification experiments using optimum medium
composition as described above were carried out in triplicates. The results showed the
antimicrobial activity (Table 4.5.3) was enhanced by 1.25 folds in S. aureus, 1.28 folds
(S. epidermidis), 1.6 folds (K. pneumoniae 1), 1.37 folds (C. albicans), 1.38 folds
(MRSA).
Results
111
Figure 4.5.3.1: Contour Plot of Staphylococcus aureus
Figure 4.5.3.2 Contour Plot of Staphylococcus epidermidis.
22
24
26
28
30
32
2 1 0
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Dextrose
Hold values: starch: 2.0
Ye
ast e
xtr
act
20
22
24
26 28
30
2 1 0
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Dextrose
Hold values: starch: 2.0
Ye
ast e
xtr
act
Results
112
Figure 4.5.3.3 Contour Plot of Klebsiella pneumoniae 1.
Figure 4.5.3.4 Contour Plot of Candida albicans.
27
29
31
33
35
37
2 1 0
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Dextrose
Hold values: starch: 2.0
Ye
ast e
xtr
act
13
15
17
19 21
23
2 1 0
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Starch
Ye
ast e
xtr
act
Hold values: Dextrose: 2.0
Results
113
Figure 4.5.3.5 Contour Plot of Methicillin resistant Staphylococcus aureus (MRSA).
Table 4.5.3: Result of Box-Benhken design experiment for antimicrobial potential of
Aspergillus wentii
Std Order Dextrose Starch
Yeast
extract S. aureus S. epidermidis K. pneumoniae C. albicans MRSA
(g/100ml) Zone of Inhibition (mm)
1 0 0 0.4 12 14 12 16 15
2 2 0 0.4 15 17 12 20 20
3 0 2 0.4 29 31 23 36 36
4 2 2 0.4 31 33 24 38 38
5 0 1 0 12 14 12 17 18
6 2 1 0 20 22 16 25 25
7 0 1 0.8 12 14 12 18 18
8 2 1 0.8 21 23 15 26 25
9 1 0 0 12 14 12 16 15
10 1 2 0 22 24 21 27 28
11 1 0 0.8 14 16 12 15 15
12 1 2 0.8 20 22 15 24 25
13 1 1 0.4 26 27 17 30 30
14 1 1 0.4 25 27 17 30 29
15 1 1 0.4 25 27 17 30 29
16 1 1 0.4 24 27 16 30 29
17 1 1 0.4 25 25 16 30 29
21
26
31
36
2 1 0
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Starch
Hold values: Dextrose: 2.0
Ye
ast e
xtr
act
Results
114
4.5.4 Statistical optimization of carbon and nitrogen sources of Aspergillus terreus
S. aureus (G1)
7.63+0.38 X1+0.94 X2+9.0 X3-0.06 X12+0.15 X2
2-2.04 X3
2-0.0 X1X2-0.44 X1X3-0.44
X2X3
Yeast extract significantly affect the antimicrobial activity. Linear and squared effect of
yeast extract was highly significant with P value ≤ 0.005. R2 value 95.6% (Fig. 4.5.4.1).
S. epidermidis (G2 )
15.4-0.61 X1-1.27 X2+3.66 X3-0.08 X12+0.35 X2
2-1.24 X3
2-0.0 X1X2+0.44 X1X3+0.0
X2X3
Squared effect of starch and yeast extract was significant with P value ≤ 0.05. R2 value
91.0 % (Fig. 4.5.4.2).
K. pneumoniae 1 (G3 )
19.79+0.33 X1-3.50 X2-1.44 X3-0.24 X12+0.64 X2
2-0.08 X3
2-0.0 X1X2+0.44 X1X3+0.88
X2X3
Linear effect of starch was significant with P value ≤ 0.05. Similarly squared effect of
starch and interactive effect of starch and yeast extract was significant with P value ≤
0.05. R2 value 90.9 % (Fig. 4.5.4.3).
C. albicans (G4)
21.77+1.83 X1-4.38 X2-4.55 X3-0.14 X12+0.96 X2
2+0.31 X3
2-0.55 X1X2+0.22 X1X3+1.55
X2X3
Starch significantly affects the antimicrobial activity. Linear and squared effect of starch
was highly significant with P value ≤ 0.005. Interactive effect of starch and yeast extract
was highly significant with P value ≤ 0.005. Similarly, linear effect of yeast extract and
interactive effect of dextrose and starch was significant with P value ≤ 0.05 (Fig.
4.5.4.5).
MRSA (G5)
17.89+0.58 X1-2.11 X2-2.61 X3+0.03 X12+0.58 X2
2+0.13 X3
2-0.44 X1X2+0.22
X1X3+0.88 X2X3
Results
115
Squared effect of starch was highly significant with P value ≤ 0.005. Similarly, linear
effect of starch was significant with P value ≤ 0.05. Interactive effect of dextrose and
starch; starch and yeast extract was significant with P value ≤ 0.05. R2 value 93.8% (Fig.
4.5.4.4).
4.5.4.1 Validation of results
Thus from the overall assessment 1% Dextrose, 4% Starch and 1.5% Yeast
extract and (1%) peptone in YPDS medium may be regarded as the optimized
conditions for antimicrobial activity The F value and R2 value showed that the model
correlated well with measured data and was statistically significant. To confirm the
adequacy of the model the verification experiments using optimum medium
composition as described above were carried out in triplicates. The results showed the
antimicrobial activity (Table 4.5.4) was enhanced by 1.1-1.2 folds.
Figure 4.5.4.1: Contour Plot of Staphylococcus aureus.
Figure 4.5.4.2 Contour Plot of Staphylococcus epidermidis.
Results
116
Figure 4.5.4.3: Contour Plot of Klebsiella pneumoniae 1.
Figure 4.5.4.4: Contour Plot of MRSA.
Figure 4.5.4.5: Contour Plot of C. albicans.
Results
117
Table 4.5.4: Result of Box –Benhken design experiment for antimicrobial activity of
Aspergillus terreus
Std Order
Dextrose Starch Yeast
extract S. aureus S. epidermidis K. pneumonia C. albicans MRSA
(g/100ml) Zone of Inhibition (mm)
1 1 1 1.25 16 17 17 17 15
2 4 1 1.25 15 16 16 1\9 17
3 1 4 1.25 19 18 19 22 19
4 4 4 1.25 18 17 18 19 17
5 1 2.5 0.5 15 16 16 17 16
6 4 2.5 0.5 14 13 14 17 15
7 1 2.5 2 18 17 17 17 16
8 4 2.5 2 15 16 17 18 16
9 2.5 1 0.5 12 14 16 19 16
10 2.5 4 0.5 17 16 17 19 17
11 2.5 1 2 16 17 17 17 15
12 2.5 4 2 19 19 22 24 20
13 2.5 2.5 1.25 17 16 18 18 16
14 2.5 2.5 1.25 17 17 17 18 16
15 2.5 2.5 1.25 16 16 16 17 16
16 2.5 2.5 1.25 17 17 16 17 15
17 2.5 2.5 1.25 17 16 16 17 15
After optimization of the medium, and the physiochemical parameters, for the
extraction and purification of antimicrobial compounds from all the four selected fungi,
all the four selected fungal isolates were grown according to their optimized conditions
and the culture filtrates were treated with different solvents viz. petroleum ether,
chloroform, ethyl acetate and butanol. The organic layer of different solvents were
separated out and then evaporated to dryness in vacuum and the resulting solids were
reconstituted in DMSO which was then checked for their antimicrobial potential.
Results
118
4.6 Solvent extraction and Antimicrobial activity
4.6.1 Solvent extraction and Antimicrobial activity of Penicillium spp.
Two phase aqueous extraction of culture broth obtained from both the fungi
revealed butanol to be the best solvent to elute the components responsible for
antimicrobial activity followed by diethyl ether, ethyl acetate, chloroform and hexane.
(Table 4.6.1). Butanolic extract from Penicillium expansum gave better results and
inhibited all the microorganisms except C. tropicalis with inhibition zone ranging from
15-48 mm as compared to aqueous extract which was active against five
microorganisms (Table 4.6.1.1). S. epidermidis remained the most sensitive followed by
S. aureus. In both the cases the inhibition zone increased by 8 mm in comparison to
aqueous extract. However, this enhancement was maximum in K. pneumoniae where
the activity increased to 45mm as compared to that of aqueous extract (27mm). The
order of antimicrobial activity was butanol > diethyl ether > ethyl acetate > chloroform
> hexane. Similarly, butanolic extract of Penicillium citrinum inhibited all the tested
microorganisms with the inhibition zone ranging from 15-32 mm. C. tropicalis was
found to be resistant to butanolic extract of Penicillium citrinum. The activity was
increased not only in terms of zone of inhibition but the spectrum as well. The order of
antimicrobial activity of different solvent extract was butanol > diethyl ether > ethyl
acetate > chloroform > hexane (Table 4.6.1.2).
Results
119
Table 4.6.1.1: Solvent extraction and antimicrobial activity of Penicillium expansum
Solvents But DE EA Chl Hex
Microorganism Zone of inhibition (mm)
E. faecalis 21±0.707 14±0.707 0 0 0
S. aureus 47±0 20±0 40±0 22±0 0
S. epidermidis 48±0 35±0 33±0.0 20±0.0 15±1.41
E. coli 15±0 13±0.707 14±0.707 0 0
K .pneumoniae1 45±0.707 35±0.707 37.5±0.707 24±0 20±0.0
K. pneumoniae 2 12±1.4 0 0 0 0
P. aeruginosa 27±0 20±0.707 0 18±0 0
Sh. Flexneri 19±0.707 14±0 0 0 0
Salm. Typhimurium 1 15±0 15±0 0 0 0
Salm. Typhimurium 2 26±0.707 12±1.41 0 0 0
C. albicans 32±0.707 15±1.4 15±0.707 0 0
C. tropicalis 0 0 0 0 0
MRSA 30±0.707 14±0.707 0 0 0
But- Butanol, DE- Diethyl ether, EA- Ethyl acetate, Chl- Chloroform, Hex- Hexane
Values are expressed in terms of mean ± Standard deviation
Table 4.6.1.2: Solvent extraction and antimicrobial activity of Penicillium citrinum
Solvents But DE EA Chl Hex
Microorganism Zone of Inhibitions (mm)
E. faecalis 16±1.4 0 0 0 0
S. aureus 32±0 17.5±0.707 11.5±0.707 20±0 14±0
S. epidermidis 31±0.707 18±0 13±0.0 21.5±0.707 13.5±0.707
E. coli 15±0.7 0 0 0 0
K. pneumoniae 1 16±0.0 11±0 13±1.4 15±0 12±0
K. pneumoniae 2 13±0.7 0 0 0 0
P. aeruginosa 20±1.4 18±1.4 0 0 0
Sh. Flexneri 17±0.7 17±0.7 0 0 0
Salm. Typhimurium 1 13±0 12±0 0 0 0
Salm. Typhimurium 2 22±0.7 18±0.7 0 0 0
C. albicans 28±0.7 19±1.4 0 0 0
MRSA 25±1.4 0 0 0 0
But- Butanol, DE- Diethyl ether, EA- Ethyl acetate, Chl- Chloroform, Hex- Hexane
Values are expressed in terms of mean ± Standard deviation
Results
120
4.6.2 Solvent extraction and antimicrobial activity of Aspergillus spp.
Two phase aqueous extraction of culture broth revealed butanol to be the best to
elute the components responsible for antimicrobial activity of Aspergillus wentii
followed by ethyl acetate, chloroform, diethyl ether, and hexane. Culture broth
extracted with different solvents was evaporated, and re-dissolved in DMSO. Butanolic
extract from Aspergillus wentii gave best results and inhibited all the microorganisms
including MRSA except C. tropicalis. S. aureus, S. epidermidis and K. pneumoniae1
were the most sensitive and the inhibitory zone ranged from 22 to 43mm in butanolic
extract of Aspergillus wentii. Hexane extract was least effective and inhibited only three
microorganisms i.e. K. pneumoniae 2, MRSA and C. albicans. As, butanol was found
to be the best organic solvent responsible for antimicrobial activity in terms of zone
size as well as number of sensitive microorganisms (Table 4.6.2.1), So it was used for
further studies . Similarly when culture broth from Aspergillus terreus was extracted
with different solvents revealed ethyl acetate to be the best solvent to elute the
components responsible for antimicrobial activity followed by butanol > chloroform >
hexane > ethyl acetate. C. albicans was found to be the most sensitive organisms. Ethyl
acetate extract showed the maximum range of zone of inhibition of 24-37mm followed
by butanol with 20mm-35mm followed by chloroform with zone of inhibition ranging
from 16-25mm. Further, Diethyl ether and hexane showed the least zone of inhibition
ranging from 13-16mm and 14-17 mm respectively. C tropicalis remained the resistant
organism throughout the study. Further ethyl acetate was selected as an organic solvent
for extraction of antimicrobial components from Aspergillus terreus for further studies
(Table 4.6.2.2).
Results
121
Table 4.7.2.1: Solvent extraction and antimicrobial activity of Aspergillus wentii
Solvents But DE EA Chl Hex
Microorganisms Zone of Inhibition (mm)
E. faecalis 25±0 0 16±0.57 14±0.57 0
S. aureus 43±0 0 0 0 0
S. epidermidis 42±0.57 0 0 0 0
E. coli 22±1 0 16±0.57 0 0
K. pneumoniae 1 42±0.57 11±0.57 19±1 15±0.57 0
K. pneumoniae 2 14±0 12±0.57 0 20±0 14±0.57
P. aeruginosa 17±0.57 14±0.57 0 0 0
Sh. Flexneri 22±0.57 0 0 0 0
Salm. Typhimurium 1 16±0.57 0 0 0 0
Salm. Typhimurium 2 23±0.57 14±0.57 18±0.57 20±0 0
C. albicans 35±0.57 17±0.57 30±0.57 20±0.57 13±0
C. tropicalis 0 0 0 0 0
MRSA 29±0.57 15±0.57 25±0.57 22±0.57 12±0.57
But- Butanol, DE- Diethyl ether, EA- Ethyl acetate, Chl- Chloroform, Hex- Hexane
Values are expressed in terms of mean ± Standard deviation
Table 4.7.2.2: Solvent extraction and antimicrobial activity of Aspergillus terreus
Solvents But DE EA Chl Hex
Microorganisms Zone of Inhibition (mm)
E. faecalis 30.75±0.35 14.5±2.1 29.5±0.7 20.5±2.1 14.5±0.7
S. aureus 20±1.4 14.5±2.1 24±0 19.5±0.7 14.5±0.7
S. epidermidis 21.5±2.1 13.5±0.7 25±0 18±0 14.5±0.7
E. coli 23±1.4 14.5±0.7 25.5±0.7 17±1.4 14.5±0.7
K. pneumoniae 1 27±0 14.75±0.35 30.5±0.7 20±1.4 14.75±1.0
K. pneumoniae 2 25±1.4 0 25.5±0.7 13.5±2.1 0
P. aeruginosa 27.5±2.1 16±0 28.5±0.7 20.5±0.7 15±0
Sh. flexneri 27.5±0.7 16.5±0.7 27±0 16±1.4 16.5±2.1
Salm. Typhimurium 1 20±1.4 0 21.75±0.35 14.5±2.1 13.75±0.35
Salm. Typhimurium 2 22±1.4 16.5±0.7 27±0 20±0 17±0
C. albicans 35±1.4 12±1.4 37.5±0.7 25.5±0.7 11±0
C. tropicalis 0 0 0 0 0
MRSA 24.5±0.7 13.75±0.35 26.75±0.35 14.75±1.0 14±1.4
But- Butanol, DE- Diethyl ether, EA- Ethyl acetate, Chl- Chloroform, Hex- Hexane
Values are expressed in terms of mean ± Standard deviation
Results
122
After the solvent extraction, the selected solvents for each organism were used
for further antimicrobial studies and purification of antimicrobial compound.
All the fungal organic extracts, were subjected to column chromatography.
Three liters of the culture broth was extracted with equal volume of butanol (1:1) for
Penicillium expansum (HT 28), Penicillium citrinum (HT 46), Aspergillus wentii (HT
113) while 3 liters of the culture broth of Aspergillus terreus (HT 66) was extracted
with equal volume of ethyl acetate The organic layer was separated and treated with
Na2SO4 and then evaporated to dryness in vacuum and the resulting solids (4g, 4.5g, 3g
and 3.25g respectively) were subjected to column chromatography.
4.7 Isolation and purification of antimicrobial compound
4.7.1 Isolation and purification of the antimicrobial compound (KB3) from
Penicillium expansum
For the purification of antimicrobial compound from Penicillium expansum, 3
litres of culture broth were extracted ethyl acetate and the resulting solid of 3.25g was
subjected to column chromatography. Three sets of fractions (A, B,C) were obtained
from column chromatography having similar Rf value and antimicrobial activity against
various pathogenic bacterial and yeast strains such as S. aureus, S. epidermidis, K.
pneumoniae 1, MRSA, C. albicans, Salm. Typhimurium1, Salm. Typhimurium 2, Sh.
flexneri and E. coli. First set (A) showed antimicrobial activity with zone of inhibition
ranging from 12-27 mm followed by second set (B) which showed zone of inhibition
ranging from 12-14 mm and third set (C) with zone of inhibition ranging from 14-18
mm against various microbial strains (Figure. 4.7.1.1). As the first set (A) showed better
antimicrobial activity so it was pursued for further spectroscopic analysis. HPLC
(Dionex P-680) was used to further analyze the purity of the compound (A). Aqueous
acetonitrile (75 % v/v) was used as mobile phase at a flow rate 0.3 ml/min and injection
volume was 20 µl at column temperature of 25 °C. The detections were monitored at
different wavelengths (225, 250, 275 and 300 nm).
All the fractions obtained from column chromatography were pooled according
to similar pattern of chromatogram on TLC plates. The first set (A) of pooled fraction
showed a single spot on TLC with Rf value (0.77 cm). It was further subjected to HPLC
analysis to determine the purity of active compound which showed single peak at
Results
123
retention time 8.536 min (Figure 4.7.1.2). The compound (A: 60 mg) responsible for
antimicrobial activity was characterized by various spectroscopic techniques such as IR,
1H &
13C NMR and mass. IR (KBr, CHCl3): λmax = 2924, 2853, 1690, 1620, 1457,
1098, 913, 745 cm-1
; 1H NMR (400 MHz, CDCl3) = δ 8.28 (s, 1H, C5-H), 7.71 (s, 1H,
C2-H), 7.67 (d, 1H J = 8.4 Hz, C7-H), 7.39-7.28 (m, 2H, Ar-H), 7.16-7.00 (m, 2H, Ar-
H), 6.98 (d, 1H, J = 8.4 Hz, C8-H), 6.16-6.13 ( m, 2H, C1′′-H), 5.51 (s, 2H, C6′-H),
5.13-5.05 (m, 2H, C3′′-H), 3.73 (s, 3H, -OCH3), 1.86-0.83 ( m, 17H); 13
C NMR (100
MHz, CDCl3) = δ 170.2 (C=O), 159.0 (Ar-CH), 146.0 (Ar-CH), 141.0 (Ar-CH), 137.0
(Ar-CH), 134 (olefinic–CH), 134.2 (q), 128 (Ar-CH), 126 (q), 124.9 (Ar-CH), 123.8
(Ar-CH), 112.1 (olefinic–CH), 65.4 (-OCH3), 52.0 (-OCH2), 42.7 (CH2), 31.4 (CH2),
30.1 (CH2), 27.1 (CH3), 22.7 (CH3), 20.1 (CH3); HR-MS (TOF, ESI): m/z: calculated
for C28H34O4: 434.2457; found: 434.1868 [M] +
.
Figure 4.7.1.1: Antimicrobial activity of fractions from column chromatography by
agar well diffusion assay (Penicillium expansum)
Figure 4.7.1.2: HPLC analysis of purified compound (KB 3)
0
5
10
15
20
25
30
A B C
Zo
ne
of
inh
ibit
ion
(m
m)
Three different sets of fractions
S.aureus
S.epidermidis
E.coli
K.pneumoniae 1
Sh.flexneri
Salm.Typhimurium 1
Salm. Typhimurium 2
C.albicans
MRSA
Results
124
Figure 4.7.1.3: 1H NMR spectrum of compound KB 3.
Figure 4.7.1.4: 13
C NMR spectrum of compound KB 3.
Results
125
Figure 4.7.1.5: IR spectrum of compound KB 3.
Figure 4.7.1.6: Mass spectrum of compound KB 3
Results
126
O
O
OCH3
O
6-[1,2-Dimethyl-6-(2-methyl-allyloxy)-hexyl]-3-(2-methoxy-phenyl)-chromen-4-one
1
2
34
5
6
7
8
1'2'
3'4'
5'6'
1''2''
3''
Figure 4.7.1.7: Structure of purified compound (KB 3) isolated from Penicillium
expansum
The structure of compound KB 3 is elucidated on the basis of spectroscopic
techniques. Proton NMR (Figure. 4.7.1.3) revealed that C5-H appears as singlet at
chemical shift 8.28 ppm and another singlet of C2-H appear at 7.71 ppm. The signal of
proton C-7 observed as doublet at 7.67 ppm with coupling constant 8.4 Hz. The other
aromatic protons signal appears as multiplet at 7.39-7.28 ppm and 7.16-7.00 ppm. The
methoxy group in the proton NMR appears as a singlet at 3.73 ppm and alkenic protons
of C1’’and C3’’ appear as multiplet at the range of at 6.16-6.13 and at 5.13-5.05 ppm.
The signal of C6’-H appears as a singlet at 5.51 ppm and the aliphatic protons appear as
multiplet at the range of 1.86-0.83 ppm. 13
C NMR (Figure. 4.7.1.4) showed the carbonyl
peak of chromone ring appear at 170.2 ppm and other resonances of aromatic and
alkenic carbon appear at the range from 159.0-112.1 ppm and the aliphatic resonances
appear at 65.4-22.7. The carbonyl group of compound further confirmed by IR
spectrum (Figure. 4.7.1.5) which showed sharp band at 1665 cm-1
and C-O stretching
appears at 1457cm-1
. On the basis of these observations the purified compound is
proposed to be 6-[1, 2-Dimethyl-6-(2-methyl-allyloxy)-hexyl]-3-(2-methoxy-phenyl)-
chromen-4-one (KB 3), which was further corroborated by mass spectra (Figure.
4.7.1.6, 4.7.1.7) showed peak corresponding to 434.1868 (M+).
4.7.2 Isolation and purification of antimicrobial compound KB 4 from
Penicillium citrinum
For the extraction and purification of active group/component from Penicillium
citrinum (HT-46), three liters of the culture broth was extracted with equal volume of
butanol (1:1). All the fractions (120) obtained from column chromatography were
pooled according to similar pattern of chromatogram on TLC plates. Three sets of
fractions (A, B and C) were obtained from column chromatography having similar Rf
Results
127
value and antimicrobial activity against various pathogenic bacteria and yeast strains
such as S. aureus, S. epidermidis, K. pneumoniae1, MRSA, C. albicans, Salm.
Typhimurium1, Salm. Typhimurium 2, Sh. flexneri and E. coli. The second set (B) of
pooled fraction showed a single spot on TLC with Rf value (0.65cm). It was further
subjected to HPLC analysis to determine the purity of active compound which showed
single peak at retention time 8.643 min (Figure 4.7.2.2). However, Set A and C also
showed antimicrobial activity but some impurities. First set (A) showed antimicrobial
activity with zone of inhibition ranging from 20-23 mm followed by second set (B)
which showed zone of inhibition ranging from 17-20 mm against various microbial
strains (Figure 4.7.2.1). As the second set (B) showed single spot on TLC, it was
pursued for further spectroscopic analysis. The compound (KB 4) 63 mg responsible for
antimicrobial activity was characterized by various spectroscopic techniques such as IR,
1H &
13C NMR and mass. Colour of compound: Yellowish brown: KB 4 ( 63 mg);
IR (KBr, CHCl3): λmax = 2940, 2915, 2876, 1666, 1651, 1535, 1454, 1373, 1284, 1161,
1107, 1072, 968, 756 cm-1
; 1H NMR (400 MHz, CDCl3): δ 9.35 (s, 1H, C8-H), 8.26-
8.12 (m, 2H, C6-H & C3-H), 7.61 (d, 1H, J = 8.0 Hz, C5-H), 7.39-7.20 (m, 2H, C4′-H
& C6′-H), 6.91-6.84 (m, 2H, C5′-H & C7′-H), 6.76-6.67 (m, 3H, -CH3), 4.37 (s, 3H,
OCH3), 2.04 (t, 2H, J = 5.2 Hz, C1′-H), 1.92-1.87 (m, 2H, C2′-H), 1.40-1.24 (m, 2H,
C3′-H); 13
C NMR (100 MHz, CDCl3): δ 170.2 (C=O), 169.1 (olefinic CH), 166.4
(arom. q), 163.7 (olefinic CH), 160.1 (arom. q), 147.9 (C4), 125.6 (olefinic CH), 124.5
(olefinic CH), 121.9 (olefinic CH), 118.8 (arom. CH), 116.2 (arom. CH), 67.0 (OCH3),
57.2 (q), 45.8 (CH2), 44.5 (CH2), 27.8 (CH3), 21.9 (CH3), 15.1 (CH2); HR-MS (TOF,
ESI): m/z: calcd for C21H26O2 + [Na]: 333.1933; found: 333.1766 [M + Na]
+.
Results
128
Figure 4.7.2.1: Antimicrobial activity of fractions from column chromatography by
agar well diffusion assay (Penicillium citrinum)
Figure 4.7.2.2: HPLC analysis of compound KB 4
0
5
10
15
20
25
A B C
Zo
ne
of
inh
ibit
ion
(m
m)
Three sets of fractions
E.coli
K.pneumoniae 1
Sh.flexneri
Salm.typhimurium 1
Salm.typhimurium 2
C.albicans
MRSA
Results
129
Figure 4.7.2.3: 1H NMR spectrum of compound KB 4.
Figure 4.7.2.4: 13
C NMR spectrum of compound KB 4.
Results
130
Figure 4.7.2.5: IR spectrum of compound KB 4.
Figure 4.7.2.6: Mass spectrum of compound KB 4.
Results
131
O1
2 3
4
1'
2'
3'
4'
5'
6'
7'
8'
H3CO
5
67
8
7-Methoxy-2,2-dimethyl-4-octa-4′,6′-dienyl-2H-napthalene-1-one (KB 4)
Figure 4.7.2.7: Structure of purified compound (KB 4) isolated from Penicillium citrinum
The assigned structure of compound KB 4 present was based on detailed
spectroscopic analysis. The 1H NMR spectrum (Figure. 4.7.2.3) of compound showed
aromatic and olefinic proton resonances in the region of 9.35-6.67, the signal of C8-H
appeared as singlet at 9.35 downfield due to anisotropic effect and hydrogen bonding
with nearing carbonyl group. The aliphatic multiplet was also observed in the region of
1.92-1.87 and 1.40-1.24. The 13
C NMR spectrum (Figure. 4.7.2.4) showed aromatic and
olefinic carbon resonances in the region of 169.1-166.2. The signals of aliphatic carbons
appeared at the region of 57.2-15.1 and signal of carbonyl group appeared at 170.2,
which further analyzed by stretching appeared in IR spectrum (Figure. 4.7.2.5) at 1666
cm-1
. Further, structure of compound KB 4 was corroborated by mass spectrum (Figure.
4.7.2.6; 4.7.2.7) which showed a molecular ion peak at m/z 333.1766 [M + Na]
+.
4.7.3 Isolation and purification of antimicrobial compound from Aspergillus wentii
For the purification of antimicrobial compound from Aspergillus wentii 3 litres
of culture broth were extracted ethyl acetate and the resulting solid of 3.25g was
subjected to column chromatography. All the fractions obtained from column
chromatography were subjected to assess their antimicrobial effect using agar disc
diffusion assay. All the fractions obtained from the column chromatography were
subjected to TLC and the active fractions (fraction no. 40-50) showed antimicrobial
activity against S. epidermidis, K. pneumoniae1, MRSA, C. albicans, Salm.
Typhimurium 2 with zone of inhibition ranging from 18-25 mm (Figure. 4.7.3.1). The
Results
132
fractions having the same Rf values (0.8 cm) were pooled and again loaded to column.
In the second column with total 55 fractions, fraction no. 20 to 29, showed
antimicrobial activity (ranging from 18 to 20 mm) having same Rf values (0.7cm) were
again pooled and concentrated for further checking its activity and TLC. One single
band was observed on TLC under and iodine chamber with Rf value (0.7cm). The active
fraction was subjected to HPLC analysis and the single peak of compound indicates its
purification. The retention time for the compound was 8.924 (Figure 4.7.3.2) and
further analyzed for NMR, IR and mass. 1H NMR of this unknown compound showed
doublets at δ 7.62 and 6.80, respectively with J = 8.0 Hz, and having protons which
showed cis coupling and signal downfield due to hydrogen bonding with carbonyl
group. Proton NMR (Figure. 4.7.3.3) also showed the broad signals at δ 9.37 and 8.89
which may be due to phenolic –OH groups and it was further confirmed through IR
spectrum which showed broad band at 3228 cm-1
. In proton NMR spectrum, the
presence of one singlet at δ 8.22 attributed to –NH group which revealed through IR
spectrum showed C-N stretching at 1437 cm-1
. Proton NMR showed some resonances at
alkenic region, indicating the presence of some alkenic proton in unknown compound.
Figure 4.7.3.1: Antimicrobial activity of fractions from column chromatography by
agar well diffusion assay (Aspergillus wentii)
0
5
10
15
20
25
30
A B
Zo
ne
of
inh
ibit
ion
(m
m)
Two sets of fractions
S.epidermidis
K.pneumoniae 1
Salm.Typhimurium 2
C.albicans
MRSA
Results
133
Figure 4.7.3.2: HPLC analysis of purified compound KB2
Figure 4.7.3.3: 1H NMR spectrum of compound KB 2.
Results
134
Figure 4.7.3.4: 13
C NMR spectrum of compound KB2.
Figure 4.7.3.5: IR spectrum of compound KB2.
RC SAIF PU, Chandigarh
Spectrum Name: Harpreet GNDU-1.sp Description: S-6
Date Created: fri apr 27 14:56:14 2012 India Standard Time (GMT+5:30)
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0
44.0
46
48
50
52
54
56
58
60
62
64
66
68
70
72
74
76
78
79.7
cm-1
%T
3238.062957.95
2928.22
2871.80
1668.90
1437.81
1325.25
1215.76
1101.75
1011.95
964.36
745.38
702.85
620.57
426.38
Results
135
Figure 4.7.3.6: Mass spectrum of compound KB2.
O
N
H
H3C
H
OHHO
3-(4-Hydroxy-phenyl)-N-[2-(4-hydroxy-phenyl)-propenyl]-acrylamide
Fig 4.7.3.7: Structure of purified compound (KB 2) isolated from Aspergillus wentii
13C NMR (Figure 4.7.3.4) of unknown compound showed some resonances at
aromatic and alkenic region, and showed peaks at δ 75.5 and 70.5 due to –OH groups
and one signal at 22.2 due to -methyl group. 13
C NMR also showed the peak at δ 178.3
due to (C=O) group which was further confirmed by its IR spectrum (Figure 4.7.3.5)
that showed carbonyl stretching at 1668 cm-1
and this carbonyl group is amide group
because C-N stretching also appeared in IR spectrum at 1437 cm-1
. On the basis of these
observations the compound was determined to be 3-(4-Hydroxy-phenyl)-N-[2-(4-
hydroxy-phenyl)-propenyl]-acrylamide (KB 2) and it further confirmed through its
mass spectrum (Figure 4.7.3.6; 4.7.3.7) which showed the mass ion peak 317. 9 (M +
Na)+ which correspond to the mass of this compound.
m/z100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
%
0
100
HARPREET 8 29 (0.538) Cm (25:60) TOF MS ES+ 1.98e4288.3
19813
227.16107
211.13134119.1
2846
106.11336
120.11402
130.1974
261.13581
249.11777
228.1795
283.11640
270.3723
316.39110
289.33963
437.28464
317.32053
415.21067356.1
739334.1614
438.22237
475.3673
Results
136
4.7.4 Isolation and purification of antimicrobial compound (KB 1) from
Aspergillus terreus
For the purification of antimicrobial compound from Aspergillus terreus, 3 litres
of culture broth were extracted ethyl acetate and the resulting solid of 3.25g was
subjected to column chromatography. A total of 130 fractions were collected and the
fraction size kept to 20 ml. All the fractions obtained from column chromatography
were pooled according to similar pattern of chromatogram on TLC plates. The first set
(A) of pooled fraction showed a single spot on TLC with Rf value (0.65cm). It was
further subjected to HPLC analysis to determine the purity of active compound, which
showed single peak at retention time 8.643 min (Figure 4.7.4.2). Three sets of fractions
(A, B and C) were obtained from column chromatography having similar Rf value and
antimicrobial activity against various pathogenic bacteria and yeast strains such as S.
aureus, S. epidermidis, K. pneumoniae1, MRSA, C. albicans, Salm. Typhimurium 1,
Salm. Typhimurium 2, Sh. flexneri and E. coli. Set C was not active against any
microorganism used so it was not perused further. First set (A) showed antimicrobial
activity with zone of inhibition ranging from 16-25 mm followed by second set (B),
which showed zone of inhibition ranging from 17-25 mm against various microbial
strains (Figure 4.7.4.1). First set (A) was pursued for further spectroscopic analysis such
as IR, 1H &
13C NMR and mass. colour of compound KB 1: white: (yield 65mg); IR
(KBr, CHCl3): λmax = 2958, 2920, 2890, 1643, 1600, 1581, 1489, 1462, 1361, 1284,
1188, 1122, 1076, 964, 744 cm-1
; 1H NMR (400 MHz, CDCl3): δ 7.73-7.67 (m, 2H, Ar-
H), 7.54-7.50 (m, 2H, Ar-H), 4.24 (q, 2H, J = 6.4 Hz, -OCH2), 4.08 (d, 2H, J = 6.8 Hz,
C1′-H), 1.73-1.68 (m, 4H, 2 x CH2), 1.47-1.41 (m, 4H, 2 x CH2), 1.31 (s, 3H, -CH3),
1.27 (t, 3H, J = 5.8 Hz, -CH3), 1.25 (s, 3H, -CH3), 0.99-0.82 (m, 3H); 13
C NMR (100
MHz, CDCl3): δ 165.2 (C=O), 144.7 (arom. q), 130.9 (arom. CH), 128.8 (arom. CH),
123.9 (arom. q), 65.5 (OCH2), 31.9 (CH2), 31.4 (CH2), 30.5 (CH3), 30.2 (CH2), 29.7
(CH3), 29.4 (CH2), 22.7 (CH2), 19.2 (CH2), 14.1 (CH), 13.7 (CH3); HR-MS (TOF,
ESI): m/z: calcd for C18H28O3 + [Na]: 315.1712; found: 315.1482 [M + Na]
+.
Results
137
Figure 4.7.4.1: Antimicrobial activity of fractions from column chromatography by
agar well diffusion assay (Aspergillus terreus)
Figure 4.7.4.2: HPLC analysis of compound KB 1
0
5
10
15
20
25
A B C
Zo
ne
of
Inh
ibit
ion
s (m
m)
Three sets of fractions
S.aureus
S.epidermidis
E.coli
K.pneumoniae 1
Sh.flexneri
Salm.Typhimurium 1
Salm.Typhimurium 2
C.albicans
MRSA
Results
138
Figure 4.7.4.3: 1H NMR spectrum of compound KB1.
Figure 4.7.4.4: 13
C NMR spectrum of compound KB 1.
Results
139
Figure 4.7.4.5: 135
DEPT spectrum of compound KB 1.
Figure 4.7.4.6: IR spectrum of compound KB1.
Results
140
Figure 4.7.4.7: Mass spectrum of compound KB 1.
O
O
O
1
2
3
4
5
6
1'
2'
3'
4'
5'
6'8'7'
4-(2-Methyl-octyloxy)-benzoic acid ethyl ester (KB1)
Figure 4.7.4.8: Structure of purified compound (KB 1) isolated from Aspergillus terreus
The assigned structure of compound KB 1 was based on detailed spectroscopic
analysis. The signals of 1H NMR (Figure 4.7.4.3) spectrum revealed, besides aromatic
proton resonances in the region of 7.73-7.67 and 7.54-7.50, aliphatic multiple was
also observed in the region of 1.73-1.68 and 1.47-1.41. The quartet appeared at 4.24
with J = 6.4 Hz and triplet at 1.27 with J = 5.8 Hz represent the ester group which
Results
141
further analyzed by carbonyl stretching appeared in IR spectrum (Figure 4.7.4.6) at
1728 cm-1
and resonance observed in 13
C NMR (Figure 4.7.4.4; 4.7.4.5) at 165.2.
Further, structure of compound KB 1 was corroborated by mass spectrum (Figure
4.7.4.7; 4.7.4.8) which showed a molecular ion peak at m/z 315.1482 [M + Na]
+.
4.8 Minimum inhibitory concentration (MIC)
Minimum inhibitory concentration of the selected fungal organic extracts and
the purified compounds isolated from all the four fungi was worked out by agar dilution
method.
4.8.1 Minimum inhibitory concentration (MIC) of butanolic extract and purified
compound of Penicillium expansum
Minimum inhibitory concentration was worked out for butanolic extract of
fungus (Penicillium expansum) as well as for the compound (KB 3) by agar dilution
method (Table 4.8.1.1). The MIC values were strain dependent. The butanolic extract
was prepared at varying concentration ranging from (0.1-20mg/ml) Butanolic extract
showed significant antimicrobial activity with MIC 0.1 mg/ml against S. aureus, S.
epidermidis and K. pneumoniae 1, followed by MIC 0.5 mg/ml against C. albicans and
MRSA, and MIC 0.7 mg/ml against P. aeruginosa and Salm. Typhimurium 2. The
butanolic extract also showed good inhibitory activity against Sh. flexneri and E.
faecalis with MIC 1 mg/ml, followed by MIC value of 10 and 20 mg/ml against E. coli,
Salm. Typhimurium 1 and K. pneumoniae 2, respectively. Similarly, the pure compound
was prepared ranging from 0.0005mg-0.015mg/ml. MIC of purified compound (KB3)
against C. albicans showed maximum sensitivity with 0.5 µg/ml as compared to
standard antibiotics amphotericin B (99 µg/ml) followed by MRSA, K. pneumoniae1
and S. epidermidis with MIC values 1 µg/ml in all the three microorganisms
respectively which was found to be comparable with gentamicin as the MIC of
gentamicin against these three microorganisms was found to be 0.19 µg/ml, 0.19 µg/ml
and 1 µg/ml respectively. However, MIC of gentamicin against E. coli, Sh. flexneri and
Salm. Typhimiurium2 was lower than purified compound which showed MIC of 15
µg/ml, 15 µg/ml and 10 µg/ml respectively.
Results
142
Table 4.8.1.1: Comparison of MIC of butanolic extract of Penicillium expansum and its
purified compound (KB3) with standard antibiotics
Microorganisms
MIC of butanolic
extract
MIC of
compound
(KB 3)
MIC of
Gentamicin
MIC of
Amphotericin B
(mg/mL) (µg/mL) (µg/mL) (µg/mL)
E. faecalis 1b ND 10
a ND
S. aureus 0.1c 2
b 1
a ND
S. epidermidis 0.1b 1
a 1
a ND
E. coli 10c 15
b 1
a ND
K. pneumoniae 1 0.1c 1
b 0.19
a ND
K. pneumoniae 2 20b ND 1
a ND
P. aeruginosa 0.7b ND 10
a ND
Sh. flexneri 1c 15
b 2
a ND
Salm. Typhimurium 1 10b ND 1
a ND
Salm. Typhimurium 2 0.7c 10
b 2
a ND
C. albicans 0.5c 0.5
a ND 99
b
MRSA 0.5c 1
b 0.19
a ND
The value represent mean of three values; different superscripts (a, b, c) show statistical
significant (P<0.05) difference between columns.
4.8.2 Minimum inhibitory concentration of butanolic extract and purified
compound of Penicillium citrinum
Minimum inhibitory concentration was worked out for butanolic extract of
Penicillium citrinum as well as for the compound by agar dilution method (Table
4.8.2.1). Butanolic extract was prepared at varying concentration ranging from 0.1-
20mg/ml and showed significant antimicrobial activity with MIC 0.1 mg/ml against S.
aureus, and K. pneumoniae 1, followed by MIC 0.2 mg/ml against S. epidermidis and
C. albicans, 0.5 mg/ml against MRSA, 0.7 mg/ml against Salm. Typhimurium 2 and P.
Results
143
aeruginosa 1 mg/ml. The butanolic extract also showed good inhibitory activity against
Sh. flexneri and E. faecalis with MIC 5 mg/ml, followed by MIC value of 10 mg/ml and
20 mg/ml against Salm. Typhimurium 1 and K. pneumoniae 2, respectively. MIC of
purified compound against C. albicans and K. pneumoniae 1 showed maximum
sensitivity with 1 µg/ml followed by MIC 2 µg/ml against S. aureus and S. epidermidis,
whereas against MRSA 5 µg/ml and Salm. Typhimurium showed highest MIC (10
µg/ml). The purified compound showed MIC of 1 µg/ml against C. albicans whereas
MIC of 99 µg/ml was observed with amphotericin B. In some cases gentamicin showed
almost comparable results with the purified compound. MIC of gentamicin against S.
aureus and S. epidermidis was 1 µg/ml whereas purified compound showed MIC of 2
µg/ml. However, gentamicin showed MIC of 0.19 µg/ml against K. pneumoniae1 and
MRSA whereas compound showed MIC of 1 and 5 µg/ml respectively against these two
microorganisms.
Table 4.8.2.1: Comparison of MIC of butanolic extract of Penicillium citrinum and its
purified compound (KB 4) with standard antibiotics.
MIC
Butanolic
extract
MIC of
compound
(KB 4)
MIC of
gentamicin
MIC of
amphotericin B
E. faecalis 5b ND 10
a ND
S. aureus 0.1c 2
b 1
a ND
S. epidermidis 0.2c 2
b 1
a ND
E. coli 10b ND 1
a ND
K. penumoniae 1 0.1c 1
b 0.19
a ND
K. penumoniae 2 20b ND 1
a ND
P. aeruginosa 1b ND 10
a ND
Sh. flexneri 5b ND 2
a ND
Salm. Typhimurium 1 15b ND 1
a ND
Salm. Typhimurium 2 0.7c 5
b 2
a ND
C. albicans 0.2b 1
a ND 99
d
MRSA 0.5c 5
b 0.19
a ND
The value represent mean of three values; different superscripts (a, b, c) show statistical
significant (P<0.05) difference between columns.
Results
144
4.8.3 Minimum inhibitory concentration of butanolic extract and purified
compound of Aspergillus wentii
The butanolic extract was prepared at varying concentration ranging from 0.016
mg-8 mg/ml. K. pneumoniae 1 and S. epidermidis were found to be most sensitive and
inhibited at (0.016 mg/ml) followed by S aureus (0.1mg/ml), C. albicans (0.5 mg/ml),
E. faecalis and MRSA (1mg/ml) Salm. Typhimurium 2 and E. coli (5 mg/ml), Sh.
flexneri (6 mg/ml) while P. aeruginosa and Salm. Typhimurium 1 gave the highest MIC
values (18 mg/ml). Purified compound was prepared at varying concentration ranging
from 0.006 mg/ml-0.02 mg/ml. MIC of purified compound against S. epidermidis, C.
albicans and MRSA was 6 µg/ml, 20 µg/ml and 20 µg/ml, respectively.
4.8.4 Minimum inhibitory concentration of ethyl acetate extract and purified
compound of Aspergillus terreus
Minimum inhibitory concentration was worked out for ethyl acetate extract of
fungus (Aspergillus terreus) as well as for the compound (KB 1) by agar dilution
method (Table 4.8.4.1). Ethyl acetate extract showed significant antimicrobial activity.
C. albicans was found to be the most sensitive with MIC of 0.05mg/ml followed by K.
pneumoniae 1, MRSA, P. aeruginosa, Salm. Typhimurium 2 and E. faecalis with MIC of
0.1 mg/ml. S. flexneri and E. coli showed MIC of 0.25 mg/ml followed by S. aureus and
S. epidermidis with 0.5 mg/ml. Salm. Typhimurium 1 showed the highest MIC of 0.7
mg/ml.
Similarly MIC of purified compound also varied with the organism tested. C.
albicans was found to be the most sensitive with MIC 0.5 µg/ml, followed by K.
pneumoniae 1, MRSA, P. aeruginosa, Salm. Typhimurium 2 and E. faecalis with MIC
of 1 µg/ml. S. flexneri showed MIC of 2.5 µg/ml, whereas E. coli, K. pneumoniae 2, S.
aureus and S. epidermidis showed MIC of 5 µg/ml. C. albicans was found to be most
sensitive purified compound (KB 1) with MIC of 0.5 µg/ml whereas MIC of
amphotericin B against C.albicans was found to be 99 µg/ml. MIC of gentamicin
against K.pneumoniae1 and MRSA was 0.19 µg/ml whereas the purified compound
Results
145
showed MIC of 1 µg/ml against these two microorganisms. MIC of 1 µg/ml against
Salm. Typhimiurium2 was observed in case of purified compound KB 1 which was
found to be better than standard antibiotic gentamicin with MIC of 2 µg/ml. However,
gentamicin showed MIC of 1 µg/ml against S. aureus, S. epidermidis and E.coli
whereas the purified compound showed MIC of 5 µg/ml against all three above
mentioned microorganisms.
Table 4.8.4.1: Comparison of MIC of ethyl acetate extract and pure compound.
Microorganisms
MIC
Ethyl acetate
ex (mg/ml)
MIC of
compound
(µg/ml)
MIC of
gentamicin
(µg/ml)
MIC of
Amphotericin B
(µg/ml)
E. faecalis 0.1c 1
a 10
b ND
S. aureus 0.5c 5
b 1
a ND
S. epidermidis 0.5c 5
b 1
a ND
E. coli 0.25c 5
b 1
a ND
K. penumoniae 1 0.1c 1
b 0.19
a ND
K. penumoniae 2 0.5c 5
b 1
a ND
P. aeruginosa 0.1c 1
a 10
b ND
Sh. Flexneri 0.25b 2.5
a 2
a ND
Salm. Typhimurium 1 0.7c ND 1
a ND
Salm. Typhimurium 2 0.1c 1
a 2
b ND
C. albicans 0.05b 0.5
a ND 99
c
MRSA 0.1c 1
b 0.19
a ND
The value represent mean of three values; different superscripts (a, b, c) show statistical
significant (P<0.05) difference between columns.
4.9 Time Kill assay
4.9.1 Time Kill assay of butanolic extract and purified compound from
Penicillium expansum
The kill kinetics provide more effective efficiency of antimicrobial agents than
does the MIC. The concentrations used for the kill time study was based on
predetermined MIC. A stock solution 10 % of butanolic extract of Penicillium
Results
146
expansum and for compound KB 3, stock solution of 2.5 % (25 mg/ml) was prepared.
Complete killing of E. coli was observed at 0 h of incubation. S. aureus, S. epidermidis
and Salm. Typhimurium 1 got completely killed at 10 h. However, Salm. Typhimurium 1
showed regrowth after 24 h of incubation with 77.25 % viability, whereas Salm.
Typhimurium 2 was killed at 4 h. K. pneumoniae 1 got completely killed at 4 h, whereas
for K. pneumoniae 2, 5 % viable cells were seen after 24 h of incubation. C. albicans
and MRSA got killed at 2 h of incubation. Similarly, E. faecalis took 8 h for complete
killing. On the basis of 1x MIC of compound (KB 3), viable cell count studies were
checked. Complete killing of E. coli, Salm. Typhimurium 2, K. pneumoniae 1 and
MRSA was observed at 0 h. Sh. flexneri took 12 h for complete killing and C. albicans
got killed in 2 h. S. aureus took 6 h for complete killing, whereas S. epidermidis was
killed in 8 h (Figure 4.9.1.1-4.9.1.2).
Figure 4.9.1.1: Time Kill assay of butanolic extract from Penicillium expansum
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8 9
Pe
rce
nta
ge
Via
bil
ity
Time of incubation (hrs)
E.faecalis
S.aureus
S.epridermidis
E.coli
K.penumoniae1
K.pneumoniae 2
P.aeruginosa
Sh. flexeri
Salm. Typhimurium 1
Salm. Typhimurium2
C.albicans
MRSA
Results
147
Figure 4.9.1.2: Time Kill assay of purified compound (KB 3) from Penicillium
expansum
4.9.2 Time Kill assay of butanolic extract and purified compound from
Penicillium citrinum
A stock solution 25 mg/ml of butanolic extract of Penicillium citrinum and 10
mg/ml for purified compound was prepared respectively. Complete killing of E. coli
was observed at 0 h of incubation. K. pneumoniae 1 and C. albicans got completely
killed at 2 h. S. aureus and Sh. Flexneri killed at 10 h of incubation while S.
epidermidis, Salm. Typhimurium 1 and P. aeruginosa got completely killed at 8 h.
MRSA took 6 h for complete killing. K. pneumoniae 2 took the longest time of 24 h for
complete killing. Similarly, E. faecalis took 4 h for complete killing (Figure 4.9.2.1).
On the basis of 1x MIC of compound viable cell count studies were checked. Complete
killing of E. coli and K. pneumoniae 1 and was observed at 0 h. C. albicans got killed at
2h of incubation, whereas MRSA took 4 h when purified compound was used. Similarly,
Salm. Typhimurium 2 got killed at 4h. S. aureus took the longest time and killed at 10 h
of incubation, whereas S. epidermidis took 6 h of incubation (Figure 4.9.2.2).
0
10
20
30
40
50
60
70
80
1 2 3 4 5 6 7 8 9
Pe
rce
nta
ge
Via
bil
ity
Time of incubation (hrs)
S. aureus
S. epridermidis
E. coli
K. penumoniae1
Sh. flexeri
Salm. Typhimurium2
C. albicans
MRSA
Results
148
Figure 4.9.2.1: Time kill assay of butanolic extract from Penicillium citrinum
Figure 4.9.2.2: Time Kill studies of purified compound (KB 4) from Penicillim citrinum
0
20
40
60
80
100
120
0 2 4 6 8 10 12 14 24
Pe
rce
nta
ge
via
bil
ity
Time of incubation (h)
E. faecalis
S. aureus
S. epidermidis
E. coli
K. pneumoniae1
K. pneumoniae 2
P. aeruginosa
Sh. flexeri
Salm. Typhimurium 1
Salm. Typhimurium2
C. albicans
MRSA
0
10
20
30
40
50
60
70
0 2 4 6 8 10 12 14 24
Pe
rce
nta
ge
via
bil
ity
Time of incubation (h)
S. aureus
S. epidermidis
E. coli
K. pneumoniae1
Salm. Typhimurium2
C. albicans
MRSA
Results
149
4.9.3 Time Kill assay of butanolic extract from Aspergillus wentii
On the basis of MIC of butanolic extract of Aspergillus wentii, obtained for
different organisms they were subjected to viable cell count studies. Complete killing
of E. coli was observed at 0 hr. S. epidermidis, Salm. Typhimurium 2 and MRSA were
killed at 4h while S. aureus took 6 h for complete killing. Salm. Typhimurium 1 took the
longest time and showed 99.5 % killing at 12 h while the residual cells restarted their
growth after 12 h (Figure 4.9.3.1).
Figure 4.9.3.1: Time Kill assay of butanolic extract from Aspergillus wentii
4.9.4 Time Kill assay of ethyl acetate extract and purified compound from
Aspergillus terreus
Similarly, a stock solution 25 mg/ml of ethyl acetate extract of Aspergillus
terreus and 10 mg/ml for purified compound was prepared, respectively. Complete
killing of E. coli was observed at 0 h of incubation. C. albicans killed at 2 h of
incubation. K. pneumoniae 1, Salm. Typhimurium 1, P. aeruginosa and E. faecalis took
4 h of complete killing, whereas MRSA took 6 h of complete killing. S. epidermidis and
S. flexneri took 8 h, while S. aureus took 10 h complete killing. Salm. Typhimurium 2
and K. pneumoniae 2 took the longest time of 14 h of complete killing. Similarly, the
viable cell count studies with the purified compound revealed E. coli and C. albicans to
be killed at 0 h of incubation. E. faecalis killed at 2 h of incubation and E. faecalis took
0
20
40
60
80
100
120
0 2 4 6 8 10 12 14 24
Per
cen
tage
Via
bil
ity
Time of incubation (hrs)
S. aureus
S. epridermidis
E. coli
K. pneumoniae1
Sh. flexeri
Salm. Typhimurium1
Salm. Typhimurium2
C. albicans
MRSA
Results
150
4 h of complete killing. Salm. Typhimurium 1, S. flexneri, MRSA, P. aeruginosa took 4
h for complete killing. S. aureus and S. epidermidis got killed at 6 h and 8h, respectively
(Figure 4.9.4.1; 4.9.4.2).
Figure 4.9.4.1: Time kill assay of ethyl acetate extract from Aspergillus terreus
Figure 4.9.4.2: Time kill assay of purified compound (KB 1) from Aspergillus terreus
0
20
40
60
80
100
120
0 2 4 6 8 10 12 14 24
Pe
rce
nta
ge
via
bil
ity
Time of incubation (h)
E.faecalis
S.aureus
S.epidermidis
E.coli
K.pneumoniae1
K.pneumoniae 2
P.aeruginosa
Sh. flexneri
Salm.Typhimurium 1
Salm.Typhimurium2
MRSA
C.albicans
0
10
20
30
40
50
60
70
80
90
0 2 4 6 8 10 12 14 24
Pe
rce
nta
ge
Via
bil
ity
Time of incubation (h)
E.faecalis
S.aureus
S.epidermidis
E.coli
K.pneumoniae1
K.pneumoniae 2
P.aeruginosa
Sh. flexneri
Salm.Typhimurium 1
Salm.Typhimurium2
MRSA
C.albicans
Results
151
4.9.5 Time kill studies of standard antibiotics
1XMIC of the standard antibiotics (Gentamicin and Amphotericin B) was used
for time kill assay. Complete killing of S. epidermidis, E. coli, Salm. Typhimurium 1,
Sh.flexneri took 2 h for complete killing. S. aureus took 4 h whereas K. pneumoniae 1
and MRSA took 6 h for complete killing. E. faecalis, P. aeruginosa killed at 12 h of
incubation by Gentamicin and Amphotericin B took 12 for complete killing of C.
albicans. Salm. Typhimurium 2 took longest time of 24 h for complete killing (Figure
4.9.5.1).
Figure 4.9.5.1: Time kill study of standard antibiotics (Gentamicin and Amphotericin B)
4.10 Post antibiotic effect
4.10.1 PAE of butanolic extract and purified compound (KB 3) from Penicillium
expansum
Butanolic extract of Peniciilium expansum induced PAE ranging from 2 - 22 h
in the microorganisms tested. Sh. flexneri 2 h (Figure 4.10.1.1), S. epidermidis 2 h
(Figure 4.10.1.2), K. pneumoniae 1-4h (Figure 4.10.1.3), Salm. Typhimurium 2 4 h
(Figure 4.10.1.4), S. aureus 6 h (Figure 4.10.1.5), MRSA 6 h (Figure 4.10.1.6), C.
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 24
Pe
rce
nta
ge
via
bil
ity
Time (h)
E. faecalis
S. aureus
S. epridermidis
E. coli
K. pneumoniae 1
K. pneumoniae 2
P. aeruginosa
Sh. flexeri
Salm. Typhimurium 1
Salm. Typhimurium2
C. albicans
MRSA
Results
152
albicans 8 h (Figure 4.10.1.7) and E. coli 22 h (Figure 4.10.1.8) . Similarly the purified
compound induced PAE of ranging from 10 h to 22 h. MRSA 10 h (Figure 4.10.1.9), K.
pneumoniae 1 20 h (Figure 4.10.1.10), E. coli (Figure 4.10.1.11), C. albicans (Figure
4.10.1.12) and S. aureus (Figure 4.10.1.13) was found to have PAE of 22 h respectively.
Figure 4.10.1.1: PAE of Sh. flexneri
Figure 4.10.1.2: PAE of S. epidermidis
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
Results
153
Figure 4.10.1.3: PAE of K. pneumoniae1
Figure 4.10.1.4: PAE of Salm. Typhimurium 2
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
Results
154
Figure 4.10.1.5: PAE of S. aureus
Figure 4.10.1.6: PAE of MRSA
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
Results
155
Figure 4.10.1.7: PAE of C. albicans
Figure 4.10.1.8: PAE of E. coli
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
Results
156
Figure 4.10.1.9: PAE of MRSA
Figure 4.10.1.10: PAE of K. pneumoniae 1
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
Results
157
Figure 4.10.1.11: PAE of E. coli
Figure 4.10.1.12: PAE of C. albicans
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
Results
158
Figure 4.10.1.13: PAE of S. aureus
4.10.2 PAE of butanolic extract and purified compound (KB 4) from Penicillium
citrinum
PAE is persistent suppression of bacterial growth after their brief exposure (1 or
2 h) to an antimicrobial agent even in the absence of host defense mechanisms. The
concentrations used in kill time assay were applied in PAE studies. Butanolic extract of
Penicillium citrinum and the purified compound induced a varied PAE amongst test
organisms and was concentration dependent. Butanolic extract of Penicillium citrinum
induced PAE ranging from 2-20 h in the microorganisms tested. Sh. flexneri 2 h (Figure
4.10.2.1), S. aureus 4h (Figure 4.10.2.2), S. epidermidis 4h (Figure 4.10.2.3), MRSA 6
h (Figure 4.10.2.4), K. pneumoniae1 6 h (Figure 4.10.2.5), Salm. Typhimurium 2 6 h
(Figure 4.10.2.6), C. albicans 8 h (Figure 4.10.2.7) and E. coli 20 h (Figure 4.10.2.8),
Similarly the purified compound induced PAE of ranging from 8 h to 22 h, S. aureus 8
h (Figure 4.10.2.9), MRSA 10 h (Figure 4.10.2.10), C. albicans 12 h (Figure 4.10.2.11)
and K. pneumoniae 1 20 h (Figure 4.10.2.12).
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
Results
159
Figure 4.10.2.1: PAE of Sh. flexneri
Figure 4.10.2.2: PAE of S. aureus
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
time (h)
treated
control
Results
160
Figure 4.10.2.3: PAE of S. epidermidis
Figure 4.10.2.4: PAE for MRSA
Figure 4.10.2.5: PAE of Salm. Typhimurium 2
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time(h)
treated
control
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
Results
161
Figure 4.10.2.6: PAE for K. pneumoniae 1
Figure 4.10.2.7: PAE for C. albicans
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
Results
162
Figure 4.10.2.8: PAE of E. coli
Figure 4.10.2.9: PAE of S. aureus
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
Results
163
Figure 4.10.2.10: PAE of MRSA
Figure 4.10.2.11: PAE of C. albicans
Figure 4.10.2.12: PAE of K. pneumoniae1
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
Results
164
4.10.3 PAE of ethyl acetate extract and purified compound (KB 1) from
Aspergillus terreus.
Ethyl acetate extract of Aspergillus terreus induced PAE ranging from 2 - 22 h
in the microorganisms tested. S. aureus 2 h (Figure 4.10.3.1), S. epidermidis 2 h (Figure
4.10.3.2), Sh. flexneri 2 h (Figure 4.10.3.3), MRSA 4 h (Figure 4.10.3.4), Salm.
Typhimurium 4 h (Figure 4.10.3.5), E. coli 6 h (Figure 4.10.3.6), K. pneumoniae 1 8 h
(Figure 4.10.3.7) and C. albicans 22 h (Figure 4.10.3.8). Similarly the purified
compound PAE of MRSA 6 h (Figure 4.10.3.9), S. aureus 10 h (Figure 4.10.3.10), E.
coli 18 h (Figure 4.10.3.11), K. pneumoniae 1 (Figure 4.10.3.12) and C. albicans
(Figure 4.10.3.13) posses PAE of 22 h.
Figure 4.10.3.1: PAE of S. aureus
Figure 4.10.3.2: PAE of S. epidermidis
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
Results
165
Figure 4.10.3.3: PAE of Sh. flexneri
Figure 4.10.3.4: PAE of MRSA
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
Results
166
Figure 4.10.3.5: PAE of Salm. Typhimurium 2
Figure 4.10.3.6: PAE of E. coli
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
Results
167
Figure 4.10.3.7: PAE of K. pneumoniae 1
Figure 4.10.3.8: PAE of C. albicans
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
Results
168
Figure 4.10.3.9: PAE of MRSA
Figure 4.10.3.10: PAE of S. aureus
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
Results
169
Figure 4.10.3.11: PAE of E. coli
Figure 4.10.3.12: PAE of K. pneumoniae1
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
Results
170
Figure 4.10.3.13: PAE of C. albicans
4.10.4 PAE of standard antibiotics
1XMIC of the standard antibiotics (Gentamicin and Amphotericin B) was used
for time Post antibiotic studies. PAE of standard antibiotic ranged from 1-23h for all
the microorganism tested with PAE of 1 h for Salm. Typhimurium1 (Figure 4.10.4.1).
Sh. flexneri (Figure 4.10.4.2), S. epidermidis was found to possessed PAE of 2 h (Figure
4.10.4.3) followed by S. aureus with PAE of 3 h (Figure 4.10.4.4), K. pneumoniae 1
(Figure 4.10.4.5), Salm. Typhimurium 2 (Figure 4.10.4.6), E. coli (Figure 4.10.4.7) and
MRSA (Figure 4.10.4.8) possessed PAE of 4h. K. pneumoniae 2 for 5 h (Figure
4.10.4.9). Amphotericin B gave PAE of 5 h and 23 h for C. albicans and C. tropicalis
respectively.
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 24
log
10
cfu
/ml
Time (h)
treated
control
Results
171
Figure 4.10.4.1 PAE of Salm. Typhimurium1
Figure 4.10.4.2 PAE of Sh. flexneri
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6 7 8 9 24
Log
10
CF
U/m
L
Time of incubation (h)
Treated
Control
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6 7 8 9 24
Log
10
CF
U/m
L
Time of incubation (h)
Treated
Control
Results
172
Figure 4.10.4.3 PAE of S. epidermidis
Figure 4.10.4.4 PAE of S. aureus
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6 7 8 9 24
Log
10
CF
U/m
L
Time of incubation (h)
Treated
Control
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6 7 8 9 24
Log
10
CF
U/m
L
Time of incubation (h)
Treated
Control
Results
173
Figure 4.10.4.5 PAE of K. pneumoniae 1
Figure 4.10.4.6 PAE of Salm. Typhimurium2
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6 7 8 9 24
log
10C
FU
/ml
Time of incubation (h)
TREATED
CONTROL
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6 7 8 9 24
Log
10
CF
U/
mL
Time of incubation (h)
TREATED
CONTROL
Results
174
Figure 4.10.4.7 PAE of E. coli
Figure 4.10.4.8 PAE of MRSA
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6 7 8 9 24
Log
10
CF
U/m
L
Time of incubation (h)
TREATED
CONTROL
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6 7 8 9 24
Log
10
CF
U/M
l
Time of incubation (h)
TREATED
CONTROL
Results
175
Figure 4.10.4.9 PAE of K. pneumoniae 2
Figure 4.10.4.10 PAE of C. albicans
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6 7 8 9 24
Log
10
CF
U/m
L
Time of incubation (h)
TREATED
CONTROL
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6 7 8 9 24
Log
10
CF
U/m
L
Time of incubation (h)
TREATED
CONTROL
Results
176
4.11 Comparison of standard antibiotics with solvent extracts and purified
compounds:
4.11.1 Comparison of standard antibiotics with purified compounds by time kill
assay:
Figure 4.11.1.1: Comparison of time kill assay of purified compounds KB1, KB3 and
KB 4 with standard antibiotics (Gentamicin and Amphotericin B).
Comparison of Time kills studies of gentamicin and amphotericin B with
purified compounds (KB 1, KB3 and KB4). Complete killing of E. coli at 0 h by KB 1,
KB 3 and KB 4 whereas gentamicin took 2h for complete killing. Similarly S. aureus
took 4 h for complete killing by gentamicin whereas KB1, KB 3, and KB 4 took 6 and
10 h for complete killing respectively. S. epidermidis took 2 h for complete killing by
gentamicin whereas KB 1, KB 3 and KB 4 took 8 h, 10 h and 6 h respectively. K.
pneumoniae 1 got killed at 0 h of incubation by KB 1, KB 3 and KB4 respectively
whereas it got killed at 6 h by gentamicin. KB 3 took 0 h for killing Salm . Typhimurium
whereas gentamicin took 24 h for complete killing of Salm. Typhimurium2. Similarly
MRSA got killed at 0 and 4 h by all the three isolated compounds (KB 1, KB 3 & KB 4)
whereas gentamicin took 6 h for complete killing. C. albicans got killed at 12 h by
gentamicin whereas 0-4 h of incubation was enough for all three isolated compounds.
0
5
10
15
20
25
30
Kil
lin
g t
ime
(in
Hrs
)
S. aureus
S. epidermidis
E. coli
K. pneumoniae1
Salm. Typhimiurium2
C. albicans
MRSA
Results
177
4.11.2 Comparison of standard antibiotics with purified compounds by Post
antibiotic studies
PAE of E. coli for compound isolated from KB 4, KB 3 and KB 1 was found to
be for 22 h , 22 h and 18 h respectively whereas for Gentamicin PAE for E. coli was
found to be 4h. Similarly PAE for K. pneumoniae 1 for compound isolated from
Penicillium expansum (KB 3), Penicillium citrinum (KB 4) and Aspergillus terreus
(KB 1) was found to be 20 h, 20 h and 22 h respectively whereas gentamicin gave PAE
of 4 h in case of K. pneumoniae 1. PAE of purified compounds isolated from
Penicillium expansum, Penicillium citrinum and Aspergillus terreus for MRSA was
found to be 10 h, 10 h and 6 h whereas gentamicin gave PAE of 4 h for MRSA. PAE for
C. albicans for the purified compounds isolated from Penicillium expansum,
Penicillium citrinum and Aspergillus terreus was found to be 12 h, 22 h, 22 h
respectively whereas for standard antibiotics amphotericin B gave PAE of 5 h for C.
albicans (Figure 4.11.2.2).
Figure 4.11.2.2: Comparison of PAE of standard antibiotics with the purified
compounds
0
5
10
15
20
25
PA
E (
in H
rs)
E. coli
K. pneumoniae1
C. albicans
MRSA
Results
178
4.12 Thermostability
4.12.1 Thermostability of the purified compound isolated from Penicillium
expansum (KB 3)
No loss in activity was observed at 50 °C against S. aureus, K. pneumoniae 1,
Salm. Typhimurium 2, C. albicans and MRSA, whereas only 2 %, 3 % and 23 % loss
was observed at 50 °C for E. coli, S. epidermidis and Sh. flexneri, respectively.
Similarly, no loss was observed till 80 °C against S. aureus, Salm. Typhimurium 2 and
C. albicans while the maximum loss they suffered was 16 %, 8 % and 9 % at 100 °C,
respectively. Further, treatment at 100 °C resulted in maximum loss of 34 % against Sh.
flexneri and 12 % against K. pneumoniae 1, 23 % against S. epidermidis while
maximum loss of 46 % was demonstrated against E. coli (Figure 4.12.1.1).
Figure 4.12.1.1 Thermostability of the purified compound KB 3
4.12.2 Thermostability of the purified compound isolated from Penicillium
citrinum (KB 4)
No loss in activity was observed with E. coli, Salm. Typhimurium 1, C. albicans, MRSA
and P. aeruginosa, whereas only 5 % loss was observed with S. aureus and Sh. flexneri,
0
5
10
15
20
25
30
35
40
45
50 60 70 80 90 100
Per
cen
tage
loss
in
act
ivit
y
Tempeature in ˚C
S.aureus
S.epridermidis
E.coli
K.penumoniae1
Sh. flexeri
Salm. Typhimurium2
C.albicans
MRSA
Results
179
respectively and 9.5 % with S. epidermidis at 50 °C. C. albicans suffered no loss in
activity at 90 °C, whereas 5.75 and 1.5 % loss was observed with MRSA and P.
aeruginosa, respectively, whereas only 2 % loss was observed at 100 °C with C.
albicans and P. aeruginosa and 5.75 % with MRSA, respectively. A maximum of 71 %
loss in activity was observed with Sh. flexneri, 42 % with E. coli, 39 % with Salm.
Typhimurium 1, 28 % with S. epidermidis, 25 % with S. aureus and 18 % loss with k.
pneumoniae at 100 °C (Figure 4.12.2.1).
Figure 4.12.2.1 Thermostability of the purified compound KB 4
4.12.3 Thermostability of the purified compound isolated from Aspergillus terreus
(KB 1)
Similarly, compound KB 1 from Aspergillus terreus , demonstrated no loss in
antimicrobial activity against K. pneumoniae 1, C. albicans and MRSA at 50 °C,
whereas only 2 % loss was observed for S. aureus, S. epidermidis and E. coli,
respectively at 50 °C. Sh. flexneri suffered a maximum loss of 49 % at 100 °C and a
minimum loss of 2 % and 4% loss in activity were observed for C. albicans and MRSA,
respectively (Figure 4.12.3.1).
0
10
20
30
40
50
60
70
80
50 60 70 80 90 100
Per
cen
tage
loss
in
act
ivit
y
Temperature ( ˚ C )
S.aureus
S.epidermidis
E.coli
K.pneumoniae1
P.aeruginosa
Sh. flexneri
Salm.Typhimurium2
MRSA
C.albicans
Results
180
Figure 4.12.3.1 Thermostability of the purified compound KB 1
4.13 Mechanism of action
4.13.1 Membrane integrity assay by ethidium bromide uptake
Ethidium bromide is a membrane impermeable dye which cannot diffuse
through intact cell membranes. Once the dye passes through the compromised
membranes, it intercalates into double stranded nucleic acids which results into
enhanced fluorescence in the visible region. The test organism i.e. MRSA and E. coli
were treated with purified compounds (KB1 and KB4) and subsequently exposed to
ethidium bromide. Examination of these organisms under confocal scanning laser
microscope (CSLM) revealed varying fluorescence intensities. MRSA gave the highest
fluorescence when treated with standard antibiotic gentamicin followed by both the
compounds where fluorescence was a slight lower than standard antibiotic gentamicin
as compared to control where the cells were less stained by EtBr (Figure 4.13.1.1).
Similarly when E. coli cells were treated with both the compounds there was a change
in the morphology of the cells and the fluorescence intensity was high as compare to the
standard antibiotic Gentamicin. Morphologically E. coli cells when treated with both
the compounds the cells enlarge from its normal shape and the cells are most vulnerable
as higher intensity was recorded in case of E .coli cells whereas when E. coli cells are
treated with standard antibiotics the fluorescence intensity was lower than the purified
compounds. In case of control for E. coli cells the fluorescence was too low (Figure
4.13.1.2).
0
10
20
30
40
50
60
50 60 70 80 90 100
Per
cen
tage
loss
in
act
ivit
y
Temperature ( ˚ C )
S.aureus
S.epidermidis
E.coli
K.pneumoniae1
Sh. flexneri
Salm. Typhimurium2
MRSA
C.albicans
Results
181
(a) (b)
(c) (d)
Figure 4.13.1.1: Ethidiium bromide uptake by MRSA treated with purified compounds
KB1, KB 4 and standard antibiotic gentamicin. (a) MRSA cells untreated; (b) MRSA
cells treated with gentamicin; (c) MRSA cells treated with KB 1; (d) MRSA cells
treated with KB 4
Results
182
(a) (b)
(c) (d) .
Figure 4.13.1.2: Ethidiium bromide uptake by E.coli treated with purified compounds
KB1, KB 4 and standard antibiotic Gentamicin. (a) E. coli cells untreated; (b) E. coli
cells treated with Gentamicin; (c) E. coli cells treated with KB 1; (d) E. coli cells treated
with KB 4
Results
183
4.14 Toxicity testing
In order to carry out the safety evaluation, the solvent extracts as well as all the
purified compounds isolated from the selected fungi were assayed for their cytotoxic
and mutagenic effect.
4.14.1 Mutagenicity testing using Ames test
All the solvent extracts viz. butanolic extract of Penicillium expansum,
Penciliium citrinum, Aspergillus wentii and ethyl acetate extract of Aspergillus terreus
as well as the purified compounds (KB 3, KB 4, KB 2 and KB 1) were subjected to
mutagenicity testing through Ames test. The number of revertant colonies in the
positive control was numerous in all the cases, whereas the bacteria incubated with the
solvent extracts and the purified compounds did not show any revertant colonies (Figure
4.14.1.1; 4.14.1.2; 4.14.1.3; 4.14.1.4; 4.14.1.5; 4.14.1.6).
Results
184
Figure 4.14.1.1: Ames test for purified compound KB 1
Figure 4.14.1.2: Ames test for purified compound KB 2
Results
185
Figure 4.14.1.3: Ames test for purified compound KB 3
Figure 4.14.1.4: Ames test for purified compound KB 4
Results
186
Figure 4.14.1.5: Control (Sodium Azide) Ames test (Disc method)
Figure 4.14.1.6: Control (Sodium Azide) Ames test (Tube method)
Results
187
4.14.2 Cytotoxicity testing using MTT assay
MTT assay is a colorimetric assay which is based on the capacity of mitochondrial
succinate dehydrogenase enzymes in living cells to reduce the yellow water soluble
substrate MTT into an insoluble, purple colour formazan product which is measured
spectrophotometrically. Since, reduction of 3-[(4,5-dimethylthiazol-2-yl)-2,5-diphenyl]
tetrazolium bromide (MTT) can only occur in metabolically active cells, where MTT is
converted to insoluble formazan crystals that are dissolved in DMSO and the absorbance of
purple coloured solutions directly represents the viability of the cells. The absorbance
values of positive control (untreated cells) were compared with the absorbance of tested
extracts/compounds. 95.4 % and 96% viable cells were observed with butanolic extract and
purified compound KB 2 of Aspergillus terreus, showing both to be non-cytotoxic.
Similarly, butanolic extract of Penicillium expansum and its isolated compound KB 3
showed 95.4 % and 98 % viability. The butanolic extract obtained from Penicillium
citrinum and its purified compound KB 4 showed 91.3 % and 90 % viable cells. Aspergillus
terreus showed 86.9 % and 85.4 % viability in ethyl acetate extract and the purified
compound KB 1, respectively (Figure 4.14.2.1).
P.exp- Penicillium expansum, P.cit- Penicillium citrinum, A.we-Aspergillus wentii,
A.te- Aspergillus terreus
Figure 4.14.2.1 Cytotoxicity testing of solvent extract and purified compounds of all
the four fungi
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
P.exp P.cit A. we A. te
Ab
sorb
an
ce a
t 5
90
nm
Fungi
Solvent extract
Purified compound
positive control
negative control
Results
188
4.15 Cytotoxic activity against some human cancer cell lines
Cytotoxicity assays are widely used by the pharmaceutical industry to screen for
cytotoxicity. Researchers can either look for cytotoxic compounds, if they are interested
in developing a therapeutic that targets rapidly dividing cancer cells, for instance; or
they can screen "hits" from initial high-throughput drug screens for unwanted cytotoxic
effects before investing in their development as a pharmaceutical. The sulforhodamine
B (SRB) assay was developed by Skehan and colleagues to measure drug-induced
cytotoxicity and cell proliferation for large-scale drug-screening applications. All the
four compounds were tested for their cytotoxicity and were tested against human cancer
cell lines. Both investigational compounds are endowed with valuable cytotoxic
potential against all tested human cancer cell lines (Table 4.15.1). Against leukemia
cancer cell line (THP-1), compounds KB 1 and KB 4 were found to display moderate
cytotoxicity with IC50 = 28. In the case of lung cancer cell line (A549), compound KB 1
showed excellent inhibitory activity with IC50 = 10, whereas, compound KB 4 displayed
low to moderate cytotoxicity with IC50 > 50. Against prostate cancer cell line (PC-3),
compounds KB 1 exhibited inhibitory potential with IC50 > 50 and compound KB 4
found to display considerable inhibitory activity with IC50 = 31. Furthermore, in the
case of breast cancer cell line (MCF-7) compound KB 1 showed significant cytotoxic
potential with IC50 = 19, whereas compound KB 4 exhibited promising activity with
IC50 = 35.
Results
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Table 4.15.1: IC50 value of purified compounds against cancer cell lines.
Compounds
Conc
(µg/ml)
A549 THP-1 PC-3 Colo-205
Lung Leukemia Prostate Colon
% Growth Inhibition
KB1 1 26 21 0 0
5 30 30 0 10
10 45 52 0 28
30 60 73 40 80
50 77 88 65 96
IC50 28 10 >50 19
KB4 1 25 0 10 0
5 28 10 27 20
10 31 22 38 39
30 55 39 45 50
50 75 65 70 72
IC50 28 >50 31 35
5-FU 1 72 74 - -
Adriamycin 1 67 70 - 80
Mitomycin-c 1 - 67 71 -
4.15.1 Flowcytometry analysis of nuclear DNA
Analysis of nuclear DNA by flow cytometry is interesting in fundamental
research and has broadly contributed to improved knowledge on cell DNA content and
their distribution in the various phases of cycle. DNA amount in cells is often the single
parameter measured for cell cycle studies by flow cytometry (Figure 4.15.1.1). In order
to obtain a linear relationship between cellular fluorescence intensity and DNA amount,
analyses are performed with fluorescent molecules that bind specifically and
stoichiometrically to DNA. Some dyes possess an intercalative binding mode such as
propidium iodide etc, whereas others such as Hoechst 33342, DAPI etc. possess an
affinity for DNA A-T rich region.
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190
To analyses the nuclear DNA content, lung cancer cells (2x106 cells/ml/well)
were treated with compounds (KB 1 and KB 4) for 24 h at concentrations 10 and 20
µM. The hypo diploid sub-G1 DNA fraction (< 2n DNA) was found to increase from 26
% to 55 % for KB-1 and 37 % to 58 % for KB 2 in a concentration dependent manner
(Figure. 76). These results indicate that compounds KB 1 &2 induce apoptosis in lung
cancer (A549) cells. (Figure 4.15.1.2)
Results
191
Figure 4.15.1.2: Flowcytometric analysis of nuclear DNA in lung cancer cells