effect of gamma radiation on the microbial synthesis of

Minoufiya University

Genetic Engineering & Biotechnology

Research Institute (GEBRI)

Industrial Biotechnology Department

Effect of Gamma Radiation on the

Microbial Synthesis of Metal Nanoparticles

By

Farag Marzouk Marea Mosalam (B. Sc. of Pharmacy, Al-Azhar University (assiut), 2008)

A thesis submitted in partial fulfillment of the requirements

governing the award of the degree of

Master of Science

In Genetic Engineering and Biotechnology

Department of Industrial Biotechnology

(Fermentation department)

2013

I

TABLE OF CONTENTS

Page number

ABSTRACT…………………………………………………….......................... VII

ACKNOWLEDGEMENTS………………………………….......... ……………… IX

LIST OF TABLES……………………………………………… ……………….. . X

LIST OF FIGURES…………………………………………..................................... VII

LIST OF ABBREVIATIONS…………………………...………………………… XVI

English summary ……………………………………………………………………... 119

Arabic summary……………………………………………………………………... 139

Chapter I: LITERATURE REVIEW…………………….

1

1.1. Monascus purpureus………………………………………………………….. 1

1.1.1 Effect of nutritional and environmental on pigments production ……. 3

1.1.2. Monascus purpureus pigments and their structures…………………… 3

1.2. Nanoparticles ………………………………………………………………… 5

1.2.1. Definition………………………………………………......................... 5

1.2.2. Strategies of Nanoparticles synthesis…………………………………. 5

1.2.3. Methods of Metal nanoparticles……………………………………….. 6

1.2.4. Advantage of biological than chemical and physical synthesis……… 7

1.2.5. Problems of biological nanoparticle synthesis……………..................... 7

1.2.6. Classification of metal nanoparticles biosynthesis techniques…………. 8

1.2.7. Advantages of extracellular biosynthesis…………………................... 9

1.2.8. Metal nanoparticles characterization……………………………………… 10

1.3. Radiation………………………………………….…………………………….. 1

1.3.1. Definition ………………………………………………………………….. 10

1.3.2. Types of Radiation ……………………………………………………… 10

1.3.3. Types of Ionizing Radiation……………………….................................... 11

II

1.3.4. Radiation exposure………………………………….…………………… 12

1.3.5. Units of radiation dose……………………………...…………………… 13

1.3.6. Radiation and metal nanoparticles…………………….............................. 14

1.3.7. Advantages of synthesized metal nanoparticles using irradiation

techniques…………………………………………………………………

15

Chapter II: Parameters optimization for Monascus purpueurs pigments

production and silver nanoparticles synthesis ……............................... 19

2.1. Abstract ……………………………………………………………………….. 19

2.2. Introduction……………………………………………………………………. 19

2.3. Material and methods…………………………………………………………. .22

2.3.1. Strain and Culturing Conditions …………………................................. 22

2.3.2. Reagents………………………………………………………………… 23

2.3.3. Pigments extraction and determination………………………………….. 23

2.3.4. Parameters optimization for pigments production using Plackett-Burman

experimental design……….………................................................................... 23

2.3.5. Silver Nanoparticle (Ag NPs) synthesis from fungal biomass and

Supernatants ……………………………………………………………………… 26

2.3.6. Measurement of antioxidant activity……………........................................ 27

2.3.7. Characterization of silver nanoparticles………………………………… 27

2.3.7.1. UV-VIS spectral analysis …………………................................... 27

2.3.7.2. Dynamic light scattering (DLS)……………………………………. 28

2.3.7.3. Fourier Transform Infrared Spectroscopy (FT-IR)……………… 29

2.4. Results and discussion…………………………………………………………

29

2.4.1. Effect of Parameters optimization on pigments production ………… 29

2.4.2. Spectral analysis of extracellular pigments…………………………… 30

2.4.3. Silver nanoparticles (Ag NPs) synthesis………………………………. 32

III

2.4.3.1 Silver nanoparticles (Ag NPs) synthesis by M. purpureus supernatants… 32

2.4.3.2 Silver nanoparticles (Ag NPs) synthesis by M. purpureus using of

fungal biomass……………………………………………………………..

32

2.4.4. Characterization of silver nanoparticles………………………………. 32

2.4.4.1. UV. Spectral analysis………....................................................... 32

2.4.4.2. Dynamic light scattering (DLS) analysis……..………………….. 34

2.4.4.3. FT-IR spectra of M.purpureus supernatant analysis…… 35

2.4.5. Relationship between optical densities, DLS size and antioxidant activity

(DPPH scavenging) ………………..................................................................

36

2.5. Conclusion ………………………………………………………………………..

37

Chapter III: Silver nanoparticles synthesis without gamma irradiation

39

3.1. Abstract………………………………………………………………………… 39

3.2. Introduction ………………………………………..………………………….. 40

3.3. Materials and Methods………………………………........................................ 41

3.3.1. Chemicals………………………………………………..…………………… 41

3.3.2. Microorganism…………………………………….…………………….. 41

3.3.3. Supernatant Preparation………………………………………………… 42

3.3.4. Nitrate Reductase Assay ………………………………………………... 42

3.3.5. Measurement of antioxidant activity………………………………….... 42

3.3.6. Measurement of reducing activity……………………..………………… 42

3.3.7. Pigments extraction, analysis and incubation with AgNO3……………. 43

3.3.8. Silver Nanoparticle (Ag NPs) synthesis………………………………… 43

3.3.9. Effect of pH on Ag NPs synthesis and particle sizes…………………… 44

3.3.10. Effect of silver nitrate concentration……………..……………………. 44

3. 3.11. Characterization of silver nanoparticles………………………………. 44

3.3.11.1. UV-VIS spectral analysis ……………………............................. 44

IV

3.3.11.2. Dynamic light scattering (DLS)……………………………….. 45

3.3.11.3. Transmission Electron Microscopy (TEM)……………………… 45

3.3.11.4. Fourier Transform Infrared Spectroscopy (FT-IR)…………… 45

3.3.11.5. Energy Dispersive X- ray (EDX)……………………………… 45

3.3.11.6. X-ray diffraction (XRD)……………………………………….. 46

3.3.12. Antimicrobial activity………………………………………………………. 47

3.4. Results and Discussions………………………………………………………..

48

3.4.1. Ag NPs colloids synthesis………………………………………………. 48

3.4.2. Effect of time on Ag NPs synthesis…………………………………….. 50

3.4.3. Effect of pH on the Ag NPs synthesis…………………………………… 50

3.4.4. Effect of silver nitrate concentration on Ag NPs synthesis…………….. 52

3.4.5. Role of M.purpureus pigments on Ag NPs synthesis ………………….. 53

3.4.6. Nitrate Reductase activity……………………………………………….. 55

3.4.7. Reducing power and DPPH Free Radical Scavenging Activity of M.

purpureus supernatant and Ag NPs…….…………………………........

55

3.4.8. Characterization and stability study of the silver nanoparticles

Particle size and distribution ……………………................................... 56

3.4.8.1. Transmission Electron Microscopy (TEM)……………………….. 57

3.4.8.2. Dynamic light scattering (DLS)…………………………………. 57

3.4.8.3. EDX analysis of Ag-NPs……………………………………….. 59

3.4.8.4. X-ray diffraction (XRD) analysis of Ag NPs …………………. 59

3.4.8.5. FT-IR spectra of silver nanoparticles analysis ………………… 62

3.4.9. Antimicrobial activity of synthesized Ag NPs by M. purpureus

supernatant…………………………………………………………………

65

3.5. Conclusion………………………………………………………………………..

68

V

Chapter IV: Silver nanoparticles synthesis using Monascus purpueurs

supernatant under gamma irradiation effect……………………………………

70

4.1. Abstract ……………………………………………………................................ 70

4.2. Introduction …………………………………………………………………… 70

4.3. Materials and methods…………………………………………………........... 76

4.3.1. Supernatant preparation………………………………………………… 76

4.3.2. Synthesis of Ag NPs colloids by gamma irradiation …………………….. 76

4.3.2.1. Gamma irradiation source…………………………………………….. 76

4.3.2.2. Radiation processing ………………………………………………….. 76

4.3.2.2.1. Ex situ radiation process…………………………………………. 76

4.3.2.2.2. In situ radiation process…………………………………………. 76

4.3.3. Assay of antioxidant activity ……………………………………………. 77

4.3.4. Characterization of silver nanoparticles…………………………………… 77

4.4. Results and discussions ……………………………………………………….

78

4.4.1. In Situ radiation process……………………………………………………… 78

4.4.1.1. Mechanism of reduction……………………………………………. 78

4.4.1.2. Characterization of synthesized Ag NPs by in situ irradiation

process……….…………………………………………………..…

83

4.4.1.2.1. Transmission electron microscope (TEM) analysis……………….. 83

4.4.1.2.2. Dynamic light scattering analysis (DLS)……………………….. 86

4.4.1.2.3. Fourier Transform Infrared Spectroscopy (FT-IR) analysis………. 90

4.4.1.2.4. X-ray diffraction (XRD) analysis of Ag-NPs synthesized by M.

purpureus supernatant prepared by in situ irradiation process……

92

4.4.2. Ex situ radiation process……………………………………............................. 94

VI

4.4.2.1. Mechanism of reduction………………………………………….. 94

4.4.2.2. Characterization of synthesized Ag NPs by ex situ radiation

process……………………………………………………………………….

100

4.4.2.2. 1. Transmission electron microscope (TEM) analysis…………….. 100

4.4.2.2.2. Dynamic light scattering analysis (DLS)………………………… 103

4.4.2.2. 3. Fourier Transform Infrared Spectroscopy (FT-IR) analysis……. 107

4.4.2.2.4. X-ray diffraction (XRD) analysis of synthesized Ag NPs by ex situ

radiation process………………………………………………..

109

4.4.3. EDX analysis of synthesized Ag NPs by gamma irradiation………………… 111

4.4.4. Effect of Time on synthesis and stability of Ag NPs colloids.………………….. 112

4.4.4.1. Synthesis by in situ irradiation process ……………………………. 112

4.4.4.2. Synthesis by ex situ radiation process………................................. 113

4.4.5. DPPH Free Radical Scavenging Activity of irradiated M. purpureus

supernatant, AgNO3 and Ag NPs ……......................................................

115

4.4.6. Comparison between DPPH scavenging activity and size of synthesized Ag

NPs by gamma irradiation ……………………………………………………. 116

4.5. Conclusion………………………………………………………………….

117

Chapter V: REFERENCES………………………………………………………..

121

VII

Abstract

The present study demonstrates an unprecedented green process for the

synthesis of spherical shaped and stabilized Ag NPs using Monascus

purpureus supernatant via two methods; Non-irradiated and irradiated

condition.

The objective of the study can be achieved in three main sections:

Part I describes the factors affecting M. purpueurs pigments production

and silver nanoparticles synthesis.

Part II of the research involves the silver nanoparticles synthesis by M.

purpueurs supernatant without gamma irradiation.

Part III of the research involves the silver nanoparticles synthesis by M.

purpueurs supernatant under gamma irradiation effect.

In Part I, the main achievements and key factors affecting M. spurpueurs

pigments production were discussed.

A sequential optimization strategy based on statistical experimental

designs was employed to enhance the production of M.purpueurs

pigments. Plackett-Burman design was used to screen the bioprocess

parameters which significantly influencing M. purpueurs pigments

production.

In Part II, AgNO3 Aqueous solution was treated with M. purpureus

supernatant for synthesis of silver nanoparticles (Ag-NPs). The Nano

metallic dispersions were characterized by surface Plasmon absorbance

measuring at 420nm for Ag Nanoparticles. Transmission electron

microscopy, Dynamic light scattering (DSL) approved particle size in

rang from 1nm up to 8nm. XRD analysis of the silver nanoparticles

confirmed the formation of metallic silver. Further analysis was carried

out by Fourier Transform Infrared Spectroscopy (FTIR), which indicated

VIII

abounding of silver Nanoparticles with M.purpureus supernatant

molecules and provides evidence for the presence of proteins as possible

biomolecules responsible for the capping agent who helps in increasing

the stability of the synthesized silver nanoparticles and EDX Spectra

confirmed the presence of Nano crystalline silver. This small synthesized

silver nanoparticles size it showed antibacterial and antifungal activities.

In Part III Aqueous dispersion of highly stable silver nanoparticles were

synthesized using gamma irradiation of M.purpureus supernatant

molecules as reducing and stabilizing agent. The size of the silver

nanoparticles can be turned by controlling the radiation dose and mixing

method of AgNO3 solution with M. purpureus supernatant (irradiation

process).The obtained Ag nanoparticles were stable for over 3 months at

room temperature

IX

ACKNOWLEDGMENT

First and forever, ultimate thanks are due to ALLAH, who blessed me

with kind professors and colleagues, gave me the support to prepare this

thesis and without their helps; this work could not be achieved.

I wish to express my gratitude to my principal supervisor, Dr. Ashraf

F. El-baz, for his valuable suggestions, ideas, guidance, support and

intellectual advice during the courses of my post graduate studies. He is

the most enjoyable person to work with, and he also gave me a lot of

personal help beyond academic research.

I would like to give a special thanks to Dr. Ahmed Ibrahim El-batal, as

he has engaged me in many helpful discussions, provided me with

important advices and nanotechnology application, information and

measurements.Curve and also the opportunity to work in Nanotechnology

Unit at National Center for Radiation Research and Technology, Nasr

City, Cairo, Egypt. Atomic authority, Egypt.

My deepest thanks and appreciation to Dr. Ahmed A.Tayel for his

valuable suggestions, advice and help during my whole research

I am grateful to the official reviewers of this thesis for their constructive

criticism and suggestions for improving this thesis. My thanks go to all

my colleagues in the laboratory for providing a friendly environment to

work in and for giving me a memorable time in my life.

I really appreciated my deep and sincere gratitude goes to my family

especially my father, my late mother and my lovely brothers. Last but

definitely not least, my gratitude is beyond words for their moral support

and patience during my studies I couldn’t have done it without all of

them.

X

LIST OF TABLES

Table number page

Table 1: Plackett-Burman experimental design screen trials for

important nutrients and conditions influencing

production of pigments by M. perpureus………………

24

Table 2: M. purpureus pigments optical density (OD) expressed

as units of absorbance (λUA)……………………

30

Table 3: Mean diameter size of synthesized silver nanoparticles

………………………………………………………

35

Table 4: Relationship between optical densities (OD), DLS size

and DPPH scavenging activity of synthesized Ag

NPs…………………………………………………

37

Table5: Optical density of M. purpureus produced Pigments

…………………………………………

55

Table 6: Difference in reducing power and antioxidant activities

of M.purpureus supernatant after and before Ag NPs

synthesis……………………………………………… 56

Table7: XRD analysis of synthesized Ag NPs from M.

purpureus supernatant………………………………...

61

Table 8: Wavenumber of characteristic bonds of M.purpureus

supernatant and Ag NP ……………………………

64

Table 9: Antimicrobial activity of synthesized Ag NPs by M.

purpureus supernatant against different microbial

strains…………………………………………………..

67

Table 11: wavenumber of characteristic bonds and

corresponding comments for M. purpureus supernatant

and synthesized Ag NPs by in situ irradiation………

92

Table 12: XRD analysis of synthesized Ag NPs by in situ

XI

irradiation at 5kGy………………………………….... 94

Table 13: Characterization of synthesized Ag NPs by ex situ

radiation process at different doses of γ-radiation……

107

Table14:Wavenumber of characteristic bonds and corresponding

comments for M.purpureus supernatant and

synthesized Ag NPs colloids by ex situ radiation

process at dose 25kGy …………………………….

109

Table 15: XRD analysis of synthesized Ag NPs by ex situ

radiation at radiation dose 25kGy ………………….

111

Table 16: DPPH Free Radical Scavenging Activity of irradiated

M. purpureus supernatant, AgNO3 and Ag NPs ……

116

Table 17: Relationship between DPPH scavenging activity and

size of synthesized Ag NPs by in situ and ex situ

irradiation process ……………………………………

117

XII

LIST OF FIGURES

Figure Page

Fig. 1: Monascus purpureus pigments structures ………………….. 4

Fig. 2: Monascus purpureus new red pigments structures ……………. 5

Fig .3:UV–Vis spectra of Ag NPs formed after 48h of Monascus

purpureus supernatants incubated with AgNO3, Trials number 5,

18, 19, 27, 32, 38, 63, 64, 69, 74, and 75…………

33

Fig.4: UV–Vis spectra of incubated aqueous extract of fungal biomass

(A), Macerated mycelia (B) and trial number 19 supernatant

(C) with silver nitrate ….........................................................

34

Fig.5: FTIR spectra of M. purpureus supernatant…………………… 36

Fig. 6: Image of color change after Ag NPs formation …………… 49

Fig.7: UV-VIS spectra of Ag NPs formed after incubation of M.

purpureus supernatant with AgNO3 after 72h.....................

49

Fig.8: UV-VIS spectra of Ag NPs formed by M. purpureus

supernatant at different incubation time……………………

51

Fig.9: Effect of pH on Ag NPs synthesized by M. purpureus

supernatant expressed as UV-VIS spectra …….................

52

Fig.10: Effect of AgNO3 concentration on Ag NPs synthesized by M.

purpureus supernatant expressed as UV-VIS spectra………

54

Fig.11: UV-visible spectra of synthesized Ag NPs by M. purpureus

supernatant and extracted pigments……………………….

54

Fig.12: TEMN Micrograph of synthesized Ag NPs colloids by M.

purpureus supernatants……………………………………

58

Fig.13: DLS graph of synthesized Ag NPs by M. purpureus

supernatant……………………………...................................

58

Fig.14: EDX spectra of synthesized Ag NPs by M.purpureus

supernatant……………………………………………….

60

XIII

Fig .15: X-ray diffraction patterns of synthesized Ag NPs by

M.purpureus supernatant………………………………..

61

Fig.16: FT-IR spectra of M.purpureus supernatant and Ag NPs

colloids……………………………………………… 63

Fig .17: Image of Ag NPs synthesized by in situ irradiation process at

doses ranged from 1kGy to 25kGy …………

81

Fig .18: UV-Vis spectra of synthesized Ag NPs by in situ irradiation

process at doses ranged from 1kGy up to25kGy …………

83

Fig .19: TEM micrograph of synthesized Ag NPs by in situ irradiation

at 5kGy, mean diameter (8.5nm)..………………………

84


at 10kGy, mean diameter (18nm)……………..……………

84


at 15kGy, mean diameter (30.5nm)……………………….

85


at 20kGy, mean diameter (51nm).……………..................

85


at 25kGy, mean diameter (72nm).………….……….………

85

Fig .24: DLS graph of synthesized Ag NPs by in situ irradiation of at

5kGy …………………………………………………..…..

87

Fig .25: DLS graph of synthesized Ag NPs by in situ irradiation at

10kGy …...................................................................

88


15kGy ………………………………………………….

88

Fig.27: DLS graph of synthesized Ag NPs by in situ irradiation at

20kGy …………………………………………………..

89


25kGy …………

89

XIV

Fig .29: FT-IR spectra of M. purpureus supernatant (irradiated at

5kGy) (A) and synthesized Ag NPs at 5kGy (B) by in situ

irradiation at 5kGy …………………………………………

91

Fig.30: X-ray diffraction patterns of synthesized Ag NPs by in situ

radiation process of M. purpureus supernatant …

93

Fig .31: Image of synthesized Ag NPs by ex situ radiation at different

doses of irradiation ranged from 1kGy to 25kGy

………………………………………………………………

96

Fig .32: UV–Vis spectra of synthesized Ag NPs by ex situ radiation

…………………………………………………………….…

99

Fig .33: UV–Vis spectra of synthesized Ag NPs by ex situ radiation at

25kGy and 30kGy …………………………….……………

99

Fig .34: TEM micrograph of synthesized Ag NPs by ex situ radiation

at 1kGy, mean diameter (24nm). ………………………

100


at 5kGy, mean diameter (19.5nm)…………………………

101


at 10kGy, mean diameter (12.5nm).……… …………......

101


at 15kGy, mean diameter (8.2nm).… ………………….

102


at 20kGy, mean diameter (8nm).……………………….....

102


at 25kGy, mean diameter (7.3nm).…………………………..

103

Fig .40: DLS graph of synthesized Ag NPs by ex situ radiation at

1kGy………………………............................................

104


5kGy ………………….…………………………………

104

XV


10kGy ………………………………………………………

105


15kGy.......................................................................................

105


20kGy ………………………………………………

106


25kGy ………………….…………………………………

106

Fig .46: FT-IR spectrum of M. purpureus supernatant (A) and Ag NPs

colloids (B) synthesized by ex situ radiation process at dose

25kGy ………………………………….………………….

108

Fig .47: XRD patterns of synthesized Ag NPs by ex situ radiation

process …………………………………………………….

110

Fig .48: EDX spectra of synthesized Ag NPs by gamma irradiation … 112

Fig .49: UV-spectra of synthesized Ag NPs by in situ irradiation at

dose of 5kGy after 30miutes and 3months ……………

113

Fig .50: UV-spectra of synthesized Ag NPs by ex situ irradiation

process at dose 25kGy within a time range from 12hr to 72hr

……………………………………………………………….

114

Fig .51: UV-spectra of synthesized Ag NPs by ex situ irradiation

process at dose 25kGy after 72h and 3months……………. 115

XVI

LIST OF ABBREVIATIONS

Ag NPs: Silver nanoparticles

SPR: Surface Plasmon resonance

DPPH: 1, 1-Diphenyl-2-picrylhydrazyl

DLS: Dynamic light scattering

TEM: Transmission Electron Microscopy

FT-IR: Fourier Transform Infrared Spectroscopy

EDX: Energy Dispersive X- ray

XRD: X-ray diffraction

CNSC: Canadian Nuclear Safety Commission

FDA: Food and Drug Administration

NCRRT: National Center for Radiation Research and

Technology

Deg: degree

FWHM: full width at half Maximum

Chapter I: LITERATURE REVIEW

1

LITERATURE REVIEW

1.1. Monasus purpureus

Monascus is an ascomycete's fungus traditionally used for the production

of food colourants, fermented foods and beverages in southern China,

Taiwan, Japan, Thailand, Indonesia and the Philippines. Species of

Monascus are mainly employed in the production of red rice (Hesseltine,

1965), (Lin, 1973) and food colouring pigments (Wong & Koehler,

1983).

This fungus divided into six species namely, M. ruber, M. pilosus, M.

purpureus, M. floridans, M. pallens and M. sanguineus. The species with

the greatest significance in the food industry are M. ruber, M. purpureus

and M. pilosus (Martınkova & Patakova, 1999).

The fungus Monascus has been traditionally used in East Asia for the

production of fermented rice which served as a red food colourant.

Nowadays, this natural dye is also used in European countries. Most

studies have addressed the production of red orange oligoketides by the

genus Monascus (Evans, & Wang, 1984) and their biological activities

(Martfnkovi et al., 1995).

M. purpureus has been used commercially to produce valuable secondary

metabolites; ankaflavin and monascin (yellow pigments), monascorubrin

and rubropunctanin (orange pigments), monascorubramine and

rubropuctamine (red pigments) ( Wong et al.,1981), as well as

antihypercholesterolemic agent, monacolin K (lovastatin), hypotensive

agent, Gamma amino butyric acid (GABA) ( Su et al .,2003), antioxidant

compounds, dimerumic acid (Aniya et al.,1999) and an antibacterial

compound, 3-hydoxy-4-methoxy-benzoic acid (Wu et al., 2000). Red

mold rice also contains unsaturated fatty acids that may also help to

reduce serum lipids (Wang et al., 1997), total cholesterol and triglyceride


2

levels (Wong et al., 1981). Red mold rice is also contains phytosterols

such as beta sitosterol and campesterol (Heber et al., 1999) which are

interfere with cholesterol absorption in the intestine (Moghadasian &

Frohlich, 1999).The combination of such dietary sterols with statin drugs

has been suggested as a more effective means of lowering cholesterol

than statin alone (Plat & Mensink, 2001).

Research on the production of pigments obtained from natural sources

has been stimulated by a marked growth in the market for colorants and

the fact that the majority of synthetic red and yellow dyes have been

prohibited by the Food and Drug Administration (Klatii & Bauernfeind,

1981). Carotenoids are widely distributed in nature and are among the

pigments of greatest economic interest. These pigments are responsible

for the yellow and red coloring of a number of fruits and vegetables,

flowers, crustaceans and microorganisms (Johnson & Schroeder, 1996).

The alternative route for the production of natural food colorants is

through the application of biotechnological tools to microorganisms.

Microalgae and several classes of fungi are produce a wide range of

excreted water soluble pigments, but the low productivity of algal

cultures is a significant bottle neck for their commercialization (Hejazi &

Wijffels, 2004).

Pigments of basidiomycetous fungi have been used in the past for dying

wool and silk (Bessette & Besette, 2001), but such fungi are difficult to

grow under laboratory and industrial large scale conditions. Our attention

is now drawn to the ascomycetous fungi. The use of Monascus pigments

in food has been carried out traditionally in the Orient for hundreds years

(Teng & Feldheim, 2001).

The appropriate use of fermentation physiology together with metabolic

engineering could allow the efficient mass production of colorants from

fungi (Nielsen et al., 2002). With recent advances in gene technology,


3

attempts haven been made to create cell factories for the production of

food colorants through the heterologous expression of biosynthetic

pathways from either already known or novel pigment producers

(Lakrod et al., 2003).

Monascus pigment fermentations have been performed mainly in solid

cultures; however production yields have been too low to allow industrial

scale production to make it economical. As a means of increasing the

pigment yield, much research has focused on submerged cultures (Johns

& Stuart., 1991).

1.1.1. Effect of nutritional and environmental on pigments

production

Studies on red pigment synthesis by various strains of M. purpureus in

submerged cultures revealed that the yield is affected by medium

composition, pH and agitation (Hamdi et al., 1996). Particularly,

composition of pigments varies significantly depending on the types of

nutrient available such as nitrogen sources and the strain used (Miyake et

al., 2008).

1. 1.2. Monascus purpeurus pigments and their structures

Six major water insoluble Monascus pigments have been reported; two

yellow pigments (Monascin and Ankaflavin), two orange pigments

(Rubropunctatin and Monascorubrin) and two red pigments

(Rubropunctatamine and Monascorubramine) (Blanc et al., 1994).The

yellow pigments, (Monascin and Ankaflavin) contain the same

chromophoric system, and show differences only in the length of the

saturated side chain linked to the carbonyl function (Figure. 1). The same

structural relationship was also observed for the two orange Monascus


4

pigments and the two red nitrogen containing analogues (Teng &

Feldheim, 1998).

Also in submerged fermentation (SmF) Monascus purpureus produce

new red pigment, the molecular formula C21H29NO5 (Figure.2. A); the

structure of the pigment has numerous similarities with two classical red

pigments, rubropunctamine (Figure.2.B) and monascorubramine

(Figure.2.C), but differs with hydroxyl alkane substitutions at C-3 and

C-9 positions that are unique from the red pigments reported earlier

(Mukherjee & Kuma, 2010)

Fig (1): Monascus purpureus pigments structures (Teng & Feldheim.,

1998)


5

Fig (2): Monascus purpureus red pigment structures (Fig. 2A) new red

pigments rubropunctamine (Fig.2B) and monascorubramine (Fig.2 C)

(Mukherjee & Kuma, 2010)

1.2. Nanoparticles

1.2.1. Definition

Nanotechnology refers to the study of compounds of 100nanometers or

smaller in one dimension. Nanotechnology has a variety of applications

in fields such as optics, electronics, biomedicine, magnetics, mechanics,

catalysis, energy science, etc. Thus, developing different branches of

nanotechnology confidently results in developing the related sciences,

and is a consequential goal of scientific word (Mohanpuria et al., 2008).

1.2.2. Strategies of Nanoparticles synthesis

Nanoparticles are commonly synthesized using two strategies; Top-down

and bottom-up.

In top-down approach, the bulk materials are gradually broken down to

Nano sized materials.


6

Bottom- up approach, atoms or molecules is assembled to molecular

structures in nanometer range. Bottom-up approach is commonly used for

chemical and biological synthesis of nanoparticles.

Metal nanoparticles are very fine and strong particles which have many

applications in different fields like medical imaging (Lee et al., 2008)

,drug delivery, Nano composites , bio labeling , biocide or antimicrobial

agents (Kirchner et al., 2005), filters, sensors, nonlinear optics,

hyperthermia of tumors (Pissuwan et al., 2006), intercalation materials

for electrical batteries (Joerger et al., 2001), optical receptors (Dahan et

al., 2003), catalysis in chemical reactions (Králik et al., 2000) and (Nam

& Lead, 2008)

The wide range of applications of nanoparticles is due to their unique

optical, thermal, electrical, chemical, and physical properties are due to a

combination of the large proportion of high energy surface atoms

compared to the bulk solid (Panigrahi et al., 2004).

1.2.3. Methods of metal nanoparticles synthesis

Metal nanoparticles are characterized by different chemical composition,

shape, size, and monodispersity. To alter these characteristics, three main

methods of synthesis techniques have been developed (Yashhiro, 2006):

1. Chemical synthesis in which use chemical agents for reduction of

metals

2. Physical synthesis

3. Biological synthesis in which used microorganisms for synthesis of

nanoparticles and this process is eco-friendly process and referred to as

green technology.

In the Biological synthesis refers to the phenomena which take place by

means of biological processes or enzymatic reactions and can be used to

obtain better metal nanoparticles from microbial cells (Mandal et al.,


7

2006). In fact microorganisms can survive and grow in high metal ion

concentration due to their ability to fight the stress. The mechanisms

include: efflux systems; alteration of solubility and toxicity via reduction

or oxidation; bio absorption; bioaccumulation; extracellular complexion

or precipitation of metals; and lack of specific metal transport systems

(Bruins et al., 2000). The microbial cell can reduce the metal ions by use

of specific reducing enzymes like NADH-dependent reductase or nitrate

dependent reductase (Mandal et al., 2006). Microbial resistance to high

metal concentration is being considered in fields like bioleaching (Olson

et al., 2003) and bioremediation of waters (Satinder et al., 2006).

1.2.4. Advantage of biological than chemical and physical synthesis

Biosynthesis is considered better than chemical and physical synthesis

because;

1. Biological nanoparticle synthesis would have greater commercial

application, energy costs and high production rate in comparison with

other methods (Mukherjee et al., 2002).

2. Large scale production by chemical and physical methods usually

results in particles larger than several micrometers while the biological

synthesis can be successfully used for production of small nanoparticles

in large scales operations (Klaus et al., 1999).

3. It is a clean, nontoxic and eco-friendly method (Senapati et al., 2005).

4. Physical methods need high temperature and chemical methods need

high pressure which is a harder situation to provide (Bansal et al., 2004).

1.2.5. Problems of biological nanoparticles synthesis

The biological nanoparticles are not monodispersible and the rate of

production is slow. These problems can overcome by strain selection,

optimizing conditions such pH, incubation temperature, incubation time,


8

concentration of metal ions and the amount of biological material (Bansal

et al., 2004). There are also possibilities of producing genetically

engineered microbes that overexpress specific reducing agents and

thereby, controlling the size and shape of biological nanoparticles

(Mohammadian et al., 2007) .

1.2.6. Classification of metal nanoparticle biosynthesis techniques

Metal nanoparticle biosynthesis techniques are classified as either

1. Extracellular biosynthesis

2. Intracellular biosynthesis

In extracellular biosynthesis of metal nanoparticles, the reduction does

not take place in microbial growth phases. In this approach, biological

reagents required for the bio reduction are presented in bio liquids

(Kalishwaralal et al., 2008).

In extracellular biosynthesis, two different preparation methods are

used: rapid and slow synthesis. The former could be done in a few

minutes, while the latter is requires several hours or even days. As an

example, forming silver nanoparticles in 5minutes by culture supernatant

of Klebsiella pneumonia is classified as a rapid synthesis (Shahverdi et

al., 2007), while its formation in 24 hours by mycelia of Phaenerochaete

chrysosporiom has been classified as a slow synthesis (Vigneshwaran et

al., 2006).

Intracellular biosynthesis means in vivo synthesis in cells, which is a

time limiting factor. Detoxification process of hazardous materials which

are mediated by some enzymatic reactions may be involved in bio

reduction of metals and their deposition inside of cells. In this method,

nanoparticles should be separated from the cells after getting synthesized

by designed method (Ahmad et al., 2003).


9

1.2.7. Advantages of extracellular biosynthesis

Extracellular biosynthesis, in comparison, has two main advantages;

First, nanoparticles are formed inside the biomass in the intracellular,

needed to additional step of processing to release the nanoparticles from

the biomass by ultrasound treatment or by reaction with suitable

detergents; in extracellular biosynthesis, however this step is no longer

necessary. Second, the extracellular biosynthesis is a cheaper and simpler

downstream processing. Because of these advantages and achieving

nanoparticles in the biomass is not feasible in the intracellular method.

Much focus has been given to the development of an extracellular process

for biosynthesis of metal nanoparticles (Dahan et al., 2003)

1.2.8. Metal nanoparticles characterization

The formation of metal nanoparticles could be easily monitored by visual

observation of the solution, as well as by recording of the UV–Vis spectra

of the reaction mixture. Highly dispersed metal solutions are intensely

colored and these colors depend on the particle’s size, shape and

composition and the presence of adsorption layers and their structure

(Krutyakov et al., 2008).The absorption spectra of many metallic

nanoparticles are characterized by a strong broad absorption band that is

absent in the bulk spectra. This band is called the surface Plasmon

resonance (SPR) (Mulvaney, 1996); TEM and XRD measurements

confirmed the formation of metal nanoparticles (Binupriya et al., 2010).

1.3. Radiation

1.3.1. Definition

Radiation is defined as energy in transit and comprises electromagnetic

rays; such as X-rays or gamma rays and particulate radiation such as


10

neutrons, alpha particles and heavily charged ions. Radiation affects

people by depositing energy in body tissues. The extent of the damage

depends upon the total amount of energy absorbed, the time period and

dose rate of exposure and the particular organ(s) exposed (Yarmonenko,

1988).

1.3.2. Types of Radiation

According to energy emitted is divided into two types

A. Non-ionizing radiation has enough energy to excite molecules and

atoms causing them to vibrate faster which is obvious in a microwave

oven, where the radiation causes water molecules to vibrate faster

creating heat.

b. Ionizing radiation has more energy than non-ionizing radiation;

enough to cause chemical changes by breaking chemical bonds. This

effect can cause damage to living tissues. The ionizing radiations of

primary concern are alpha and beta particles, gamma and x rays.

Different types of ionizing radiations cause similar kinds of damage but

all cells in the living body do not respond to radiations to the same

degree. Apparent differences in radiosensitivity of different cell

populations were recognized early in the history of radiobiology and a

law was formulated that "The radiosensitivity of cells is directly

proportional to their reproductive activity and inversely proportional to

their degree of differentiation (Purohit et al., 2009).

Ionizing radiation is defined as a specific form of radiation that

possesses sufficient energy to remove electrons from the atoms. This

process is called ionization and it is the reason for the name "ionizing

radiation." When this energy is received in appropriate quantities and

over a sufficient period of time it can result damage (Borek et al., 1993)


11

According to (Harikumar & Kuttan, 2004) radiation is used

therapeutically for investigating different types of diseases and treatment

of various types of malignancies.

Radiation is known as a producer of reactive oxygen species (ROS).

When water which constitutes around 80% of the cell is exposed to

ionizing radiation, decomposition occurs through which a variety of ROS,

such as the superoxide radical O2, the hydrogen peroxide (H2O2) and the

hydroxyl radical (OH•) are generated (Avunduk et al., 2000).The emitted

electron reacts with other water molecule.

1.3.3. Types of Ionizing Radiation

According to Cember; There are 5 types of ionizing radiation (Cember,

1996), known as:

1-Alpha Radiation: Alpha particles consist of two protons and two

neutrons, and carry a positive charge. Alpha particles are barely able to

penetrate skin and can be stopped completely by a sheet of paper.

2-Beta Radiation: Beta radiation consists of fast moving electrons

ejected from the nucleus of an atom. More penetrating than alpha

radiation, beta radiation is stopped by a book or human tissue.

3-Gamma Radiation: Gamma radiation is a very penetrating type of

radiation. It is usually emitted immediately after the ejection of an alpha

or beta particle from the nucleus of an atom. It can pass through the

human body, but is almost completely absorbed by denser materials such

as concrete or lead.

4-X-rays: X-rays are a form of radiation produced mainly by artificial

means rather than by naturally occurring radioactive substances.

5-Neutrons: Less common, neutron radiation occurs when neutrons are

ejected from the nucleus by nuclear fission and other processes. The

nuclear chain reaction is an example of nuclear fission.


12

1.3.4. Radiation exposure:

Chronic radiation exposure

Exposure to ionizing radiation over an extended period of time is called

chronic exposure. The natural background radiation is chronic exposure,

but a normal level is difficult to determine due to variations. Geographic

location and occupation often affect chronic exposure. Chronic exposure

is continuous or intermittent exposure to low doses of radiation over a

long period of time. With chronic exposure, there is a delay between the

exposure and the observed health effect. These effects can include cancer

and other health outcomes such as benign tumors, cataracts, and

potentially harmful genetic effects (Shapiro, 2002)

Acute radiation exposure

Acute radiation exposure is an exposure to ionizing radiation which

occurs during a short period of time. Acute exposure is exposure to a

large, single dose of radiation, or a series of moderate doses received

during a short period of time. Large acute doses can result from

accidental or emergency exposures or from specific medical procedures

(radiation therapy).

For approved medical exposures, the benefit of the procedure may

outweigh the risk from exposure. In most cases, a large acute exposure to

radiation causes both immediate and delayed effects. Delayed biological

effects can include cataracts, temporary or permanent sterility, cancer,

and harmful genetic effects. For humans and other mammals, acute

exposure to the whole body, if large enough, can cause rapid

development of radiation sickness, evidenced by gastrointestinal

disorders, bacterial infections, hemorrhaging, anemia, loss of body fluids,

and electrolyte imbalance. Extremely high dose of acute radiation


13

exposure can result in death within a few hours, days, or weeks (Shapiro,

2002)

Effect of radiation on cell

Damage to a cell can come from direct action or indirect action of the

radiation.

Direct action occurs when the radiation interacts directly with cell's

essential molecules. The radiation energy may damage cell components

such as the cell walls or the deoxyribonucleic acid (DNA).

Indirect action occurs when radiation interacts with water molecules

which are representing 80% of cells composition. The energy absorbed

by the water molecules can result in the formation of free radicals. Free

radicals are molecules that are highly reactive due to the presence of

unpaired electrons, which result when water molecules are split. Free

radicals may form compounds, such as hydrogen peroxide.

1.3.5. Units of radiation dose

According to the Canadian Nuclear Safety Commission (CNSC, 2002),

the scientific unit of measurement for radiation dose, commonly referred

to as effective dose, is the milli sievert (mSv). Other radiation dose

measurement units include rad, rem, Roentgen, Sievert, and Gray.

Because different tissues and organs have varying sensitivity to radiation

exposure, the actual radiation risk to different parts of the body from an

x-ray procedure varies. The term effective dose is used when referring to

the radiation risk averaged over the entire body. The effective dose

accounts for the relative sensitivities of the different tissues exposed.

More importantly, it allows for quantification of risk and comparison to

more familiar sources of exposure that range from natural background

radiation to radiographic medical procedures.


14

Gray (Gy). The unit of absorbed dose, One gray = 1 J/kg = 100 rad.

Rad. The unit of absorbed dose, One rad = 100 erg/g = 0.01Gy.

Sievert (Sv). The unit of dose equivalent, equal to absorbed dose in gray

multiplied by the quality factor. One Sv = 100 rem.

Rem. The conventional unit of dose equivalent, One rem = 0.01 Sv.

1.3.6. Radiation and metal nanoparticles

Radiation is one of the important methods for the synthesis of metal

clusters under ambient conditions and has important advantages as

compared to the chemical reduction method, for example: it does not

require the addition of reducing agents, thus without using excessive

reducing agents or producing undesired by products of reductant,

reducing species are uniformly distributed in the solution; the overall

process can be performed at room temperature (Jurasekova et al.,

2011).The radiolytic method does not require the addition of reducing

agents because the metal reduction is performed by radical species

formed after interaction of ionizing radiation with the solvent. In fact, the

interaction of high energy radiation with a solution of metal ions induces

ionization and excitation of the solvent and leads to the formation of

radiolytic species (Eq. (1)). In particular, the solvated electron, e-aq, and

hydrogen atom, •H, produced by radiolysis of water, are strong reducing

agents capable to reduce metal ions to lower valences and finally to metal

atoms (Kumar, 2002)

H2O → e-aq + OH

• + H

•

The progressive extent of the reduction is accurately controlled by the

dose absorbed and the reduction rate by the dose rate. At quite high dose

as in pulse radiolysis, an instantaneous distribution of the reducing agent

and then of atoms are obtained through the solution (Choi et al., 2003).

The physical and chemical properties of metal nanoparticles are reported


15

to be strictly dependent on their size, shape and morphology (Belloni,

1998) Therefore, parameters such as i.e. irradiation dose, dose rate, and

metal concentration, which can influence nucleation, growth and

aggregation mechanisms, need to be investigated in the synthesis of metal

clusters by radiolytic methods.

1.3.7. Advantages of synthesized metal nanoparticles using

irradiation techniques

The radiation induced synthesis is one of the most promising strategies

because there are some important advantages to the use of the irradiation

techniques (Remita et al., 1996), as compared to conventional chemical

and photochemical methods; the process is simple and clean, the gamma

ray irradiation has harmless feature, controlled reduction of metal ions

can be carried out without using excess reducing agent or producing

undesired oxidation products of the reductant, the method provides metal

nanoparticles in fully reduced, highly pure and highly stable state, no

disturbing impurities like metal oxide are introduced.

Radiolytic reduction generally involves radiolysis of aqueous solutions

that provides an efficient method to reduce metal ions and form homo and

hetero nuclear clusters of transition metals. In the radiolytic method,

aqueous solutions are exposed to gamma rays creating solvated electrons,

e-aq. These solvated electrons, in turn, reduce the metal ions and the metal

atoms eventually coalesce to form aggregates as depicted by following

reactions (Marignier et al., 1985).

H2O Radiation

e-aq, H3O

+, H

•, H2, OH

•, H2O2

e- aq + M

m+ → M

(m−1) +


16

e- aq + M

m+ → M

0

nM0 → M2 → · · ·M n· · · → M agg

Due to its unique advantages, the irradiation based strategy as a worth

tool has been extensively used to prepare Nano scale particles and

materials during the past 20years.

The solution reaction growth technique (Radiation technique) is the

cheapest and most convenient approach for the synthesis of metal

Nanoparticles. However, Nanoparticles are not thermodynamically stable

in the solution due to their high surface energy. These Nanoparticles tend

to aggregate to form larger particle and thus, losing their special

properties. In order to avoid aggregation, suitable capping agents or

stabilizing agents can be used to passivate the nanoparticles aggregation.

Many capping agents have been introduced which include organic agents,

such as thiolphenol, thiourea, mercapto acetate and dodecanethiol which

are toxic and will pollute the environment if a great amount of

nanoparticles is synthesized other commonly used capping agent are

surfactants, polymers, copolymers and recently there has been an

increasing demand for "green” capping agents, for example starch,

calixarene, cyclodextrin and chitosan laurate (Huang et al., 2009(.

The radiolytic method is suitable for generation of metal particles,

particularly silver, in solution (Remita et al, 2005).The amount of

zerovalent nuclei can be controlled by varying the dose of the irradiation

(Eisa et al, 2011).


17

Radiation approaches for nanoparticles synthesis

1. ex situ irradiation techniques

2. In situ irradiation techniques

In the ex situ approach, polymerization of monomers and formation of

metal nanoparticles were separately performed, and then they were

mechanically mixed to form Nanocomposites.

In the in situ methods, metal nanoparticles are generated inside a polymer

matrix by decomposition. A commonly employed in situ method is the

dispersion process, in which the solutions of the metal precursor and the

protective polymer are combined and the reduction is subsequently

performed in solution (Eksik et al., 2010).

In situ nanoparticle generation can be attained by using three approaches:

1- The nanoparticles reduction in solution (Khanna et al., 2005).

2- The ions can be absorbed within a preformed polymer film immersed

in metal solution and then reduced (Gaddy et al., 2004).

3- The NPs are nucleated and grown in the solid state (Porel et al., 2005)

Silver NP nucleation and growth can be easily followed by absorption

spectroscopy, since the surface Plasmon resonance (SPR) band is

sensitive to particle size (Kelly et al., 2003), morphology, dispersity,

dielectric properties of the supporting medium and aggregation (Sun &

Xia, 2003) .

The irradiation, as a new method had been extensively used to prepare

Nano scale clusters and materials; the most studies of irradiation have

been focus on the synthesis of metallic clusters and crystals (Yang et al,

2002).A commonly employed in situ method is the dispersion process, in

which the solutions of the metal precursor and the protective polymer are

combined, and the reduction is subsequently performed in solution

(Yeum et al., 2005)


18

The radiation induced Au NPs synthesis which involves radiolysis of

aqueous solution that provides an efficient method to reduce metal ions;

Gamma irradiated aqueous extract of fermented soybean and garlic was

used for rapid preparation of Au NPs combining both effects of radiolytic

reactions by radiation and stabilization by bioactive components of

fermented extract (El-Batal et al., 2013)

Gamma irradiation potentiates the productivity of the enzyme to its

maximum value post exposure to 0.7kGy. This enhancement of enzyme

production might have been due to either, an increase in the gene copy

number or the improvement in gene expression, or both; A gradual

decrease in the enzyme activity after exposure to the different doses of 1,

1.5 and 2 kGy was observed. The complete inhibition of the enzyme

activity obtained at dose 2kGy. This could be explained by damage or

deterioration in the vitals of the microorganism as radiation causes

rupturing in the cell membrane .This major injury to the cell allows the

extracellular fluids to enter in to the cell. Inversely, it also allows leakage

out of ions and nutrients which the cell brought inside (El-Batal et al.,

2013)

The size of the nanoparticles resulted from gamma irradiation increased

and this correlation could be efficiently used for the preparation of silver

nanoparticles with controlled size simply by changing the starting AgNO3

concentration in the tested range; Gamma irradiation induced strategy

also offered silver nanoparticles with narrower sized distribution (Li et

al., 2007)

Chapter II: Parameters optimization for Monascus purpueurs

pigments production and silver nanoparticles synthesis

91

Parameters optimization for Monascus purpueurs pigments

production and silver nanoparticles synthesis.

2.1. Abstract

Natural colorants derived from polyketide compounds of Monascus

purpureus have been extensively used in pharmaceuticals and foods to

replace the synthetic colorants. Plackett-Burman experimental design was

used to screen important nutrients influencing the production of pigments

by M. purpureus under submerged fermentation (SmF) and silver

nanoparticles synthesis. Seven parameters; inoculum age, yeast extract,

starch, pH, glucose, aeration and incubation period were screened. The

fungal M. purpureus was grow under Eighty two different trials on

submerged fermentation and their supernatants and biomass was

incubated with AgNO3 solution (1mM).Three types of pigments were

extracted from M. purpureus supernatant. The optimum condition for

pigments production are at a pH 5.5, 10days inoculum age, 50/250ml

medium/flask volume(aeration), 5g/l yeast extract, 20g/l starch, 15g/l

glucose and the incubation period 7days that show optical density

0.681UA at λ400nm, 0.962UA at λ 470nm and 2.542UA at λ 500nm and

it’s the best medium supernatant produce silver nanoparticles. The M.

purpureus biomass has no ability to produce silver nanoparticles.

2.2. Introduction.

Monascus is an ascomycete's fungus traditionally used for the production

of food colourants, fermented foods and beverages in southern China,

Taiwan, Japan, Thailand, Indonesia and the Philippines. Species of

Monascus are mainly employed in the production of red rice (Hesseltine,



02

1965), (Lin, 1973) and food colouring pigments (Wong and Koehler,

1983).

The genus is divided into six species namely, M. ruber, M. pilosus, M.

purpureus, M. floridans, M. pallens and M. sanguineus, the species with

the greatest significance in the food industry are M. ruber, M. purpureus

and M. pilosus (Martınkova and Patakova, 1999).

The fungus Monascus has been traditionally used in East Asia for the

production of fermented rice which served as a red food colourant.

Nowadays this natural dye is also used in European countries. Most

studies have addressed the production of red orange oligoketides by the

genus Monascus (Evans and Wang, 1984) and their biological activities

(Martfnkovi et al., 1995).

M. purpureus has been used commercially to produce valuable secondary

metabolites viz., ankaflavin and monascin (yellow pigment),

monascorubrin and rubropunctanin (orange pigment), monascorubramine

and rubropuctamine (red pigment) ( Wong et al.,1981), as well as,

antihypercholesterolemic agent monacolin K (lovastatin), hypotensive

agent, gamma amino butyric acid (GABA) ( Su et al.,2003), antioxidant

compounds, dimerumic acid (Aniya et al.,1999) and an antibacterial

compound, 3-hydoxy-4-methoxy-benzoic acid (Wu et al., 2000).

Red mold rice also contains unsaturated fatty acids that may also help to

reduce serum lipids (Wang et al., 1997), total cholesterol and triglyceride

levels (Wong et al., 1981). Red mold rice also contains phytosterols such

as betasitosterol and campesterol (Heber et al., 1999), which are known

to interfere with cholesterol absorption in the intestine (Moghadasian

and Frohlich, 1999). The combination of such dietary sterols with statin

drugs has been suggested as a more effective means of lowering

cholesterol than statin alone (Plat and Mensink, 2001).



09

Research on the production of pigments obtained from natural sources

has been stimulated by a marked growth in the market for colorants and

the fact that the majority of synthetic red and yellow dyes have been

prohibited by the Food and Drug Administration (FDA) (Klatii and

Bauernfeind, 1981). Carotenoids are widely distributed in nature and

among the pigments of greatest economic interest. These pigments are

responsible for the yellow and red coloring of a number of fruits and

vegetables, flowers, crustaceans and microorganisms (Johnson and

Schroeder, 1996).

An alternative route for the production of natural food colorants is

through the application of biotechnological tools to microorganisms.

Microalgae and several classes of fungi are known to produce a wide

range of excreted water soluble pigments but the low productivity of algal

cultures is a significant bottle neck for their commercialization (Hejazi

and Wijffels, 2004).

Pigments of basidiomycetous fungi have been used in the past for dying

wool and silk (Bessette and Besette, 2001) but such fungi are difficult

to grow under laboratory and industrial large scale conditions. Our

attention now is drawn to the ascomycetous fungi, the use of such fungi

to colour foodstuffs is not a novel practice.

The use of Monascus pigments in food has been carried out traditionally

in the Orient for hundreds of years (Teng and Feldheim, 2001).

Considering the apparent heat and pH stability of the Monascus

derivatives during food processing taken together with the socio climatic

independence of such a readily available raw material.

Fungi seem to be well worth further exploration as an alternative source

of natural colorants. The appropriate use of fermentation physiology



00

together with metabolic engineering could allow the efficient mass

production of colorants from fungi (Nielsen et al., 2002).

With recent advances in gene technology, attempts have been made to

create cell factories for the production of food colorants through the

heterologous expression of biosynthetic pathways from either already

known or novel pigment producers (Lakrod et al., 2003).

2.3. Material and methods.

2.3.1. Strain and Culturing Conditions

Fungal culture of M. purpureus NRRL 1992 was obtained from the

National Center for Radiation Research and Technology (NCRRT),

Cairo, Egypt, maintained and sub cultured on potato dextrose agar

(PDA)with the following composition g/l: potato extract; 200, dextrose;

20 and agar; 20.

Plackett-Burman experimental design was used to screen important

nutrients influencing the production of pigments by M. purpureus under

SmF. Seven parameters; inoculum age, yeast extract, starch, pH, glucose,

aeration and incubation period were screened.

Preparation of inoculum

To fully sporulated agar slope culture (7-14day), 10ml of sterile saline

0.9% was added. Then the spores were scrapped under aseptic conditions.

The spore suspension obtained was used as the inoculum (1.5×105

spores/ml). Each flask was inoculated with 1ml of spore's suspension and

incubated at 30ºC at 150rpm.



02

2.3.2. Reagents

Silver nitrate, yeast extract, sulphanilamide, NEED (N (1naphthyl)

ethylene diamine dihydrochloride), DPPH (1, 1-Diphenyl-2-

picrylhydrazyl) from (sigma Aldrich), Starch, agar and glucose From

ADWIC. All other reagents were of analytical grade and used as

received.

2.3.3. Pigments extraction and determination

The supernatants (50ml) were extracted with ethanol 90% for 1h on a

rotary shaker (100 rpm) at room temperature and then filtered through a

Preweighed Whatman GF: C disc (Fenice et al., 2000). The supernatants

were made up to 100ml using the ethanol 90%.

Pigment concentrations were estimated using spectrophotometer (UV-Vis

spectrometer (JASCO-Japan-model 560) at 400, 470 and 500nm for

yellow, orange and red pigments; these wavelengths represent absorption

maxima for yellow, orange and red pigments respectively (Johns et al.,

1991). Pigment production (units of absorbance, UA) was calculated by

the absorbance values.

2.3.4. Parameter optimization for pigments production using

Plackett-Burman experimental design

Monascus purpureus under submerged fermentation, seven parameters;

inoculum age (7, 10 and 14day), yeast extract (2.5, 3.75 and 5g/l), starch

(20, 40 and 60g/l), pH (5.5, 6.25 and 7), glucose (15, 22.5 and 30g/l),

aeration (50/250, 75/250ml and 100/250ml (Medium/flask volume ml/ml) aeration,

and incubation period (7, 8.5 and 10days) were screened using Plackett-

Burman experimental design show in (table 1) and The inoculated flasks

(1.5×105 spores/ml) each flask was inoculated with 1ml of spore's

suspension] were incubated at 30°C on a rotatory shaker at 150rpm.



02

Table (1): Plackett-Burman experimental design screen trials for

important nutrients and conditions influencing the production of

pigments by M. purpureus

Trial

number

inoculum age

(day)

Yeast extract

(g/l)

Starch

(g/l)

pH Aeration

(Medium/flask

volume

ml/ml)

Glucose

(g/l)

Incubation

Period

(day)

1 14 2.5 60.0 5.5 100/250 15.0 10.0

2 7 2.5 60.0 5.5 50/250 30.0 10.0

3 7 2.5 20.0 7.0 50/250 15.0 7.0

4 14 2.5 20.0 5.5 50/250 15.0 10.0

5 7 2.5 20.0 7.0 100/250 15.0 10.0

6 7 5.0 60.0 7.0 50/250 15.0 7.0

7 14 5.0 60.0 5.5 100/250 30.0 10.0

8 7 5.0 60.0 7.0 50/250 30.0 10.0

9 14 5.0 20.0 7.0 100/250 30.0 10.0

10 14 2.5 20.0 5.65 100/250 30.0 10.0

11 7 2.5 20.0 7.0 50/250 30.0 10.0

12 7 5.0 20.0 7.0 50/250 30.0 7.0

13 14 2.5 20.0 7.0 50/250 30.0 10.0

14 7 2.5 20.0 5.5 100/250 15.0 7.0

15 7 5.0 60.0 5.5 50/250 30.0 7.0

16 14 5.0 20.0 5.5 100/250 15.0 10.0

17 7 5.0 60.0 5.5 50/250 15.0 10.0

18 7 3.75 40.0 6.25 75/250 15.0 8.5

19 10 5.0 20.0 5.5 50/250 15.0 7.0

20 7 5.0 60.0 7.0 100/250 15.0 10.0

21 14 5.0 20.0 5.5 50/250 30.0 10.0

22 7 3.75 40.0 6.25 75/250 22.5 8.5

23 10 2.5 60.0 7.0 50/250 15.0 10.0

24 14 2.5 20.0 5.5 100/250 15.0 7.0

25 14 2.5 40.0 6.25 75/250 22.5 8.5

26 14 2.5 60.0 5.5 50/250 30.0 10.0

27 14 3.75 40.0 6.25 75/250 22.5 8.5

28 14 2.5 20.0 7.0 100/250 15.0 10.0

29 14 2.5 60.0 7.0 50/250 15.0 10.0

30 7 5.0 20.0 5.5 50/250 15.0 7.0

31 14 5.0 20.0 7.0 50/250 15.0 10.0

32 7 3.75 40.0 6.25 75/250 22.5 8.5

33 10 5.0 20.0 7.0 100/250 30.0 10.0

34 7 5.0 60.0 5.5 50/250 15.0 10.0

35 14 3.75 40.0 5.5 75/250 22.5 8.5

36 10 2.5 60.0 7.0 100/250 15.0 7.0

37 14 3.75 40.0 6.25 75/250 22.50 10.0

38 10 2.5 20.0 5.5 50/250 30.0 7.0

39 7 5.0 60.0 7.0 100/250 30.0 7.0

40 7 2.5 20.0 7.0 100/250 30.0 7.0

41 14 2.5 60.0 5.5 100/250 30.0 7.0



02

42 14 2.5 20.0 5.5 100/250 30.0 10.0

43 7 2.5 60.0 7.0 100/250 15.0 7.0

44 7 3.75 20.0 6.25 75/250 22.5 8.5

45 10 5.0 20.0 7.0 100/250 15.0 7.0

46 14 3.75 40.0 7.0 75/250 22.5 8.5

47 10 3.75 40.0 6.25 75/250 22.5 8.5

48 10 5.0 20.0 5.5 100/250 30.0 7.0

49 10 5.0 60.0 5.5 50/250 30.0 7.0

50 7 5.0 40.0 6.25 75/250 22.5 8.5

51 14 5.0 20.0 7.0 50/250 30.0 7.0

52 7 2.5 60.0 5.5 100/250 15.0 10.0

53 14 5.0 20.0 5.5 100/250 15.0 10.0

54 7 2.5 20.0 5.5 50/250 15.0 10.0

55 7 5.0 60.0 5.5 100/250 30.0 10.0

56 7 2.5 60.0 5.5 50/250 15.0 7.0

57 14 5.0 60.0 7.0 50/250 30.0 10.0

58 7 2.5 60.0 7.0 50/250 30.0 7.0

59 7 5.0 60.0 5.5 100/250 15.0 7.0

60 10 3.75 40.0 6.25 50/250 22.50 8.5

61 14 2.5 20.0 5.5 50/250 30.0 7.0

62 14 5.0 60.0 7.0 50/250 15.0 7.0

63 14 2.5 60.0 7.0 100/250 30.0 10.0

64 10 3.75 40.0 6.25 100/250 22.5 8.5

65 10 3.75 60.0 6.25 75/250 22.5 8.5

66 7 2.5 60.0 7.0 100/250 30.0 10.0

67 7 2.5 20.0 7.0 100/250 30.0 7.0

68 14 5.0 20.0 5.5 50/250 30.0 10.0

69 7 5.0 60.0 7.0 100/250 15.0 10.0

70 14 5.0 60.0 5.5 100/250 15.0 7.0

71 14 2.5 60.0 5.5 50/250 15.0 7.0

72 14 5.0 20.0 7.0 50/250 15.0 10.0

73 14 2.5 60.0 7.0 50/250 30.0 7.0

74 10 3.75 40.0 6.25 75/250 30.0 8.5

75 7 2.5 60.0 5.5 100/250 30.0 7.0

76 10 3.75 40.0 6.25 75/250 22.5 7.0

77 7 5.0 20.0 5.5 100/250 30.0 7.0

78 7 3.75 40.0 6.25 75/250 22.5 8.5

79 7 5.0 20.0 7.0 100/250 15.0 7.0

80 14 5.0 60.0 7.0 100/250 30.0 7.0

81 14 2.5 20.0 7.0 50/250 15.0 7.0

82 10 3.75 40.0 6.25 75/250 22.5 8.5



02

2.3.5. Silver Nanoparticle (Ag NPs) synthesis from fungal biomass

and supernatants

The fungal culture (eighty-two different media) was centrifuged at

5.000rpm for 15minutes and their supernatants and biomass (mycelia) were

used for silver nanoparticles synthesis as follow:

1. Using fungal biomass

The fungus mycelia after separation by centrifugation of eighty-two

different media are treated with silver nitrate (1mM) solution by two

processes.

1.1. The fungus mycelia washed thrice with deionized water, the washed

mycelia were suspended in deionized water (50ml) and kept on rotary

shaker for 24h at 180rpm. The aqueous extract obtained after separation of

mycelial suspension by centrifugation (aqueous extract of fungal biomass)

was challenged with 50mL of 1mM silver nitrate solution (prepared in

deionized water) and incubated in shaker at 180rpm in dark conditions at

25°C for 72h. Simultaneously a positive control of incubating the fungal

supernatant with deionized water was maintained under same conditions.

1.2. The fungal mycelia were challenged with 50mL of 1mM silver nitrate

solution (prepared in deionized water) and incubated in shaker at 180rpm in

dark conditions at 25°C for 72h. Simultaneously a positive control of

incubating the fungal mycelia with deionized water was maintained under

same conditions.



02

2. Using fungal supernatants

The fungus supernatants obtained by centrifugation at 5.000rpm for

15minutes of eighty-two different media were treated with silver nitrate

solution (1mM).

Aqueous silver nitrate solution 1mM (50ml) was mixed with Monascus

purpureus supernatants (50ml) and agitated at 150rpm on 25°C. Control

(without silver nitrate, only supernatant for each one) was also run along

with the experimental flask. The reaction between this supernatants and

Ag+ ions was carried out in dark for 24h.The reduction of silver ions was

monitoring by measuring the UV-VIS spectrum of solution.

2.3.6. Measurement of antioxidant activity

The method of Shimada (Shimada et al., 1992).was used to determine

the antioxidant activity of M. purpureus supernatant and Ag NPs

colloids.4.0 ml of different supernatants of M. purpureus and Ag NPs

colloids was added to methanol solution of 10mM DPPH (1.0 ml) in

separate tubes. The mixture was shaken and left to stand at room

temperature for 30 min. The absorbance of the resulting solution was than

measured spectrophotometrically at 517nm. The inhibitory percentage of

DPPH was calculated According to the following equation:

Scavenging effect (%)

= [1- (absorbance of sample/absorbance of control)] ×100%

2.3.7. Characterization of silver nanoparticles

2.3.7.1. UV-VIS spectral analysis

Preliminary characterization of the silver nanoparticles was carried out

using UV–visible spectroscopy (JASCO-Japan-model V- 560) at a



02

resolution of 1nm. Noble metals such as silver (Ag) nanoparticles exhibit

unique and tunable optical properties on account of their surface Plasmon

resonance (SPR) dependent on shape, size and size distribution of the

nanoparticles (Tripathy et al., 2010). The reduction of silver ions was

monitored by measuring the UV–visible spectra of the solutions from 300

to 800nm. The supernatant was used as blank.

2.3.7.2. Dynamic light scattering (DLS)

Average particle size and size distribution was determined by the

dynamic light scattering (DLS) technique (PSS-NICOMP 380-ZLS,

USA). Before measurements the samples were diluted 10times with

deionized water. 250μl of suspension were transferred to a disposable low

volume cuvette. After equilibration to a temperature of 25°C for 2 min,

five measurements were performed using 12runs of 10s each.

2.3.7.3. Fourier Transform Infrared Spectroscopy (FT-IR)

To determine Fourier transform infra-red (FTIR) pattern of the sample

fungal supernatant was diluted with potassium bromide in the ratio of

1:100 and after drying (room temperature ) compressed to form a disc.

The discs were later subjected to FTIR spectroscopy measurement. These

measurements were recorded on a JA SCO FT-IR -3600 and spectrum

was collected at a resolution of 4cm-1

in wave number region of 400 to

4000cm-1

. FT-IR measurements were carried out in order to obtain

information about chemical group's present fungal supernatant.



01

2.4. Results and discussion

2.4.1. Effect of Parameters optimization on pigments production

Table (2) shows Plackett-Burman experimental design screen trials for

varying conditions effect on the pigments production of M. purpureus.

Studies on Pigment synthesis by various strains of M. perpureus in

submerged cultures revealed that the yield is affected by medium

composition, pH and agitation (Hamdi et al., 1996). Particularly,

composition of pigments varies significantly depending on the types of

nutrient available, such as nitrogen sources and the strain used (Miyake

et al., 2008).

At pH higher than 8.5 and lower than 5.5 red pigment production

decreased noticeably. Pigment production was observed on 3rd

day after

incubation and continued to accumulate throughout the fermentation

period. Maximum pigment yield was observed on the 10th day after

incubation. Mycelial growth increased with time and reached to its

maximum on 6th

day of incubation. When growth and pigment production

were examined at different glucose concentrations (15-30g/l), Up to

30g/l, increasing glucose concentration increased both biomass and

pigment production but above this concentration biomass and pigment

production were drastically reduced due to Crabtree effects, which

generally occur in yeast batch fermentation with high sugar

concentrations and inhibit respiratory enzyme and increase ethanol

production. Chen and Johns also reported that a high glucose

concentration (50 g/l) lead to low growth rates, pigment synthesis and

considerable ethanol production.



22

2.4.2. Spectral analysis of extracellular pigments.

The optical density (OD) was measured at 400, 470 and 500nm

(wavelength which represents the absorption maxima for yellow, orange

and red pigments, respectively) thus yielding called yellow, orange and

red pigment production expressed in units of absorbance (λUA) at a given

wavelength (λ) shows in (table. 2). Maximum pigments production

medium conditions are (10days inoculum age, 5g/l yeast extract, 20g/l

starch, pH 5.5, 50/250ml Medium/flask volume (aeration), 15g/l glucose and

Incubation period is 7days.

Table 2: M. purpureus pigments optical density (OD) expressed as

units of absorbance (λUA)

Trial

number

inoculum

age

(day)

Yeast

extract

(g/l)

Starch

(g/l)

pH Aeration

(Medium/

flask volume

ml/ml)

Glucose

(g/l)

Incubation

Period

(day)

400nm

(Yellow

pigment)

(OD)

470nm

(Orange

pigment

(OD)

500nm

Red

pigment

(OD)

1 14 2.5 60.0 5.5 100/250 15.0 10.0 0.121 0.203 0.98

2 7 2.5 60.0 5.5 50/250 30.0 10.0 0.08 0.248 1.002

3 7 2.5 20.0 7.0 50/250 15.0 7.0 0.151 0.274 1.05

4 14 2.5 20.0 5.5 50/250 15.0 10.0 0.154 0.274 0.945

5 7 2.5 20.0 7.0 100/250 15.0 10.0 0.32 0.433 1.556

6 7 5.0 60.0 7.0 50/250 15.0 7.0 0.067 0.258 1.132

7 14 5.0 60.0 5.5 100/250 30.0 10.0 .014 0.273 0.782

8 7 5.0 60.0 7.0 50/250 30.0 10.0 0.145 0.208 0.344

9 14 5.0 20.0 7.0 100/250 30.0 10.0 0.1032 0.198 0.942

10 14 2.5 20.0 5.65 100/250 30.0 10.0 0.183 0.265 0.980

11 7 2.5 20.0 7.0 50/250 30.0 10.0 0.1955 0.131 0.97

12 7 5.0 20.0 7.0 50/250 30.0 7.0 0.0751 0.215 0.762

13 14 2.5 20.0 7.0 50/250 30.0 10.0 0.1902 0.245 0.879

14 7 2.5 20.0 5.5 100/250 15.0 7.0 0.1424 0.1615 0.2459

15 7 5.0 60.0 5.5 50/250 30.0 7.0 0.108 0.1399 0.2459

16 14 5.0 20.0 5.5 100/250 15.0 10.0 0.135 0.237 0.723

17 7 5.0 60.0 5.5 50/250 15.0 10.0 0.196 0.202 0.518

18 7 3.75 40.0 6.25 75/250 15.0 8.5 0.279 0.397 1.2243

19 10 5.0 20.0 5.5 50/250 15.0 7.0 0.681 0.962 2.542

20 7 5.0 60.0 7.0 100/250 15.0 10.0 0.1229 0.2439 1.005

21 14 5.0 20.0 5.5 50/250 30.0 10.0 0.027 0.312 1.0102

22 7 3.75 40.0 6.25 75/250 22.5 8.5 0.175 0.275 0.985

23 10 2.5 60.0 7.0 50/250 15.0 10.0 0.160 0.132 0.568

24 14 2.5 20.0 5.5 100/250 15.0 7.0 0.14 0.155 0.2696

25 14 2.5 40.0 6.25 75/250 22.5 8.5 0.029 0.213 0.935

26 14 2.5 60.0 5.5 50/250 30.0 10.0 0.157 0.312 0.724

27 14 3.75 40.0 6.25 75/250 22.5 8.5 0.234 0.343 1.03

28 14 2.5 20.0 7.0 100/250 15.0 10.0 0.148 0.099 0.208

29 14 2.5 60.0 7.0 50/250 15.0 10.0 0.1349 0.149 0.792

30 7 5.0 20.0 5.5 50/250 15.0 7.0 0.258 0.326 0.54



29

31 14 5.0 20.0 7.0 50/250 15.0 10.0 0.162 0.1708 0.962

32 7 3.75 40.0 6.25 75/250 22.5 8.5 0.348 0.545 1.664

33 10 5.0 20.0 7.0 100/250 30.0 10.0 0.182 0.105 0.2556

34 7 5.0 60.0 5.5 50/250 15.0 10.0 0.161 0.164 0.191

35 14 3.75 40.0 5.5 75/250 22.5 8.5 0.082 0.202 0.823

36 10 2.5 60.0 7.0 100/250 15.0 7.0 0.154 0.137 1.823

37 14 3.75 40.0 6.25 75/250 22.50 10.0 0.172 0.203 1.002

38 10 2.5 20.0 5.5 50/250 30.0 7.0 0.355 0.54 1.747

39 7 5.0 60.0 7.0 100/250 30.0 7.0 0.172 0.023 1.02

40 7 2.5 20.0 7.0 100/250 30.0 7.0 0.1251 0.1435 0.389

41 14 2.5 60.0 5.5 100/250 30.0 7.0 0.1403 0.1581 0.2614

42 14 2.5 20.0 5.5 100/250 30.0 10.0 0.108 0.201 0.321

43 7 2.5 60.0 7.0 100/250 15.0 7.0 0.0324 0.051 0.89

44 7 3.75 20.0 6.25 75/250 22.5 8.5 0.152 0.207 0.762

45 10 5.0 20.0 7.0 100/250 15.0 7.0 0.169 0.178 0.698

46 14 3.75 40.0 7.0 75/250 22.5 8.5 0.085 0.136 0.742

47 10 3.75 40.0 6.25 75/250 22.5 8.5 0.172 0.185 0.927

48 10 5.0 20.0 5.5 100/250 30.0 7.0 0.098 0.125 0.872

49 10 5.0 60.0 5.5 50/250 30.0 7.0 0.139 0.15 0.923

50 7 5.0 40.0 6.25 75/250 22.5 8.5 0.127 0.1299 0.178

51 14 5.0 20.0 7.0 50/250 30.0 7.0 0.125 0.182 0.723

52 7 2.5 60.0 5.5 100/250 15.0 10.0 0.029 0.172 0.832

53 14 5.0 20.0 5.5 100/250 15.0 10.0 0.1354 0.1525 0.562

54 7 2.5 20.0 5.5 50/250 15.0 10.0 0.172 0.192 1.02

55 7 5.0 60.0 5.5 100/250 30.0 10.0 0.025 0.102 0.723

56 7 2.5 60.0 5.5 50/250 15.0 7.0 0.103 0.172 0.924

57 14 5.0 60.0 7.0 50/250 30.0 10.0 0.025 0.103 0.723

58 7 2.5 60.0 7.0 50/250 30.0 7.0 0.127 0.163 0.932

59 7 5.0 60.0 5.5 100/250 15.0 7.0 0.148 0.163 0.735

60 10 3.75 40.0 6.25 50/250 22.50 8.5 0.105 0.137 0.872

61 14 2.5 20.0 5.5 50/250 30.0 7.0 0.026 0.105 0.523

62 14 5.0 60.0 7.0 50/250 15.0 7.0 0.118 0.127 0.725

63 14 2.5 60.0 7.0 100/250 30.0 10.0 0.300 0.41 1.277

64 10 3.75 40.0 6.25 100/250 22.5 8.5 0.263 0.358 2.214

65 10 3.75 60.0 6.25 75/250 22.5 8.5 0.032 0.054 0.725

66 7 2.5 60.0 7.0 100/250 30.0 10.0 0.041 0.098 0.325

67 7 2.5 20.0 7.0 100/250 30.0 7.0 0.008 0.136 0.472

68 14 5.0 20.0 5.5 50/250 30.0 10.0 0.051 0.127 0.584

69 7 5.0 60.0 7.0 100/250 15.0 10.0 0.358 0.545 1.146

70 14 5.0 60.0 5.5 100/250 15.0 7.0 0.025 0.102 0.368

71 14 2.5 60.0 5.5 50/250 15.0 7.0 0.092 0.158 0.637

72 14 5.0 20.0 7.0 50/250 15.0 10.0 0.107 0.137 0.829

73 14 2.5 60.0 7.0 50/250 30.0 7.0 0.082 0.157 0.729

74 10 3.75 40.0 6.25 75/250 30.0 8.5 0.31 0.433 1.538

75 7 2.5 60.0 5.5 100/250 30.0 7.0 0.580 0.84 2.224

76 10 3.75 40.0 6.25 75/250 22.5 7.0 0.107 0.128 0.825

77 7 5.0 20.0 5.5 100/250 30.0 7.0 0.024 0.092 0.382

78 7 3.75 40.0 6.25 75/250 22.5 8.5 0.109 0.147 0.729

79 7 5.0 20.0 7.0 100/250 15.0 7.0 0.057 0.139 0.562

80 14 5.0 60.0 7.0 100/250 30.0 7.0 0.045 0.153 0.372

81 14 2.5 20.0 7.0 50/250 15.0 7.0 0.103 0.140 0.572

82 10 3.75 40.0 6.25 75/250 22.5 8.5 0.048 0.153 0.829



20

2.4.3. Silver nanoparticles (Ag NPs) synthesis

2.4.3.1. Silver nanoparticles (Ag NPs) synthesis by M. purpureus

supernatants

After treatment of M. purpureus supernatants obtained from Plackett-

Burman experimental design screen trials with AgNO3 solution 1mM

(v/v). Eleven trials that highly created the silver nanoparticles and show

specific absorbance peak of Ag NPs (Plackett-Burman experimental

design screen trials numbers 5, 18, 19, 27, 32, 38, 63, 64, 69,74, and 75)

2. 4.3.2 Silver nanoparticles (Ag NPs) synthesis by M. purpureus using

fungal biomass

1. Aqueous extract of fungal biomass

After treatment of M. purpureus aqueous extract of biomass obtained from

Plackett-Burman experimental design screen trials with AgNO3 solution

1mM (v/v) no one trials created the silver nanoparticles and show no

specific absorbance peak of Ag NPs.

2. Macerated mycelia with silver nitrate solution

Macerated mycelia with silver nitrate solution were centrifuged at

5.000rpm for 15minuets and this separated supernatant shows no specific

peak of Ag NPs so cannot used for Ag NPs biosynthesis.


2.4.4.1. UV.VIS Spectral analysis

Fig (3) shows the UV–Vis spectra of Ag NPs after 24h of incubation to

M. purpureus supernatants with AgNO3 solution (1mM) a single peak at

(430nm) is very sharp with high intensity in trial number nineteen; this



22

mean that the more yield of silver nanoparticles and size distribution of

the silver nanoparticles is narrow (Chen et al., 2007).

Fig (4) shows the UV–Vis spectra after 72h of incubated aqueous extract

of fungal biomass and Macerated mycelium with silver nitrate solution

show no specific peak of Ag NPs compared with trial number nineteen

that show specific peak of Ag NPs at 430nm (Sanghi and Verm, 2009)

this result confirm use of M. purpureus supernatant but cannot use M.

purpureus biomass for Ag NPs synthesis.

Fig (3): UV–Vis spectra of Ag NPs formed after 48h of M. purpureus

supernatants incubated with AgNO3. Trials number 5, 18, 19, 27, 32, 38, 63, 64,

69, 74, and 75.



22

Fig (4): UV–Vis spectra of incubated aqueous extract of fungal biomass (A),

Macerated mycelium (B) and trial number 19 supernatant (C) with silver nitrate.

2.4.4.2. Dynamic light scattering (DLS) analysis.

Table (3) shows average size of silver nanoparticles prepared by eleven

experimental that highly create silver nanoparticles; DLS analysis

confirm that the smallest particles size obtained by trials number

nineteen that also shows the highest optical density; the peak is very

sharp with high intensity than other trials; that means there is more yield

of silver nanoparticles (Chen et al., 2007).



22

Table (3) Mean diameter size of synthesized silver nanoparticles

Trials

number

λ Max (UA)

Ag NPs

DLS

Average size(nm)

19 0.88 (1-5.4) ±0.7

75 0.79 (1-6.7) ±1.2

64 0.48 (1-7.4) ±0.7

38 0.35 (1-7.8) ±0.7

32 0.23 (1-8.6) ±1.1

5 0.18 (1-9.6) ±1.9

74 0.12 (1-26.1) ±5.4

63 0.12 (1-34.9) ±6.6

18 0.08 (1-44.3) ±6.8

69 0.08 (1-60.5) ±9.3

27 0.05 (1-87.30)±18

2.4.4.3. FT-IR spectra of M. purpureus supernatant analysis

Fig (5) shows FTIR spectra of M. purpureus supernatant; exhibited the

following absorption bands: broad absorption band peaking at 3405cm-1

corresponding to OH group vibrations followed by a peak at 2923cm–1

assigned to vibration of the –CH group. Other bands in this spectrum are

observed at 1427cm-1

and 1087cm-1

due to the bond vibrations of the –

NO3 group and of the N–OH complex respectively.

The proteins present me be acts as capping agent. The carbonyl groups

from the amino acids residues and peptides of proteins have ability to

bind silver; their proteins could possibly form a coat covering the metal

nanoparticles to prevent their agglomeration and aid in its stabilization in

the medium (Balaji et al., 2009). It is also reported that proteins can bind



22

to nanoparticles either through free amine or cysteine groups in proteins

(Mandal et al., 2005).

Fig (5): FTIR spectra of M. purpureus supernatant.

2.4.5. Relationship between optical densities, DLS size and

antioxidant activity (DPPH scavenging)

Table (4) shows the relationship between optical densities (OD), DLS

size and DPPH activity of eleven trials that highly create Ag NPs. The

results confirm presence of positive relationship between the OD; DLS

average size and DPPH activity. The size of silver nanoparticles has

linear correlation with the Peak intensity, (Jaidev & Narasimha, 2010);

but the DPPH activity of M. purpureus supernatants reduced after

formation of Ag NPs colloids; decrease of M. purpureus supernatants

antioxidant activity after Ag NPs synthesis indicate to its consumption for

reduction of Ago to Ag NPs.



22

Table (4) Relationship between optical densities (OD), DLS size and

DPPH scavenging activity of synthesized Ag NPs

Trials

number

λ Max

(UA)

Ag NPs

DLS

Mean

diameter(nm)

DPPH

scavenging%

Before

DPPH

scavenging%

After

19 0.88 (1-5.4) ±0.7 74 38

75 0.79 (1-6.7) ±1.2 73 40

64 0.48 (1-7.4) ±0.7 70 42

38 0.35 (1-7.8) ±0.7 66 42

32 0.23 (1-8.6) ±1.1 65 44

5 0.18 (1-9.6) ±1.9 64 46

74 0.12 (1-26.1) ±5.4 60 50

63 0.12 (1-34.9) ±6.6 60 50

18 0.08 (1-44.3) ±6.8 58 51

69 0.08 (1-60.5) ±9.3 57 52

27 0.05 (1-87.30)±18 55 53

2.5. Conclusion

This study confirm possibility to use of Monascus purpureus supernatants

for synthesis of Ag NPs and cannot use fungal biomass, the conditions of

medium used for M. purpureus grow can effect on pigments and Ag NPs

synthesized. The size of silver nanoparticles has linear correlation with

the Peak intensity; but the DPPH activity of M. purpureus supernatants

reduced after formation of Ag NPs colloidal; decrease of M. purpureus

supernatants antioxidant activity after Ag NPs synthesis indicate to its

consumption for reduction of Ago to Ag NPs. The optimum conditions

for pigments production are at a pH 5.5, 10days inoculum age, 50/250ml



22

medium/flask volume (aeration), 5g/l yeast extract, 20g/l starch, 15g/l

glucose and the incubation period 7days.


39

Silver nanoparticles synthesis without gamma

irradiation

3.1. Abstract


production of spherical shaped silver nanoparticles (Ag NPs) synthesized

and stabilized using M. purpureus supernatant. Aqueous solutions AgNO3

were treated with M. purpureus supernatant for the formation silver

nanoparticles (Ag NPs).The Nano metallic dispersions were characterized

by surface Plasmon absorbance measuring around 420nm for Ag

nanoparticles. Transmission electron microscopy (TEM) showed the

formation of nanoparticles in the range of 2–8 nm.Dynamic light

scattering (DSL) approved particle size. XRD analysis of the silver

confirmed the formation of metallic silver. Further analysis carried out by

Fourier Transform Infrared Spectroscopy (FTIR), data indicate abounding

of Ag nanoparticles with M. purpureus supernatant molecules and

provides evidence for the presence of proteins as possible biomolecules

responsible for the stabilization and capping agent which helps in

increasing the stability of the synthesized silver nanoparticles. Stability of

the silver nanoparticles was confirmed even after four months. No nitrate

reductase activity was detected and pigments of the M. purpureus could

be the principle reducing power for the silver nanoparticles synthesis as

its reducing power was decreased after nanoparticles formation. Changes

in the pH values during nanoparticles biosynthesis leads to variations in

size of the silver nanoparticles. This small synthesized silver

nanoparticles size it showed antibacterial and antifungal activities.


40

3.2. Introduction

Reduction of metal is an important defense mechanism in

microorganisms as a way to manage metal toxicity. The inherent, clean,

nontoxic and environmentally friendly ability of eukaryotic and

prokaryotic microorganisms to form the metal nanoparticles is

particularly important in the development of Nano biotechnology. New

methods have been developed to control disparity, chemical composition,

the size, and the shape to get the best particles which can be well applied

in different fields of science. However, there is a growing need to

understand the basics of this technique to facilitate application of the new

methodology to laboratory and industrial needs (Mollazadeh, 2010).

Nanotechnology deals with the synthesis and stabilization of various

nanoparticles. Currently, there is a constant need to develop eco-friendly

processes for the synthesis of nanoparticles. The focus for this synthesis

has shifted from physical and chemical processes towards ‘green’

chemistry and bioprocesses. And various chemical and biochemical

methods are being explored for its production. Various microbes are

known to reduce metal ions to metals (Shaligram et al., 2009).

In fact microorganisms can survive and grow in high metal ion

concentration due to their ability to fight the stress. The mechanisms

include: efflux systems; alteration of solubility and toxicity via reduction

or oxidation; bio absorption; bioaccumulation; extracellular complexation

or precipitation of metals; and lack of specific metal transport systems

(Fenice et al.,2000).Synthesis of nanoparticles was found to be

intracellular; this makes the job of downstream processing difficult so

shifted to extracellular biological synthesis. Biological synthesis process

provides a wide range of environmentally acceptable methodology, low


41

cost production and minimum time required. Extracellular synthesis of

nanoparticles using cell filtrate could be beneficial over intracellular

synthesis, the fungi being extremely good candidates for extracellular

process and also environmental friendly (Natarajan et al., 2010).

Biologically synthesized silver NPs could have many applications, in

areas such as nonlinear optics, spectrally selective coating for solar

energy absorption and intercalation materials for electrical batteries, as

optical receptors, catalysis in chemical reactions, biolabeling and as

antibacterial (Cao, 2004)). Size control during synthesis of particles is an

important criterion in the area of silver nanoparticle biosynthesis.

Depending on the size of the nanoparticles, their applications branch out.

Although Ag NPs are synthesized both intra and extracellular, the latter

method of biosynthesis of nanoparticles is highly advantageous because

of ease of control over the environment, large scale synthesis and easy

downstream processing steps (Gurunathan et al., 2009).

3.3. Materials and Methods

3.3.1. Chemicals

It has been discussed previously in chapter II.

3.3.2. Microorganism

M. purpureus fungus was grown aerobically in a yeast-starch-glucose liquid

culture with the following composition g/l: yeast extract; 5, starch; 20 and

glucose; 15, pH was adjusted at 5.5 and aeration 50/250ml medium/flask

volume. The inoculated flasks were incubated on an orbital shaker at 30°C

and agitated at 150rpm.


42

3.3.3. Supernatant Preparation

After 7days of growth, the cultures were centrifuged at 5.000 rpm for 15

minutes and the supernatant was collected for further studies.

3.3.4. Nitrate Reductase Assay

Nitrate reeducates activity of the fungal broth filtrate was assayed

according to the procedure followed by Harley (Jaide & Rand, 2010).

Where 5ml of the M. purpureus broth was mixed with 5mL of the assay

mixture (30mM KNO3 in 5% propanol / 0.1M phosphate buffer of pH 7.5)

and incubated in the dark for 60min. The resulting nitrites were estimated

by adding 2.5mL of 58mM sulphanilamide – 0.05mM NEED (N-(1-

naphthyl ethylene diamine dihydrochloride ) reagent solution and the

developed pink color was measured using UV– Vis. Spectrophotometer at

540nm. Enzyme activity was finally expressed in terms of nmoles

nitrite/mL/h.

3.3.5. Measurement of antioxidant activity


3.3.6. Measurement of reducing activity

Reducing power was determined using ferric-reducing antioxidant power

assay by Apati (Apati et al., 2003). Briefly, 1ml of M. purpureus

supernatant and 1ml of Ag NPs colloidal were mixed with 2.5ml of

phosphate buffer (0.1M pH 6.6) and 2.5ml of 1% (w/v) potassium

ferricyanide. The mixture was incubated at 50°C for 20 minutes, then; 2.5

ml of 10% (w/v) tricholoracetic acid was added to terminate the reaction.

The reaction mixture was then centrifuged at 5000rpm for 10minutes.

Then, 2.5ml of upper layer is diluted with equal amount of deionized


43

water. Finally, 0.5 ml of 0.1% (w/v) ferric chloride (FeCl3) was added.

After 10minutes, the absorbance was measured at 700nm against blank of

deionized water. A higher absorbance of the mixture indicates higher

reducing activity.

3.3.7. Pigments extraction, analysis and incubation with AgNO3

M. purpureus broth filtrate was extracted with ethanol 90% for 1h on a

rotary shaker (100rpm) at room temperature, then filtered through a

reweighed Whatman GF: C disc (Fenice et al., 2000). Absorbance was

measured spectrophotometrically (JASCO-Japan-model 560) at 400, 470

and 500nm which corresponds to the maximum absorbance of yellow,

orange and red pigments respectively (Johns et al., 1991). Pigment

production (units of absorbance, UA) was calculated by the absorbance

values.

Extracted pigments solution and M. purpureus supernatant was mixed with

aqueous AgNO3 solution (1mM) at individual flasks. Ag NPs was

monitoring by measuring the UV-VIS spectrum.

3.3.8. Silver Nanoparticle (Ag NPs) synthesis.

Aqueous silver nitrate solution 1mM ( prepared in deionized water) was

mixed with M. purpureus supernatant in a 250ml Erlenmeyer flask (1%,

v/v) and agitated at 25°C and 150rpm. Control (without silver nitrate,

only supernatant) was also run along with the experimental flask. The

reaction between this supernatant and Ag+ ions was carried out in dark for

several times. The reduction of silver ions was monitoring by measuring

the UV-VIS spectrum of solution in range 300 to 800nm (Kathiresan et

al., 2009).


44

3.3.9. Effect of pH on Ag NPs synthesis and particle sizes

To obtain optimum conditions for maximum synthesis of nanoparticles

and particle size distribution, AgNO3 (1mM) was added to the

supernatant (v/v) and pH of the reaction mixture was adjusted at (3.5, 6.5

and 9.5). The pH of the incubation mixtures was adjusted using 1M

HNO3 and 1M NaOH solutions (Sneha et al., 2010).

3.3.10. Effect of silver nitrate concentration

The possibility of controlling the reaction rate and particle size was

further investigated by changing the composition of the reaction mixture.

In order to confirm, whether the concentration of AgNO3 plays an

important role in the synthesis and size reduction of nanoparticles;

different concentrations of AgNO3 were used, The maximum synthesis of

Ag NPs occurred with respect to Ag+ ion concentration in the range 0.5

up to 2.0mM. This was reflected with an increase in the absorbance at

around 420nm using UV.Visible.


3.3.11.1. UV-VIS spectral analysis

Preliminary characterization of the silver nanoparticles was carried out

using UV–visible spectroscopy (JASCO-Japan-model V- 560) at a

resolution of 1nm. Noble metals, especially silver (Ag) nanoparticles

exhibit unique and tunable optical properties on account of their surface

Plasmon resonance (SPR), dependent on shape, size and size distribution

of the nanoparticles (Tripathy et al., 2010). The reduction of silver ions

was monitored by measuring the UV–visible spectra of the solutions from

300 to 800 nm; the supernatant was used as blank.


45

3.3. 11. 2. Dynamic light scattering (DLS)


3.3.11.3. Transmission Electron Microscopy (TEM)

The particle size and shape were observed by TEM (Sanghi & Verma,

2009) (JEOL electron microscope JEM-100 CX) operating at 80kV

accelerating voltage. The prepared silver suspensions were diluted

10times with deionized water. A drop of the suspension was dripped into

coated copper grid and allowed to dry at room temperature.


To determine Fourier transform infra-red (FTIR) pattern of the sample

silver nanoparticles was diluted with potassium bromide in the ratio of

1:100 and , after drying (room temperature ), compressed to form a disc.

The discs were later subjected to FTIR spectroscopy measurement. These

measurements were recorded on a JA SCO FT-IR -3600 and spectrum

was collected at a resolution of 4cm-1

in wave number region of 400 to

4000cm-1

.

For comparison, M. purpureus supernatant was measured by the same

process. FT-IR measurements were carried out in order to obtain

information about chemical groups present around Ag NPs for their

stabilization and understand the transformation of functional groups due

to reduction process.

3.3.11.5. Energy Dispersive X- ray (EDX)

The Energy Dispersive X- ray (EDX-model-OXFORD) spectroscopy

was performed on scanning Electron Microscope( SEM- JEOL-JEM-


46

5400) equipped with an EDX detector and EDX spectrum was measured

at 10KV accelerating voltage. Solid sample was prepared by drying of

silver Nanoparticles solution on plastic disc and dried at room

temperature.

3.3.11.6. X-ray diffraction (XRD)

The reaction mixture embedded with the silver nanoparticles was placed

on glass slide to form film and dried at room temperature to use for X-ray

diffraction (XRD) analysis. XRD scans were obtained using (The XRD-

6000 series, including stress analysis, residual austenite quantitation,

crystallite size / lattice strain, crystallinity calculation, materials analysis

via overlaid X-ray diffraction patterns Shimadzu apparatus using nickel-

filter and Cu-K a target, Shimadzu Scientific Instruments (SSI), Kyoto

Japan. Founded in 1875) operating with a Cu anode at 40Kv and 50mA in

the range of 2θ value between 20° and 100° with a speed of 2°/min. Prior

to peak width measurement, each diffraction peak was corrected for

background scattering and was stripped of Kα2 portion of the diffracted

intensity. Crystallite size was calculated from Scherrer's equation (Lei &

Fan., 2006)), D = K λ / βcosθ for peak broadening from size effects

only.

Where λ is the wavelength of X-rays used (1.5418 ˚A),

β is the full width at half maximum (FWHM) intensity of the diffraction

line,

θ is the Bragg angle for the measured hKl peak and K is a constant equal

to 0.9 for Ag0.


47

3.3.12. Antimicrobial activity

The Ag NPs synthesized were tested for their antimicrobial activity by the

agar well diffusion method (Bauer et al., 1966) against different kinds of

pathogenic multidrug resistant bacteria and yeast. The tested strains

included; Staphylococcus aureus (RCMB 000106), Staphylococcus

epidermis (RCMB 010024) , Streptococcus pyrogenes (RCMB 010015),

as gram positive bacteria while, Pseudomonas aeruginosa (RCMB

010043), Escherichia coli(RCMB 010052) , Salmonella typhimurium

(RCMB 010072 ) as gram negative bacteria and Candida albicans

(RCMB 05036), Candida tropicals (RCMB 06242), & Candida glabrata

(RCMB 08375), were tested as yeast. Standardized suspension of each

tested strain (108cfu/ml and 10

6cfu/ml for bacteria and yeast,

respectively) was swabbed uniformly onto sterile Muller Hinton Agar

(MHA) (Oxoid) plates using sterile cotton swabs. Wells of 10mm

diameter were bored into the agar medium using gel puncture. Using a

micropipette, 100ul Ag NPs colloid (1mM) (prepared by Monascus

purpureus supernatant) and Monascus purpureus supernatant was added

into each well. After incubation at (37˚C for bacteria and 30˚C for yeast)

for 24h, the different levels of zone of inhibition were measured and

interpreted using the CLSI (Clinical and Laboratory Standard Institute)

zone diameter interpretive standards (CLSI, 2008).


48

3.4. Results and Discussions

3.4.1. Ag NPs colloids synthesis

The formation of silver nanoparticles by M. purpureus supernatant was

investigated. Appearance of the yellowish brown (or red) color in the

reaction vessels suggested the formation of silver nanoparticles (Bhaisa

& D’Souza, 2006). The increase in colour intensity of culture supernatant

was due to increasing number of nanoparticles formed as a result of

reduction of silver ions present in the aqueous solution (Kathiresan et

al., 2009).

Figure (6) shows color change from yellow to yellowish brown (or red)

after 72h of incubation of M. purpureus supernatant with AgNO3 solution

(1mM). The increasing in color intensity of Ag NPs solution was due to

increasing number of nanoparticles formed as a result of reduction of

silver ions present in the aqueous solution.

Figure (7) Shows the UV–Vis spectrum of Ag NPs after 72h of

incubation to M. purpureus supernatant with AgNO3 solution (1mM),

show a single peak at (430nm) is very sharp and the intensity is the

highest means there is more yield of silver nanoparticles and size

distribution of the silver nanoparticles is narrow, Ag NPs peak remained

close to 430nm even after 72h of incubation. The presence of the sharp

resonance indicates no aggregation and broad resonance indicates the

aggregation of the silver nanoparticles in the solution.


49

Fig (6): Image of color change after Ag NPs formation

Fig (7): UV-VIS spectra of Ag NPs formed after incubation of M.

purpureus supernatant with AgNO3 after 72h.


50

3.4.2. Effect of time on Ag NPs synthesis

SPR peak of silver nanoparticles at 430nm; it is well known that the size

and shape of the silver nanoparticles reflects the absorbance peak. The

number of silver nanoparticles has linear correlation with the Peak

intensity, while the size of silver nanoparticles does not have such linear

correlation (Jaidev & Narasimha, 2010), the SPR peak shifts to longer

wavelengths with increase in particle size.

Figure (8) show the UV-VIS spectrum, strong Plasmon resonance

(430nm) was observed after 72h of incubation; show optical density

(2.2nm) even after four months, the peak remained close to 430nm with

decrease in optical density to 2nm, this indicated that the nanoparticles

were well dispersed and that there was low aggregation. After 72h of

incubation no further increasing in intensity was recorded indicating

complete reduction of silver ions (Shaligram et al., 2009).

3.4.3. Effect of pH on the Ag NPs synthesis

In general, the reduction reaction of metallic ions is sensitive to the pH of

the solution as it may affect the morphology of the product via the

formation of certain species as demonstrated. The present study involved

analysis of pH dependent changes in the reaction mixture for synthesis of

Ag NPs. In this reaction, the concentration of AgNO3 was maintained at

1mM, the reaction temperature 25°C, Incubation time 72h and PH

adjusted at (3.5, 6.5, and 9.5).

Figure (9) shows the UV-VIS spectrum of Ag NPs synthesized at

different pHs, Peak area and height of the UV–visible spectrum obtained

from the reaction carried out at pH 6.5 is considerably higher than those

obtained at other pHs, indicating its higher nanoparticle productivity.


51

Also, UV absorbance at 445nm (pH 9.5) clearly indicates the increasing

in silver nanoparticles size; this is attributed to Ag NPs tend to

precipitation in alkaline pH values which could be due to the formation of

silver hydroxide (Eha et al., 2010).On the other hand acidic pH

conditions causes a low negative values of the zeta potential so the silver

tend to aggregate and precipitate (Aji et al., 2009).

Fig (8): UV-VIS spectra of Ag NPs formed by M. purpureus

supernatant at different incubation time.


52

Fig (9): Effect of pH on Ag NPs synthesized by M.purpuerus

supernatant expresed as UV-VIS spectra.

3.4.4. Effect of silver nitrate concentration on Ag NPs synthesis

Figure (10) shows the UV-VIS spectrum of Ag NPs synthesized at

different AgNO3 concentration. The maximum synthesis of Ag NPs

occurred with respect to Ag+ ion concentration in the range 0.5 up to

2.0mM. This was reflected with increasing in the absorbance at 420nm up

to a concentration of 1.0mM; however, the absorbance was found to

decrease at higher concentrations of Ag+ ions. The results clearly

indicated that a1.0mM concentration of Ag+ ions was most appropriate

for the maximum synthesis of Ag NPs from the culture supernatant of M.

supernatant. The reason for the decrease optical density with increasing

AgNO3 concentration may also be because AgNO3 forms a coat on

growing particles (Gurunathan et al., 2009).


53

3.4.5. Role of M. purpureus pigments on Ag NPs synthesis

Figure (11) shows UV-VIS spectrum of Ag NPs synthesized by both

extracted pigments of Monascus purpureus and Monascus purpureus

supernatant, SPR peak at (430nm) is the broadest with low intensity in

case of extracted pigments means the nanoparticles size distribution is

broadened and low yield of silver nanoparticles (Chen et al., 2007) and

SPR peak is very sharp with high intensity in case of M. purpureus

supernatant, means there is more yield of silver nanoparticles and size

distribution of the silver nanoparticles is narrow(Chen et al., 2007). The

study revealed that M. purpureus pigments play important role on

reduction of silver ions to metallic silver nanoparticles and role of

proteins for capping of synthesized silver nanoparticles because amount

of Ag+

ions converted to Ag0 metals is decreased but not abolished in case

of extracted pigments.

Table (5) show M. purpureus pigments production (units of absorbance,

UA) where the high absorbance value observed at 500nm that indicate

high yield of red pigments.


54

Fig (10): Effect of AgNO3 concentration on Ag NPs synthesized by

M.purpureus supernatant expresed as UV-VIS spectra.

Fig (11): UV-visible spectra of synthesized Ag NPs by M. purpureus

supernatant and extracted pigments.


55

Table (5): Optical density of M. purpureus produced Pigments

Type of Pigments

Yellow Orange Red

(UA400) (UA470) (UA500)

0.681±0.01 0.962±0.02 2.542±0.03

3.4.6. Nitrate Reductase activity

The extracellular pigments and proteins secreted by the fungus are

responsible for the reduction of Ag+ to Ag

0. Hence, the role of reeducates

in the fungal filtrate was investigated by nitrate reductase assay.

Extracellular produced nanoparticles were stabilized by the proteins and

reducing agents secreted by the fungus. Our study indicated that M.

purpureus didn’t secrete NADH-dependent nitrate reductase enzyme

which indicated that other reducing factors may be responsible for the

nanoparticles synthesis such as the M. purpureus pigments.

3.4.7. Reducing power and DPPH Free Radical Scavenging Activity

of M. purpureus supernatant and Ag NPs

Table (6) shows the difference in reducing power and antioxidant

activities after and before Ag NPs formation. The study indicates a

decrease in reducing power and antioxidant activities of M. purpureus

supernatant after Ag NPs synthesis. This may be due to reduction of Ag+

ions into Ago metals.


56

Table (6): Difference in reducing power and antioxidant activities of

M. purpureus supernatant after and before Ag NPs synthesis

M.purpureus

supernatant

Ag NPs

Colloids

loss

Reducing

Power

DPPH Free

Radical

Scavenging

Activity%

o.64

74.3

o.201

38

0.439

36

3.4.8. Characterization and stability study of the silver nanoparticles

Particle size and distribution

Size characterization and stability study of the silver nanoparticle

suspension. Volume size distribution of the silver nanoparticles

determined by dynamic light scattering and transmission electron

micrograph; TEM was used as a second technique to confirm the

nanoparticle sizes obtained by DLS and to obtain information about the

particle shape. The nanoparticle diameters observed by TEM were in

agreement with the DLS results, but no quantitative analysis was

performed with these techniques.


57


Figure (12) represents TEM micrograph of Ag NPs solution prepared by

M.s purpureus supernatant; TEM micrograph of Ag NPs was taken after

72h of reaction time. Interestingly, the Ag NPs synthesized were more or

less uniformly shaped and sized. The particle size was found to be

between 2.5 and 7.5nm with average size 4.2nm. Few irregularly shaped

Ag NPs were also noticed. The size of some particles is irregular, which

could be due to different phases of growth of nanoparticles. These

variations in shape and size of nanoparticles synthesized by biological

systems are common (Binupriy et al, 2010), but the difference is very

meager compared to other reports on biological synthesis of Ag NPs. The

biosynthesized silver nanoparticles were previously characterized using

TEM by several investigators (Balaji et al, 2009).


Figure (13) represents DLS graph of Ag NPs solution prepared by M.

purpureus supernatant. DLS graph of Ag NPs was taken after 72h of

reaction time. The volume distribution of the hydrodynamic size of the

nanoparticles showed two peaks. The first peak (approximately 100% of

the particle volume) particle size was found to be between 1nm and 7.4

nm±1nm; the second peak represents approximately 0.0% of the total

volume of nanoparticles had its maximum size 54.3±7.4nm.


58

Fig (12): TEM Micrograph of synthesized Ag NPs colloids by M.

purpureus supernatants.

Fig (13): DLS graph of synthesized Ag NPs by M. purpureus

supernatant.


59

3.4.8.3. EDX analysis of Ag-NPs

Energy dispersive x-ray spectroscopy (EDX) was used to confirm that the

observed granules in the solution indeed consisted of silver.

Figure (14) shows the EDX analysis revealed, the optical absorption

peak was observed at approximately 3keV; which is typical for the

absorption of metallic silver Nano crystallites due to surface Plasmon

resonance (Kalimuthu et al., 2008).

3.4.9.4. X-ray diffraction (XRD) analysis of Ag NPs

In order to verify the results of the UV–Vis spectral analysis, the sample

of the Ag+ ions exposed to the supernatant of the fungus was examined by

XRD pattern.

Figure (15) shows the XRD patterns obtained for Ag NPs synthesized

using fungus M. purpureus supernatant. A number of clear peaks

corresponding to the (111), (20 0), (220) and (311) sets of lattice planes

are observed which may be indexed based on the FCC (face centered

cubic) structures of silver (JCPDS file no. 03-0921). The XRD pattern

thus clearly shows that the Ag NP formed by the reduction of Ag+ ions

by fungus M. purpureus supernatant are crystalline in nature. The mean

diameter of Ag NPs was calculated from the XRD patterns using the

following Scherrer equation:

D = K λ / Β cosθ


60

Where ‘λ’ is wave length of X-Ray (0.1541nm) K is a shape factor (0.9

for Ag nanoparticles), ‘β’ is FWHM (full width at half Maximum), ‘θ’ is

the diffraction angle and ‘D’ is diameter size. The calculated size details

are in Table (7). Size increased in compere with TEM and DLS may be

due to agglomeration of Ag NPs and precipitation of stabilizing agents

after drying.

Fig (14): EDX spectra of synthesized Ag NPs by M. purpureus

supernatant.


61

Fig (15): X-ray diffraction patterns of synthesized Ag NPs by M.

purpureus supernatant.

Table (7): XRD analysis of synthetized Ag NPs from M. purpureus

supernatant

2θof the intense

peak

(deg)

hK1 FWHM of

Intense peak

(β) radians

Diameter Size

(D)

nm

38 (111) 0. 012 12

44 (200) 0. 016 9.5

64 (220) 0.012 13.4

77 (311) 0.0154 11.5

Mean diameter size 11.6


62

3.4.8.5. FT-IR spectra of silver nanoparticles analysis

Figure (16) shows FTIR spectra of both M. purpureus supernatant and

Ag NPs while the wave numbers of characteristic bands and

corresponding comments for M. purpureus supernatant and Ag NPs thin

films are listed in Table (8).

The FTIR spectrum of M. purpureus supernatant exhibited the following

absorption bands: broad absorption band peaking at 3405cm-1

,

corresponding to OH group vibrations, followed by a peak at 2932cm–1,


observed at1427cm-1

and 1087cm-1

, due to the bond vibrations of the –

NO3 group and of the N–OH complex, respectively.

Ag NPs spectrum exhibited a few differences in peak positions compared

to the M. purpureus supernatant, suggesting the bonding between M.

purpureus supernatant molecules and Ag NPs. One of the main

differences observed is the shift of the amide carbonyl group band of M.

purpureus supernatant from 1650cm–1

toward 1662 cm-1

in the Ag NPs

spectrum; the shift can be attributed to the change of bond character of

the N−H bond due to the nitrogen bonding with the metal (Davies, 1963).

These results show that there is strong interaction in the interfaces

between Ag NPs and M. purpureus supernatant molecules. All of the

differences exhibited in the Ag NPs spectrum compared to the spectrum

of M. purpureus supernatant indicated a bonding between nitrogen and

Ag NPs, as well as between oxygen and Ag NPs (Jovanovic et al., 2012).

The proteins present over the silver nanoparticle surface acts as capping

agent .Their study confirmed that the carbonyl groups from the amino

acids residues and peptides of proteins have ability to bind silver; their

proteins could possibly form a coat covering the metal nanoparticles to


63

prevent their agglomeration and aid in its stabilization in the medium.

The carbonyl groups of amino acid residues and peptides have strong

ability to bind to silver (Balaji et al., 2009); it is also reported that

proteins can bind to nanoparticles either through free amine or cysteine

groups in proteins (Mandal et al., 2005).

IR spectroscopic study has confirmed that the carbonyl group from

amino acid residues and peptides of proteins has the stronger ability to

bind metal, so that the proteins could most possibly form a coat covering

the metal nanoparticles (i.e. capping of Ag NP) to prevent agglomeration

of the particles and stabilizing in the medium.

Fig (16): FT-IR spectra of M. purpureus supernatant and Ag NPs

colloids.


64

Table (8): Wavenumber of characteristic bonds of M. purpureus

supernatant and Ag NPs

M. purpureus

supernatant

Wavenumber cm-1

Ag NPs

Wavenumber cm-1

Comment

34405

2923

1650

1360

1427

1280

3450

2950

1662

1373

1495

1290

OH band vibration

(Zheng and Shetty, 2000).

Corresponding to aliphatic

CH group vibration (Zheng

& Shetty, 2000 )

N-C=O group vibration

(Basavaraja et al ., 2008)

-NO3 group vibration

Tertiary nitrogen vibration

N-OH complex


65

3.4.9. Antimicrobial activity of synthesized Ag NPs by M. purpureus

supernatant

Synthesis of Nano sized particles with antibacterial properties which are

called "nanoantibiotics" is of great interest in the development of new

pharmaceutical products (Huh and Kwon, 2011). The use of Ag NPs as

an antibacterial agent is relatively new and has attracted significant

attention in recent years (Morones et al., 2005). In this study; the

antimicrobial activity of the Ag NPs synthesized against different species

of highly pathogenic multidrug resistant bacteria and yeast was

investigated, the average of particle size of Ag NPs used in this study was

2nm up to 8.5nm. The Ag NPs synthesized were proved to have

antimicrobial activity against all the tested microorganisms (Tables 9).

The diameters of bacterial inhibition zones (mm) measured for Staph.

aureus, Staphy. epidermidis, and Strepto. pyrogenes as gram positive

bacteria, P. aeruginosa, E. coli, and S. typhimurium as gram negative

bacteria for Ag NPs are around 16.2±0.44, 17.2±0.19, 15.9±0.25,

11.4±0.35, 11.2±0.58 and 15.8± 0.25(mm) respectively. The diameters

of yeast inhibition zones (mm) measured for C. allbicans, C. tropicalis

and C. glabrata for Ag NPs are around 16.7± 0.25, 18.2± 0.37 and 16.9±

0.44 respectively. The mechanism of inhibitory action of Ag NPs on

microorganisms suggests that upon treatment, DNA loses its replication

ability and expression of ribosomal subunit protein, as well as other

cellular proteins and enzymes essential to ATP production becomes

inactivated (Yamanaka et al., 2005) other studies proposed that, Ag NPs

may attach to the surface of cell membrane disturbing permeability and

respiration functions of the cell or by interfering with components of the

microbial electron transport system (Percival et al., 2005), (Ketchart et


66

al., 2012).It is possible that Ag NPs act similarly to the antimicrobial

agents used for the treatment of bacterial infections which show four

different mechanisms of action including; interference with cell wall

synthesis, inhibition of protein synthesis, interference with nucleic acid

synthesis and inhibition of metabolic pathway (Guzman et al., 2012).

Some studies have reported that, the positive charge on the Ag+ ion is

crucial for its antimicrobial activity through the electrostatic attractions

between the negatively charged cell membrane of microorganisms and

the positively charged nanoparticles (Vijayakumar et al., 2013). In

addition, Ag NPs not only interact with the surface of membrane, but can

also penetrate inside the bacteria (Sharma et al., 2009). Ag NPs have

also intensive tendency to react with sulfur and phosphorus groups, so the

cell membrane proteins containing sulfur and compounds containing

phosphorus such as DNA are likely to be the preferential sites for Ag NPs

(Dehkordi et al., 2011). Ag NPs has not been shown to cause bacterial

resistance currently complicating antibiotic therapy of bacterial

infections. This is presumably due to the fact that, unlike antibiotics, Ag

NPs do not exert their antibacterial effects in a single specific site but at

several levels such as bacterial wall, protein synthesis and DNA (i.e.

attack a broad range of targets in the organisms) which means that

microorganisms would have develop a host of mutations simultaneously

to protect themselves (Parameswari et al., 2010).

In this work, the M. purpureus culture filtrate antifungal activity against

the different Candida spp. were greatly enhanced by the action of silver

nanoparticles , where it was increased by 65.4% against C. albican , by

38% against C.tropicalis and by 34.1% against C.glabrata (Table.5).

Also the antifungal activity of the silver nanoparticles was comparable,


67

70% and 61% on comparison to amphotericin against C.tropicalis, C.

albicans and C. glabrata respectively. The results also show that the

Monascus purpureus culture filtrate activities against Gram –ve bacteria

was greatly improved by the action of the synthesized silver nanoparticles

and it was much more effective than against the Gram +ve one.

Table (9): Antimicrobial activity of synthesized Ag NPs by M.

purpureus supernatant against different microbial strains

Tested

microorganism

Inhibition zones in mm

Control sample standard

M. purpureus

filtrate

Ag NPs Antimicrobial

activity

increment

Amphotericin Sample :

standard

Fungi

Candida allbicans

(RCMB 05036)

10.1±0.58 16.7±0.25 62.7% 23.7±0.1 0.7

Candida tropicalis

(RCMB 06242)

13.5±0.44 18.7±0.37 38% 19.7±0.2 0.94

Candida glabrata

(RCMB 08375)

12.6±0.44 16.9±0.44 34.1% 25.8±0.2 0.65

Gram Positive

Bacteria

Ampicillin

Staphylococcus

aureus (RCMB

000106)

13.4±0.19 16.2±0.44 15% 27.4±0.18 0.59

Staphylococcus

epidermidis (RCMB

14.1±0.25 17.2±0.19 22% 25.4±0.3 0.68


68

NA= NO activity, data are expressed in the form of mean ±SD

3.5. Conclusion

This work provides a simple and economic synthesis of silver of 2nm up

to 8nm. Culture supernatant of M. purpureus used for green synthesis of

silver nanoparticles with silver nitrate solution at room temperature. It is

proposed that the reduction of the silver ions may plausibly be due to M.

purpureus pigments due to its antioxidant and reduction power. The

nanoparticles formed were stabilized probably by the capping of proteins

present in the culture supernatant as revealed by FT-IR spectral analysis.

The UV-VIS spectrum showed surface Plasmon resonance peak at around

430 nm; which characteristic of silver nanoparticles. The silver

nanoparticles formed with average size from 2nm to 8nm it confirmed by

010024) *

Streptococcu

spyrogenes (RCMB

010015)

14.8±0.58 15.9±0.25 7% 26.4±0.34 0.6

Gram Negative

Bacteria

Gentamicin

Pseudomonas

aeruginosa (RCMB

010043)

NA 11.4±0.35 17.3±0.15 0.56

Escherichia coli

(RCMB 010052)

NA 11.2±0.58 28.8±0.24 0.39

Salmonella

typhimurium (RCMB

010072 )

13.6±0.37 15.8±0.25 16% 22.3±0.18


69

TEM and DLS. After 72h of incubation no further increasing in intensity

was recorded indicating complete reduction of silver ions. When pH

increased from 3.5 to 9.5, maximum synthesis was observing at pH 6.5,

with the synthesis time greatly reduced and the yield of Ag NPs was

significantly enhanced. This small synthesized nanoparticles size would

be suitable for developing a biological process for mass scale production

as it showed antibacterial and antifungal activities .However, future

studies on the mode of action of this biocidal influence of this are

necessary in order to fully evaluate its possible use as a new bactericidal

material

Chapter IV: Silver nanoparticles synthesis using Monascus

purpueurs supernatant under gamma irradiation effect

07

Silver nanoparticles synthesis using Monascus purpueurs supernatant

under gamma irradiation effect

4.1. Abstract

Aqueous dispersion of highly stable silver nanoparticles were synthesized

using gamma irradiation; M. purpureus supernatant as reducing and

stabilizing agent. The formation of Nano sized silver was confirmed by

its characteristic surface Plasmon absorption peak around 430nm in UV-

VIS spectra. The size of the silver nanoparticles can be turned by

controlling the radiation dose and radiation process of AgNO3 solution

with M. purpureus supernatant (irradiation process).Dynamic light

scattering (DLS) measurement and Transmission electron microscope

(TEM) of the synthesized nanoparticles indicated that the size depend on

dose and irradiation process . The irradiation was carried out at doses

from 1 to 25kGy. XRD analysis of the silver confirmed the formation of

metallic silver. Fourier transform infr-red (FTIR) spectroscopic data

indicate abounding of Ag nanoparticles with M. purpureus supernatant.

The obtained Ag nanoparticle dispersion was stable for over 3months at

room temperature.

4.2. Introduction

Gamma radiation has been proved to be a simple and efficient method for

silver nanoparticles synthesis, requires an aqueous system, room

temperature and ambient pressure ( Li et al., 2012). The preparation and

study of metal nanoparticles are interest in both research and technology,

because of their potential applications in areas such as catalysis, Nano

electronics, optical filters, electromagnetic interference shielding and

surface Raman scattering (Ramnani et al., 2007). The surface Plasmon



07

absorbance (SPA) properties of nanoparticles have direct relationship

with the size, shape and chemical composition of nanoparticles. Intensity

and wavelength shift of SPA absorption spectrum were used to follow

growth or particle size (Naghavi et al., 2010).

Various methods for synthasis of colloidal silver nanoparticles using

metal salts as precursors have been reported namely chemical reduction,

photochemical, electrochemical, microwave processing and irradiation.

Irradiation induced reduction synthesis which offers some advantages

over the conventional methods; because of its simplicity, provides metal

nanoparticles in fully reduced, highly pure and highly stable state

(Ramnani et al., 2007).

In this method, the aqueous solution of metal salt is exposed to gamma-

rays; the species hydrated electron and hydrogen atoms arising from

radiolysis of water are strong reducing reagents and they reduce the metal

ion to zero valent state (Marignier et al., 1985).

H2O γ irradiation

e-aq, H3O

+, H

•, OH

•, H2

• , H2O2

• (1)

The radiolytic method is particularly suitable for generation of metal NPs

in solution, because the radiolytically generated species exhibit strong

reducing power and reduction of metal ions occur at each encounter

(Mollazadeh, 2010).A blue shift of the silver Plasmon absorption band

resulting from the electronic polarization of Ag particles was observed for

aqueous solution in the case of electron transfer from the free radicals

generated radiolytically or photolytically due to adsorption of ions or

molecules on metal clusters (Temgire and joshi, 2004).

The radiolytic method is suitable for generation of metal particles,

particularly silver, in solution (Remita et al, 2005).The amount of



07

zerovalent nuclei can be controlled by varying the dose of the irradiation

(Eisa et al, 2011).Generally, metal Nano composites can be obtained by

two different approaches namely; ex situ and in situ techniques.

In the ex situ approach, polymerization of monomers and formation of

metal nanoparticles were separately performed and then they were

mechanically mixed to form Nano composites.

In the in situ methods; metal nanoparticles are generated in solution.

A commonly employed in situ method is the dispersion process in which

the solutions of the metal precursor and the protective polymer are

combined and the reduction is subsequently performed in solution (Eksik

et al., 2010). In situ nanoparticle generation can be attained by using

three approaches:

1-Ag+ reduced in solution (Khanna et al., 2005).

2-The silver ions can be absorbed within a preformed polymer film

immersed in an Ag+ solution and then reduced (Gaddy et al., 2004).

3- The NPs are nucleated and grown in the solid state (Porel et al., 2005)

Silver nanoparticles nucleation and growth can be easily followed by

absorption spectroscopy since the SPR band is sensitive to particle size

(Kelly et al., 2003), morphology, disparity, dielectric properties of the

supporting medium and aggregation (Sun & Xia, 2003).

The irradiation as a new method had been extensively used to prepare

Nano scale clusters and materials, so the most studies of irradiation have

been focus on the synthesis of metallic clusters and crystals (Yang et al.,

2002).

Metal nanoparticles (NPs) have stimulate worldwide investigation

because of their remarkable physical and chemical properties relative to

their bulk solid counterparts, and their large proportion of high energy

surface atoms (Murphy, 2002).As a consequence the production of NPs



07

has attracted an enormous amount of attention in recent years. Silver NPs

(Ag NPs) have a number of superior properties and are widely used in

different fields such as in medicine because of their antibacterial

properties in electronics as thick film conductor conductivities in surface

enhanced resonance Raman scattering, in optical biosensors and in

oxidative catalysis photo catalysis and chemical analysis (Shameli et al.,

2010). In addition, Nano sized silver colloid has recently been used for

inkjet printing (Lee et al., 2005). The Ag NPs are normally short lived in

aqueous solution as they agglomerate quickly.

Numerous methods have been utilized for the synthesis and stabilization

of Ag NPs. Problems with the stability of the produced colloidal Ag NPs

dispersions have been solved by the addition of polymers and surfactants

(Ahmad et al., 2010) .Such complications do not occur if the NPs are

deposited on a stable inert carrier such as glass (Li et al., 2003).

The use of gamma irradiation in the preparation of Ag NPs has been

demonstrated to have a number of highly advantageous properties

compared with conventional chemical and photochemical methods,

namely (Shameli et al., 2010)

• The controlled reduction of silver ions can be carried out without using

of reducing agents or producing any undesired oxidation products from

the reductants.

• The method provides Ag NPs in completely reduced, extremely pure,

and very stable states.

• The reducing agent is generated uniformly in the solution.

• The process is uncomplicated and uncontaminated.

• The gamma irradiation is harmless.

• No undesirable impurities similar to silver oxide are introduced.



07

This novel method in comparison to our other works consist of controlled

reduction without any undesired oxidation products and extremely stable

colloids NPs. Very pure silver ions reduced to NPs depend on gamma

irradiation doses. Stabilizer was used to prevent the Ag NPs from

aggregation. At different gamma irradiation doses both reduction and

fragmentation of large Ag NPs were found to have occurred

simultaneously and the particle size of the Ag NPs decreasing or

increasing at different irradiation doses. With using this method; the

researchers are able to obtain Ag NPs of different sizes by controlling the

gamma irradiation dose.

AgNO3 separated into Ag+ and NO3

- ions in the aqueous solution as

shown in Equation (2). The solvated electrons, ie, e−

aq, and H atoms are

strong reducing agents; therefore, in the following steps; they easily

reduced silver ions down to the zerovalent state (Equations 3 and 4)

(Sheikh et al., 2009).

AgNO3 Ag + NO3− (2)

Ag+

+ e−aq reduction Ag

0 (3)

Ag+ + H• reduction Ag

0 + H

+ (4)

Silver atoms formed by the irradiation tended to coalesce into oligomers

(Equation 5) which progressively grew into large clusters (Equation 6).

The aqueous electrons reacted with the Ag+ clusters to form the relatively

stabilized Ag clusters (Equation 7) (Janata et al., 1994).



07

Ag 0 + Ag

+ → Ag

+2 (5)

nAg0 + Ag

+2 → (Ag) 4

+ (6)

(Ag+) n + n e

−aq → (Ag) n (7)

We presented an improved gamma radiation approach for the synthesis of

silver nanoparticles. Here we use M. purpureus supernatant which has

been proved to be biocompatible components as a template for

nanoparticles growing. The results of gamma irradiation investigate that

low and high doses of gamma irradiation have an effect on the size and

optical absorption of Ag NPs. Reduction of metal is an important defense

mechanism in microorganisms as a way to manage metal toxicity. The

inherent; clean, nontoxic and environmentally friendly ability of

eukaryotic and prokaryotic microorganisms to form the metal

nanoparticles are particularly important in the development of Nano

biotechnology. New methods have been developed to control disparity,

chemical composition, the size and the shape to get the best particles

which can be well applied in different fields of science. However, there is

need to understand the basics of this technique to facilitate application of

the new methodology to laboratory and industrial needs (Biswal et al.,

2011).



07

4.3. Materials and methods

4.3.1. Supernatant preparation

It has been discussed previously in chapter III.

4.3.2. Synthesis of Ag NPs colloids by gamma irradiation

4.3.2.1. Gamma irradiation source

The process of irradiation was carried out at the National Center for

Radiation Research and Technology (NCRRT). The facility used was

60Co-Gamma chamber 4000-A-India. Irradiation was performed using

60Co- gamma rays at a dose rate 2.9kGy/h at the time of the experiment.

In this process, 60

Co γ-rays interact with matters in the solution mainly by

photoelectric absorption and Compton scattering to produce free electrons

and also hydrated electrons induced from water radiolysis. Together,

these electrons reduce the Ag+ into Ag

0.

4.3.2.2. Radiation processing

4.3.2.2.1. Ex situ radiation process

M. purpureus supernatant and AgNO3 solution (1mM) were separately

irradiated at different doses ranged from 1 up to 25kGy and then mixed.

The irradiated supernatant was mixed with irradiated and non-irradiated

AgNO3 solution (1mM, v/v) and then incubated at room temperature in

dark.

4.3.2.2.2. In Situ radiation process

50ml of AgNO3 solution (1mM) was mixed with M. purpureus

supernatant 50ml then irradiated at different doses ranged from 1 to

25kGy and, then incubated at room temperature in dark.



00

4.3.3. Assay of antioxidant activity

The scavenging activity for DPPH free radicals was measured according

to (Zhao et al., 2006) was used to determine the antioxidant activity of

irradiated, Nan irradiated M. purpureus supernatant and Ag NPs

(prepared by in situ and ex situ irradiation process)

To 2ml of distilled water, 1ml of o.1mM DPPH solution in ethanol and

o.5ml of irradiated supernatant, Nan irradiated supernatant and Ag NPs

was added in separate tubes. The mixtures was shaken vigorously and

allowed to reach a steady state for 30min at room temperature.

Decolorization of DPPH was determined by measuring the decrease in

absorbance at 517nm and the DPPH radical scavenging effect was

calculated according to the following equation.

Scavenging rate % = [1-(A1-A2)/A0] × 100

Where A0 = absorbance of control (DPPH without supernatant)

A1 = absorbance of the reaction mixture

A2 = absorbance without DPPH (DPPH was replaced by the same

volume of distilled water)


4.3.4.1. UV-VIS spectroscopy










07

4.3.4.5. Energy Dispersive X- ray (EDX)


4.3.4.6. X-ray diffraction (XRD)


The reaction mixture embedded with the silver nanoparticles prepared by

both ex situ and in situ radiation was placed on glass slide to form film

this slides were dried at room temperature and were exposed for X-ray

diffraction (XRD) analysis.

4.4. Results and discussions

4.4.1. In Situ radiation process

4.4.1.1. Mechanism of reduction

The mechanism of radiolytic reduction of aqueous silver solution is

carried out by the hydrated electrons, free radicals and organic radicals

formed as follow;

4.4.1.1.1. Hydrated electrons and free radicals:

Aqueous solutions mixture of M.purpureus supernatant and AgNO3

(1mM) is subjected to gamma radiolysis to produce the following species

(Spinks et al., 1990)

H20 γ-radiolysis

e-aq, H3O

+, H2, H

•, OH

•, H2O2 (1)

The solvated electrons and H• atoms are strong reducing agents: and can

reduce Ag+ ions to neutral Ag

0 atoms (Sheikh et al., 2009).

Ag+ + e-aq Ag

0 (2)

Ag+ + H

• Ag

0 + H

+ (3)



07

These neutral Ag0 atoms at first dimerize when they encounter or

associate with the excess Ag and Ag+

ions (Sheikh et al., 2009)

Ag0

+ Ag0 Ag

02 (4)

Ag0 + Ag

+ Ag

+2 (5)

The charged dimer clusters Ag+

2 may further react with excess silver

cations by a cascade of coalescence processes to form trimmers, tetramer

and higher order silver ion clusters (Ag+

n +1 ) (Janata, 2003) and also the

doubly.

4.4.1.1.2. Organic radicals formed:

The mechanism of radiolytic reduction of aqueous solution is carried out

by organic radicals formed (Rao et al., 2010). M. purpureus pigments

(especially red pigments) play an important role in scavenging the free

radicals and converted into organic radicles. The arrow indicates the

segment in which it occurs resonance and organic radical formed and

R=main nucleus of M. purpureus pigments.

Red pigment structure of M. purpureus (Mukherjee et al., 2010) as

examples:



77

Suggested that the following provisional mechanism for in situ irradiation

process for reduction which is consistent with similar studies on the

irradiation reduction of Ag NPs in other solutions (Temgire et al., 2004);

the growth of silver nanoparticles by reduction of Ag+ to Ag0 is step

wise show in the following Eqs. (6), (7), (8), and (9)

R-CH-(OH)-CH3-CH3-CH3-CH3-CH4 + OH• → H20 + R -

C• - (OH)-CH3-CH3-CH3-CH3-CH4 +H2O (6)

A secondary radical is formed (R-C•

- (OH)-CH3-CH3-CH3-CH3-CH4)

which efficiently reduces the precursor metal ions Ag+

to Ag0.

R-C• - (OH)-CH3-CH3-CH3-CH3-CH4 + Ag

+ → Ag

0 + R-

CO-CH3-CH3-CH3-CH3-CH4 + H2O (7)

(R-C• - (OH)-CH3-CH3-CH3-CH3-CH4)2 + Ag 2

+ → Ag2

0 +

R-CO-CH3-CH3-CH3-CH3-CH4 + 2H2O (8)

(R-C• - (OH)-CH3-CH3-CH3-CH3-CH4) n + Ag n

+ → Agn

0 + R

-CO-CH3-CH3-CH3-CH3-CH4 + nH2O (9)

The hydroxyl radicals from the water radiolysis were scavenged

efficiently by Monascus purpureus pigments (resonance system R-CH-

(OH)-CH3-CH3-CH3-CH3-CH4) to yield a secondary radical called



77

organic radical (R-C• - (OH)-CH3-CH3-CH3-CH3-CH4) that reduce Ag+

to Ago this is coincide with similar studies on the irradiation reduction of

Ag NPs in other solutions (Mostafavi et al., 2002).

After gamma irradiation of the AgNO3 (1mM) solution the dispersion

became reddish brown and deep red depend on dose of radiation as in

figure (17), reddish brown indicating the formation of highly stable and

uniform sized silver nanoparticles (Liu et al., 2009) at 5kGy. It was

found that the Ag NPs dispersion could be stored for more than 3months

without obvious sedimentation, demonstrating a long term stability of

silver Nano particles.

Fig (17): Image of Ag NPs synthesized by in Situ irradiation process

at doses ranged from 1kGy to 25kGy.

As shown in results of the UV–Vis spectra of the Ag NPs prepared by in

Situ irradiation process at different radiation doses ranged from 1kGy up

to 25kGy figure (18). With the low irradiation dose, 1kGy does not

appear diagnostic peak of Ag NPs which indicated Ag NPs not formed.



77

On the other hand irradiation doses at 10, 15, 20 and 25KGy, resulted two

peaks around (430 and 600 nm). The peak at 430nm is the broader and its

intensity lower which means nanoparticles size distribution is broadened

and low yield of silver nanoparticles which consistent with study of

(Chen et al.,2007). While in case the peak at 600nm, SPR peak was

shifted towards longer wavelength (Red shift), its intensity was reduced

and the peak became broader. These changes which are related to the

silver particle growth (increase size) and formation of different sized

particles with Puis et al., (2011).The reason can be attributed to the

formation of large amount of radical species that causes high random

motion of solution molecules leading to Ag NPs and Ag+

aggregation and

precipitation as in Eq (10, 11)

Ag0

+ Ag0 Ag

02 (10)

Ag0 + Ag

+ Ag

+2 (11)

In this study at irradiation dose 5kGy a single peak at (430nm) with very

sharp and highest intensity which means there is more yield of silver

nanoparticles and size distribution of the silver nanoparticles is narrow.

This results also obtained by Chen et al.,(2007) . The reason can be

attributed to the formation of suitable amount of radical species that

responsible for reduction of Ag+ to Ag

o and M. purpeureus supernatant

still has a higher antioxidant activity that responsible for prevention of

Ag0 aggregation (increase size) and sedimentation.



77

Fig (18): UV-Vis spectra of synthesized Ag NPs by in situ irradiation

process at doses ranged from 1kGy up to25kGy.

4.4.1.2. Characterization of synthesized Ag NPs by in situ irradiation

process

4.4.1.2.1. Transmission electron microscope (TEM) analysis:

The images in figure (19, 20, 21, 22 and 23) represent TEM micrograph

of synthesized Ag NPs by in situ irradiation at different doses of radiation

ranged from 1kGy up to 25kGy. The concentration of Ag+ ion was fixed

at concentration 1mM when the irradiation dose increased from 5, 10, 15,

20 and 25kGy, and the average size of particles increased from 10nm

(Fig.19) to 75nm Fig (23). In fact, increasing of irradiation dose in case of

in situ irradiation process causes Ag NPs and Ag+ aggregations (large size

particles).Therefore, the number of Ag0 in the aggregations decreased

with decreasing of irradiation dose decreased up to 5KGy.



77

Fig (19): TEM micrograph of synthesized Ag NPs by in situ

irradiation at 5kGy, mean diameter (8.5nm).


irradiation at 10kGy, mean diameter (18nm).



77


irradiation at 15kGy, mean diameter (30.5nm)





77



4.4.1.2.2. Dynamic light scattering analysis (DLS)

The images in figure (24, 25, 26, 27 and 28) represent DLS graphs of Ag

NPs solution prepared by in situ irradiation process at different doses

ranged from 1 up to 25KGy. In this study when the irradiation dose

increased from 5, 10, 15, 20 to 25kGy the size distribution range and size

of the synthesized Ag NPs increased from 10.6± 1.7 (Figure. 24) to 123.7

± 34.9nm (Figure.28). Avery narrow size distribution with small size

particles obtained at irradiation dose of 5kGy. The volume distribution of

the hydrodynamic size of the nanoparticles showed two peaks (5kGy); the

first peak (approximately 100% of the particle volume) had its maximum



70

size at 10.6±1.7nm. The second peak represents approximately 0.0% of

the total volume of nanoparticles had its maximum at 78.9±43.5 nm.

Table (10) shows Ag NPs characters prepared under in situ irradiation

process, TEM, DLS and UV-λ Max; where particles size has positive

relationship with wavelength, if wavelength increased (red shift) the

particle size increased and size distribution increased.

Fig (24): DLS graph of synthesized Ag NPs by in situ irradiation of

at 5kGy.



77

Fig (25): DLS graph of synthesized Ag NPs by in situ irradiation at

10kGy.


15kGy.



77


20kGy.


25kGy.



77

Table (10): characters of Ag NPs prepared under in situ irradiation

process at different doses of gamma irradiation

Irradiation

dose

λ max

wavelength

(nm)

TEM

average

size(nm)

DLS

average

size(nm)

1kGy

5kGy

10kGy

15kGy

20kGy

25kGy

-----

430

430,600

430,600

430,600

430,600

----

8.5

18

30.5

51

72

------

10.6

31

36

89

123

4.4.1.2. 3. Fourier Transform Infrared Spectroscopy (FT-IR) analysis

In order to investigate the status of the Ag NPs in the colloid, FTIR

technique was employed to analyze the samples Ag NPs (Fig. 29B). For

comparison a sample without adding AgNO3 was prepared in the route

and thus only M. purpureus supernatant (irradiated at 5kGy) was

prepared (Figure.29A). In the IR spectrum curve of the comparison, the

frequency of the C=O stretching vibration in M. purpureus supernatant

extremely shifted 1650 cm-1

to 1660 cm-1

in case of Ag NPs . The

decreasing frequency of the C−O stretching vibration implies that M.

purpureus supernatant molecules use the oxygen atom in carbonyl to

coordinate with the metal ion in the Ag NPs solution.

Appearance of sharp peak 1200cm-1

in case of Ag NPs solution (Figure

.29B) more sharp than in case of M. purpureus supernatant (Figure.29A)

may be attributed to the contribution towards the stabilization of the Ag



77

NPs by OH bond. With the above FT–IR spectra (Figure.29A and B)

indicates the formation of a new peak near 882.0cm-1

, which may be

assigned to one of the vibration modes of Ag–O (Roy et al., 2007). At the

same time the strong absorption peak of M. purpureus supernatant at

1430cm−1

(−N−H−) was also shifted in case of the Ag NPs solution. The

shift can be attributed to the change of bond character of the N−H bond

due to the nitrogen bond interaction in the interfaces between Ag NPs and

M. purpureus supernatant molecules. It is convinced that M. purpureus

supernatant molecules played an important role in the stabilization and

formation of the Ag NPs. Corresponding comments for M. purpureus

supernatant and Ag NPs thin films are listed in Table (11).

Fig (29): FT-IR spectra of M. purpureus supernatant (irradiated at

5kGy) (A) and synthesized Ag NPs at 5kGy (B) by in situ irradiation

at 5kGy.



77

Table (11): wavenumber of characteristic bonds and corresponding

comments for M. purpureus supernatant and synthesized Ag NPs by

in situ irradiation

M. purpureus

Wavenumber

cm-1

Ag-NPs

Wavenumber

cm-1

Comment

3490

2980

1660

1495

1370

1280

3450

2970

1650

1480

1365

1290

OH band vibration

CH group vibration

N-C=O group

tertiary nitrogen

-NO3 group vibration

N-OH complex

4.4.1.2.4. X-ray diffraction (XRD) analysis of synthesized Ag NPs by

M. purpureus supernatant prepared by in situ irradiation process

Figure (30) shows the XRD patterns obtained for Ag NPs synthesized by

in situ irradiation process of fungus M. purpureus supernatant. A number

of clear peaks at 2θ about 38.0858°, 44.2100°, 64.4850°, and 77.38° in

case of in situ irradiation corresponding to the (1 1 1), (2 0 0), (2 2 0) and

(3 1 1) sets of lattice planes are observed which may be indexed based on

the FCC (face centered cubic) structures of silver (JCPDS file no. 03-

0921). The XRD pattern thus clearly shows that the Ag NP formed by the

reduction of Ag+ ions by fungus Monascus purpureus supernatant are

crystalline in nature. The mean particle diameter of Ag NPs was

calculated from the XRD pattern according to the line width of the (1 1

1), (200), (220) and (311) planes refraction peak using the following

Scherrer equation:

D = K λ / Β cosθ



77

Where ‘λ’ is wave length of X-Ray (0.1541nm) K is a shape factor (0.9

for Ag0), ‘β’ is FWHM (full width at half Maximum), ‘θ’ is the

diffraction angle and ‘D’ is diameter size. The calculated particle size

details are listed in Table (12) where the mean diameter size in case of in

situ irradiation at 5kGy size is 12.112nm increased in comparison with

TEM and DLS this may be referred to agglomeration of Ag NPs and

precipitation of stabilizing agents after drying of Ag NPs colloids.

Fig (30): X-ray diffraction patterns of synthesized Ag NPs by in situ

radiation process of M. purpureus supernatant.



77

Table (12): XRD analysis of synthesized Ag NPs by in situ irradiation

at 5kGy

2θ of the intense

peak

(deg)

hK1 FWHM of

Intense peak

(β) radians

(in situ)

Diameter Size

nm

38.0858 (111) 0.012 13.97

44.2100 (200) 0.02 8.816

64.4850 (220) 0.012 14.13

77.38 (311) 0.02 11.54

Mean diameter size 12.114

4.4.2. Ex situ radiation process

4.4.2.1. Mechanism of reduction

The mechanism of radio lytic reduction of aqueous solution is carried out

mainly by formed organic radicals,

M. purpureus supernatant molecules (especially red pigments) play an

important role in scavenging the free radicals and converted them into

organic radicles.

Suggested that the following provisional mechanism of ex situ

irradiation process for reduction which is coincide with similar studies on

the irradiation reduction of Ag NPs in other solutions (Temgire et al.,

2004); the growth of silver nanoparticles by reduction of Ag+ to Ag

0 is



77

showed step wise show in the following equations: (1), (2), (3), (4) and

(5)

H20 γ-radiolysis

e-aq H3O

+, H2, H

•, OH

•, H2O2

(1)

R-CH-(OH)-CH3-CH3-CH3-CH3-CH4 + OH• → H20 + R

-C• - (OH)-CH3-CH3-CH3-CH3-CH4 + H20

(2)

A secondary radical is formed (R-C•

- (OH)-CH3-CH3-CH3-CH3-CH4)

which efficiently reduces the precursor metal ions Ag+

to Ag0.

R-C• - (OH)-CH3-CH3-CH3-CH3-CH4 + Ag

+ → Ag

0 + R-CO

-CH3-CH3-CH3-CH3-CH4 + H20

(3)

(R-C• - (OH)-CH3-CH3-CH3-CH3-CH4)2 + 2Ag

+ → 2Ag

0 + R-CO-

CH3-CH3-CH3-CH3-CH4 + 2H20

(4)

(R-C• - (OH)-CH3-CH3-CH3-CH3-CH4) n + nAg

+ → nAg0 + R -CO-

CH3- CH3-CH3-CH3-CH4 + nH20

(5)



77

The hydroxyl radicals from the water radiolysis were scavenged

efficiently by M. purpureus supernatant molecules (resonance system R-

CH-(OH)-CH3-CH3-CH3-CH3-CH4) to yield a secondary radical called

organic radical (R-C• - (OH)-CH3-CH3-CH3-CH3-CH4), that reduce Ag

+

to Ago.

Since the electrochemical potential of the organic radical is more positive

than that of the Ag+/ hydrated electron and hydrogen atoms system so

reaction of secondary radicals formed with Ag+ ions is relatively slow

(Henglein, 1998).

After mixing of irradiated M. purpureus supernatant with AgNO3 solution

(1mM), the dispersion became yellowish brown and deep red depend on

dose of radiation as in figure (31), the yellowish brown color indicating

the formation of highly stable and uniform sized silver nanoparticles (Liu

et al., 2009).



70

Fig (31): Image of synthesized Ag NPs by ex situ radiation at

different doses of irradiation ranged from 1kGy to 25kGy.

Figure (32) shows the UV–Vis spectra of Ag NPs prepared by ex situ

irradiation process after 72h of incubation. With the low irradiation doses

1kGy and 5kGy two peaks occurs (450 and 650 nm), the peak at 450nm

is the broadest and its intensity is low; this means the nanoparticles size

distribution is broadened and low yield of silver nanoparticles.

The peak at 650nm, SPR peak was shifted towards longer

wavelength (Red shift), its intensity was reduced and the peak became

broader. These changes are related to the silver particle growth (increase

size) and formation of different sized particles (Puis et al., 2011).

With the highest irradiation dose such as 10, 15 and 20kGy there is a

single narrow peak in the UV–Vis spectra which means the size

distribution of the silver nanoparticles is narrow. The peaks intensities are

higher than that in 1kGy and 5kGy and this means there is more yields of

Filtrate Non IR 1kgray 5kgray 10kgray 15kgray 20kgray 25kgray



77

silver nanoparticles. The same results were obtained by Chen et al.,

(2007).

The peaks at doses 10, 15 and 20kGy are somewhat less broad than that

at 1kGy and 5kGy; means the nanoparticles size distribution is narrow

(Puis et al., 2011). At a very high irradiation dose that is 25kGy in this

study, the peak is very sharp and the intensity is the highest, means there

is more yield of silver nanoparticles than other doses, the reason can be

attributed to the formation of large amount of secondary radical (organic

radicals) which responsible for reduction of Ag+ to Ag

o by increase of

irradiation dose.

In this work, the maximum absorbance obtained after 72hr, the

electrochemical potential of the organic radical is more positive than that

of the Ag+, Ag

0, hydrated electron and hydrogen atoms system so reaction

of secondary radicals formed with Ag+ ions is relatively slow (Henglein,

1998).

UV–Vis spectra of synthesized Ag NPs at 30kGy show broadest peak at

450nm and the intensity is the lower than that occurred at 25kGy figure

(33); this means the nanoparticles size distribution is broadened and low

yield of silver nanoparticles. These results matched with that obtained by

Chen et al., (2007).



77

Fig (32): UV–Vis spectra of synthesized Ag NPs by ex situ radiation.

Fig (33): UV–Vis spectra of synthesized Ag NPs by ex situ radiation

at 25kGy and 30kGy.



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4.4.2.2. Characterization of Ag NPs prepared by ex situ radiation

process.

4.4.2.2. 1. Transmission electron microscope (TEM) analysis

Control on the size, morphology and distribution of nanoparticles plays

an important role in the properties of metal nanoparticles. TEM

micrographs were taken into account. Figure (34, 35, 36, 37, 38, and 39)

Represents TEM images of Ag NPs solution prepared by ex situ radiation

process at different doses ranged from 1 to 25kGy; Average diameter of

the nanoparticles depends on dose of irradiation. The particle size

distribution of the Ag NPs prepared under radiation (25kGy) exhibit a

very narrow size distribution with average size of 7.3 nm. This result

means that the size of the prepared Ag NPs gets smaller and the particle

size distribution is improved with increasing of irradiation dose.

Fig (34): TEM micrograph of synthesized Ag NPs by ex situ radiation

at 1kGy, mean diameter (24nm).



777


at 5kGy, mean diameter (19.5nm).





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at 20kGy, mean diameter (8nm).



777



4.4.2.2. 2. Dynamic light scattering analysis (DLS)

Average particle size was determined by DLS method and was found that

the particle size and size distribution depend on dose of radiation.

Figure (40, 41, 42, 43, 44, and 45) represents DLS graphs of Ag NPs

solution prepared by ex situ radiation process at different doses ranged

from 1kGy up to 25KGy.Ag NPs prepared under radiation dose (25kGy)

exhibit a very narrow size distribution with small particles size. The

volume distribution of the hydrodynamic size of the nanoparticles show

peak (approximately 100% of the particle volume) had its maximum size

at 10.8 nm. Table (13) shows Ag NPs characters prepared under ex situ

radiation process at different doses of gamma irradiation where particles

size has positive relationship with wavelength, if wavelength increased

(red shift) the particle size increased and size distribution increased.



777

Fig (40): DLS graph of synthesized Ag NPs by ex situ radiation at

1kGy.


5kGy.



777


10kGy.


15kGy.



777


20kGy.


25kGy.



770

Table (13): Characterization of synthesized Ag NPs by ex situ

radiation process at different doses of γ-radiation

Irradiation

dose

λ max

(nm)

TEM

average size

(nm)

DLS

average size

(nm)

1kGy

5kGy

10kGy

15kGy

20kGy

25kGy

450,650

450,650

430

430

430

430

24

19.5

12.5

8.2

8

7.3

29.9

24.5

11.3

11.2

11.1

10.8

4.4.2.2. 3.Fourier Transform Infrared Spectroscopy (FT-IR) analysis

FTIR spectra are presented in figure (46), while the wave numbers of

characteristic bands and corresponding comments for M. purpureus

supernatant and Ag NPs thin films are listed in Table (14).

The FTIR spectrum of M. purpureus supernatant (A) exhibited the

following absorption bands: broad absorption band peaking at 3405cm-1

,

corresponding to OH group vibrations, followed by a peak at 2923cm–1,


observed at 1373cm-1

and 1288 cm-1

, due to the bond vibrations of the –

NO3 group and of the N–OH complex, respectively. Ag NPs spectrum (B)

exhibited a few differences in peak positions compared to the M.

purpureus supernatant, suggesting the bonding between M. purpureus

supernatant and Ag NPs. One of the main differences observed is the shift

of the amide carbonyl group band of M. purpureus supernatant from

1660cm–1

toward 1670cm-1

in the Ag NPs spectrum; the shift can be



777

attributed to the change of bond character of the N−H bond due to the

nitrogen bonding with the metal. These results show that there is a strong

interaction in the interfaces between Ag particles and M. purpureus

supernatant. All these differences exhibited in the Ag NPs spectrum (B)

compared to the spectrum of M. purpureus supernatant (A) indicated the

bonding between nitrogen and Ag NPs, as well as between oxygen and

Ag NPs (Jovanovic et al., 2012).

Fig (46): FT-IR spectrum of M. purpureus supernatant (A) and Ag

NPs colloids (B) synthesized by ex situ radiation process at dose

25kGy.



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Table (14): Wavenumber of characteristic bonds and corresponding

comments for M.purpureus supernatant and synthesized Ag NPs

colloids by ex situ radiation process at dose 25kGy

M.purpureus

supernatant

wavenumber

cm-1

Ag-NPs

wavenumber

cm-1

Comment

3490

2923

1660

1427

1373

1288

3450

2915

1650

1415

1360

1270

OH band vibration

CH group vibration

N-C group vibration

Tertiary nitrogen

N-C=O group

N-OH complex

4.4.2.2.4. X-ray diffraction (XRD) analysis of synthesized Ag NPs by

ex situ radiation process.

In order to verify the results of the UV–Vis spectral analysis, the sample

of the Ag+ ions exposed to the fungal supernatant was examined by XRD

pattern. XRD patterns obtained for Ag NPs synthesized by ex situ

radiation process (Figure. 47). A number of clear peaks at 2θ are 38.42°,

44.64°, 64.53°, and 77.635° in case of ex situ irradiation corresponding to

the (1 1 1), (2 0 0), (2 2 0) and (3 1 1) sets of lattice planes are observed

which may be indexed based on the FCC (face centered cubic) structures

of silver (JCPDS file no. 03-0921). The XRD pattern thus clearly shows

that the Ag NP formed by the reduction of Ag+ ions is crystalline in

nature. The mean particle diameter of Ag NPs was calculated from the

XRD pattern according to the line width of the (1 1 1), (200), (220) and

(311) planes, refraction peak using the following Scherrer equation:



777

D = K λ / Β cosθ

Where ‘λ’ is the wave length of X-Ray (0.1541nm) K is a shape factor

(0.9 for Ag0), ‘β’ is FWHM (full width at half Maximum), ‘θ’ is the

diffraction angle and ‘D’ is particle diameter size. The calculated particle

size details are in Table (15), mean diameter size is 45nm in case of ex

situ radiation at 25kGy, size increased in compere with TEM and DLS

may be referred to agglomeration of Ag NPs and precipitation of

stabilizing agents after drying of Ag NPs colloids.

Fig (47): XRD patterns of synthesized Ag NPs by ex situ radiation

process.



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Table (15): XRD analysis of synthesized Ag NPs by ex situ radiation

at radiation dose 25kGy

2θof the intense

peak (deg)

hK1 FWHM of

Intense peak

(β) radians

(ex situ)

Diameter Size

nm

38.42 (111) 0.007 21

44.64 (200) 0.003 53.6

64.53 (220) 0.004 40.8

77.635 (311) 0.003 65

Mean diameter size 45

4.4.3. EDX analysis of synthesized Ag NPs by gamma radiation:

Energy dispersive x-ray spectroscopy (EDX) was used to confirm that the

observed granules in the solution indeed consisted of silver.

The results of EDX analysis in Figure (48) showed that an optical

absorption peak in a range from 3keV to 4keV, which is typical for the

absorption of metallic silver nanoparticles and referred to surface

Plasmon resonance (Kalimuthu et al., 2008)



777

Fig (48): EDX spectra of synthesized Ag NPs by gamma irradiation.

4.4.4. Effect of Time on synthesis and stability of Ag NPs colloids.

4.4.4.1. Synthesis by in situ irradiation process

Figure (49) shows the UV–Vis spectrum for the Ag NPs synthesized by

in situ irradiation process at 5kGy after only 30minutes of irradiation, Ag

NPs solution was diluted 50% with deionized water before UV-spectral

analysis. Strong Plasmon resonance at 430nm was observed after

30minutes of irradiation shows optical density (double dilute), even after

three months the peak remained close to 430nm with the same optical

density. This indicated that the nanoparticles were still dispersed and that

there were not much aggregation (Binupriya et al., 2010).Time

consumed to obtain maximum optical density (30minutes) is attributed to

the reduction process which was carried out by both hydrated electrons

and formed organic radicals.



777

Fig (49): UV-spectra of synthesized Ag NPs by in situ irradiation at

dose of 5kGy after 30miutes and 3months.

4.4.4.2. Synthesis of Ag NPs by ex situ radiation process.

Figure (50) shows the UV–Vis spectrum for the Ag NPs synthesized by

ex situ radiation process at 25kGy. Strong Plasmon resonance (440nm)

was observed after 72h of radiation shows optical density (2.2nm), even

after three month the peaks intensity was reduced and the peak became

broader figure(51). These changes are related to the silver particle growth

(increase size) and formation of different sized particles (Puis et al.,

2011). This may be referred to a decrease of stabilizing activity of

Monascus supernatant at high radiation dose so nanoparticles tend to

much aggregation (Binupriya et al., 2010). Time consumed to obtain

maximum optical density (72h) is attributed to the electrochemical

potential of the organic radical (secondary radical responsible for

reduction of Ag+ to Ag

0) is more positive than that of Ag

+, Ag

0, hydrated



777

electrons and hydrogen radical system. So the reaction of secondary

radicals formed with Ag+ ions is relatively slow (Henglein,1998).The

peak area and height of the UV–Vis spectrum obtained for the 25kGy in

ex situ irradiation was also comparatively higher than other radiation

doses, which confirmed the higher productivity of silver nanoparticles.

Fig (50): UV-spectra of synthesized Ag NPs by ex situ irradiation

process at dose 25kGy within a time range from 12hr to 72hr.



777

Fig (51): UV-spectra of synthesized Ag NPs by ex situ irradiation

process at dose 25kGy after 72h and 3months.

4.4.5. DPPH Free Radical Scavenging Activity of irradiated M.

purpureus supernatant, AgNO3 and Ag NPs

The study indicated decreasing of antioxidant activity of M. purpureus

supernatant by increasing of radiation dose; this is attributed to hydrated

electrons and organic radicals formed. AgNO3 (1mM) solution has

antioxidant activity about 14% that abolished by radiation table (16).



777

Table (16): DPPH Free Radical Scavenging Activity of M. purpureus

supernatant, AgNO3 and synthesized Ag NPs colloids by in situ and

ex situ radiation process

DPPH scavenging (%( Radiation Doses

Ag NPs ex

situ

Ag NPs

in situ

AgNO3

(1Mm)

M.purpureus

supernatant

------ ------ 14 88 0kGy

55 85 0 72 1kGy

38 77 0 60 5kGy

32 81 0 40 10kGy

31 84 0 35 15kGy

29 86 0 34 20kGy

16 89 0 30 25kgy

4.4.6. Comparison between DPPH scavenging activity and size of

synthesized Ag NPs by gamma irradiation

Table (17) shows the relationship between DPPH scavenging activity %

and size of Ag NPs formed by in situ and ex situ irradiation process, the

DPPH scavenging activity (%) increase with decreased of Ag NPs size.

This confirmed the antioxidant activity of Ag NPs.



770

Table (17): Relationship between DPPH scavenging activity and size

of synthesized Ag NPs by in situ and ex situ irradiation process

Radiation

Doses

DPPH scavenging (%) TEM average size(nm)

Ag NPs

in situ

Ag NPs

ex situ

Ag NPs

in situ

Ag NPs

ex situ

1kGy 85 55 ---- 24

5kGy 77 38 8.5 19.5

10kGy 81 32 18 12.5

15kGy 84 31 30 8.2

20kGy 86 29 51 8

25kgy 89 16 72 7.3

4.5. Conclusions

The preparation of silver nanoparticles was carried out by gamma

radiation under simple conditions, i.e., air atmosphere, using M.

purpureus supernatant as reducing agent and a stabilizer. The γ-ray doses

of 1kGy to 25kGy were sufficient to achieve maturely formed particles

depend on method of radiation process (in situ or ex situ process). The

obtained Nano particles were spherical with size depending on the

radiation dose and method of radiation (in case of in situ irradiation at

5kGy, the size is approximate 8.5nm and size increased with the

increasing of radiation dose up to 25kGy, size 72 nm. In case of ex situ

radiation the diameter of the particles decrease with radiation dose

increase and size is approximate 24nm at 1kGy and 7.5nm at 25kGy.



777

FTIR analysis has revealed that the interactions in Ag NPs, M. purpureus

supernatant molecules are the result of the interaction between Ag NPs

and nitrogen atoms.

The result of UV–Vis spectroscopy revealed that, the Ag NPs exhibited a

peak of the absorption band at wavelength of 430nm which confirmed the

presence of Ag NPs.

This study confirmed the antioxidant activity of Ag NPs, which increased

with decreasing of size particles.

English Summary

111

Summary

Monascus is an Ascomycota's fungus traditionally used for the

production of food colourants, fermented foods and beverages.Natural

colorants derived from polyketide compounds of Monascus purpureus

have been extensively used in pharmaceuticals and foods to replace the

synthetic colorants. Monascus purpureus fungus is well known for its

ability to produce pigments ranging from bright yellow to deep red in

color; Plackett-Burman experimental design was used to screen

important nutrient parameters influencing the production of pigments by

M. purpureus NRRL 1992 under submerged fermentation.

The fungal M. purpureus was grow under Eighty two different conditions

on submerged fermentation and their supernatants and biomass were

incubated with AgNO3 solution (1mM).

Three types pigments were extracted from M. purpureus supernatant, the

optical density (OD) was measured at 400, 470 and 500 nm (a

wavelength which represents the absorption maxima for yellow, orange

and red pigments, respectively) expressed in units of absorbance (λUA) at

a given wavelength (λ) ; the optimum conditions for pigment production

are a pH 5.5, 10 days inoculum age, 50/250ml medium /flask volume,

5g/l yeast extract, 20g/l starch, 15g/l glucose and the incubation period is

7days that shows optical density0.681UA at λ400nm, 0.962UA at λ

470nm and 2.542UA at λ 500nm; and it’s the best medium supernatant

produce silver nanoparticles; the M. purpureus biomass has no ability to

produce silver nanoparticles.


production of spherical shaped silver nanoparticles (Ag NPs) synthesized

and stabilized using M. purpureus supernatant. Aqueous solutions AgNO3

were treated with M. purpureus supernatant for the formation silver

English Summary

121

nanoparticles (Ag NPs).The Nano-metallic dispersions were

characterized by surface Plasmon absorbance measuring around 420nm

for Ag nanoparticles. Transmission electron microscopy (TEM) showed

the formation of nanoparticles in the range of 2–8 nm. Dynamic light

scattering (DSL) approved particle size; XRD analysis of the silver

confirmed the formation of metallic silver. Further analysis carried out by

Fourier Transform Infrared Spectroscopy (FTIR), data indicate abounding

of Ag nanoparticles with M. purpureus supernatant molecules and

provides evidence for the presence of proteins as possible biomolecules

responsible for the reduction and capping agent which helps in increasing

the stability of the synthesized silver nanoparticles. Monascus pigments

has important role for reduction of silver nanoparticles

Aqueous dispersion of highly stable silver nanoparticles were synthesized

using gamma radiation with M. purpureus supernatant molecules as

reducing and stabilizing agent. The size of the silver nanoparticles can be

turned by controlling the radiation dose and mixing method of AgNO3

solution with M. purpureus supernatant (radiation process in situ and ex

situ radiation).Dynamic light scattering (DLS) measurement and

Transmission electron microscope(TEM)of the synthesized nanoparticles

indicated that the size depend on dose and radiation process. The

radiation was carried out at doses from 1 to 25kGy; in case of in situ

radiation the best dose for high reduction of silver ions into nanoparticles

with smallest size is 5kGy at a dose rate 2.9kGy/h and 25kGy in case of

ex situ radiation process and when increase the radiation dose to 30kGy

the reduction of silver ions is decreased. The obtained Ag nanoparticle

dispersion was stable for over 3 months at room temperature.

Chapter V: References

121

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العربى الملخص

931

الملخص العربى

نتاج مكسبات لون الاطعمة المخمرة والمشروبات والملونات الموناسكس هو فطر يستخدم لإ

المستمدة من فطر الموناسكس لها خاصية مركبات متعددة الكيتيد والتى تستخدم على نطاق

غذية لتحل محل الملونات الصناعية .وفطر واسع فى مجال المستحضرات الصيدلانية والأ

صباغ مختلفة عن بعضها فى اللون هى أنتاج ثلاث إعلى هعرف عنة قدرت بربيورسالمونسكس

حمراء وبرتقالى وصفراء.

لتصميم التجارب لدراسة تاثير المواد الغذائية التى Blacket burmanستخدم نظام إ تم وقد

فى الوسط المحيط تحت التخمر بربيورس فطر الموناسكسصباغ بواسطة نتاج الأإتؤثر على

على اثنين بربيورسفطر الموناسكس يةنمتتم Blacket burmanالمغمور.وبناء على نتائج

ح عن الكتلة الحيوية بواسطة يرشالوثمانون تجربة تختلف كل واحدة عن الاخرى وقد تم فصل

وتم بربيورسح الموناسكس ية من رشصباغ الثلاثجهاز الطرد المركزى وقد تم فصل الأ

لصبغة لنانومتر 074لصبغة الصفراء ولنانومتر 044تقديرها بواسطة الكثافة الضوئية عند

صباغ هى لأعلى كثافة ضوئية لأن أوقد وجد .ءلصبغة الحمرالنانومتر 044البرتقالى و

الوسط نانومتر فى044عند 08000نانومتر 074عند 48960نانومتر و044عند 486.0

جرام 0 الخميرة مستخلص جرام و نشا 04جرام و00 جلوكوز جم/لتر الذى مكوناتة الغذائى

وفى فطرالملقحعمرال يامأ 04و بالنسبة لحجم القارورة( الغذائى مل )جحم الوسط04/004و

وكتلتة بربيورس ح الموناسكسييام للتحضين .وتم تحضين رشأ7و 080س الهيدروجينى الأ

. (ملى مول0 )الحيوية مع محلول نترات الفضة

لى جسيمات إيونات الفضة أختزال إعلى بربيورسح الموناسكس يثبتت التجارب قدرة رشأ

ح يستخدام رشإ تم يهن الكتلة الحيوية غير قادرة على ذلك وبناءا علأالفضة النانوية غير

ح يوبعد خلط محلول نترات الفضة مع رشجل تكوين جسيمات الفضة النانوية.أالموناسكس من

الموناسكس تم تمييز تكون جسيمات الفضة النانوية بواسطة قياس الامتصاص الموجى

ونىرلكتلإأظهر قياس المجهر أنانومتروالذى هو مميز لجسيمات الفضة النانوية وقد 004حول

(TEM) وافق ذلك تنانومترو .نانو متر الى 0من يتراوحن جسيمات الفضة النانوية لها حجم أ

الفضية كرستلاتتكوين XRDتحليل اثبت .و(DLS)وء قياس جهاز دينامكية تشتت الض

( الى وجود بروتين فى (FTIRتحليل فورية الطيفى باالاشعة تحت الحمراءاثبت و ية.النانو

ة معلقة فى المحلول ح الموناسكس والذى قد يكون مسؤل عن بقاء جسيمات الفضة النانوييرش

العربى الملخص

941

ختزال الفضة إلها دور هام فى عملية بربيورسن الصبغات التى تنتجها الموناسكس أ.وقد وجد

الى جسيمات نانوية .

ح الموناسكس وقد يشعة جاما على تخليق جسيمات الفضة النانوية بواسطة رشأثير أوتم دراسة ت

على جرعات التشعيع التى كانت تتراوح النانوية يعتمدن حجم جسيمات الفضة أثبتت التجارب أ

و جراى لكل ساعة وكذلك تعتمد على كيل089 كيلوجراى بمعدل جرعة00كيلو جراى الى 0بين

فضل أن أو بعد التشعيع .وقد وجد أح الموناسكس مع محلول نترات الفضة قبل يطريقة خلط رش

قل وقت بينما أقل حجم فى أعطت أكيلو جراى والتى 0جرعة تشعيع فى الخلط قبل التشعيع هى

يادة الجرعة الى زفضل جرعة فى حالة الخلط بعد التشعيع وعندما تم ألو جراى هى يك00

ن أفى حالة الخلط بعد التشعيع قل تكوين جسيمات الفضة النانوية. وقد وجد كيلو جرى04

ن أوقد وجد شهر.أ ةالمحلول النانوى المخلق بواسطة التشعيع ثابت كمعلق حتى بعد ثلاث

.ت للميكروباتداجسيمات الفضة النانوية لها قدرة كمض

شعة جاما على التخليق الميكروبى لجسيمات النانو المعدنيةأثير أت

رسالة مقدمة من

فرج مرزوق مرعى مسلم

(8002 سيوطأ) زهرجامعة الأ ,كلية الصيدلة ,بكالوريوس فى العلوم الصيدلانية

لحصول على درجةكمتطلب جزئى ل

الماجستير

البيوتكنولوجيا الصناعية الهندسة الوراثية والتكنولوجيا الحيوية قسم فى

(تخصص تخمرات)

قسم البيوتكنولوجيا الصناعية

ث الهندسة الوراثية والتكنولوجيا الحيويةبحو معهد

جامعــة المنوفيـة

مدينة السادات

8002

effect of gamma radiation on the microbial synthesis of

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