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

189

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.

Results

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