PRODUCTION AND DEVELOPMENT OF NUTRACEUTICAL

FROM Ganoderma lucidum BY SUBMERGE FERMENTATION

PROCESS

Thesis
Submitted in partial fulfillment of the requirements for the degree of

MASTER OF TECHNOLOGY
IN
FOOD TECHNOLOGY AND NUTRIGENOMICS

By

MD FARUQUE AHMAD

(SUPERVISOR) (CO-SUPERVISOR)

DR .BIBHU PRASAD PANDA DR. Z. R. AZAZ AHMAD AZAD

Assistant Professor Assistant Professor
F/o Pharmacy F/o F.E.I.S
Jamia Hamdard Jamia Hamdard

DEPARTMENT OF FOOD TECHNOLOGY

FACULTY OF ENGINEERING AND
INTERDISCIPLINARY SCIENCE
JAMIA HAMDARD
(HAMDARD UNIVERSITY)
NEW DELHI – 110062
2010
 DECLARATION

I hereby declare that the work entitled “Production and Development of Nutraceutical
from Ganoderma lucidum by Submerge Fermentation Process ” embodied in this
thesis was carried out by me during the academic session 2009-2010. in the Department
of Pharmaceutical Biotechnology, Faculty of Pharmacy, Jamia Hamdard University
,New Delhi, under supervision of Dr. .Bibhu Prasad Panda (Assistant Professor), Deptt.
of Pharmaceutical Biotechnology and Dr. Z. R. Azaz Ahmad Azad (Assistant Professor),
Deptt. of Food Technology .The content and source of information have been derived
from the existing literature and have been cited throughout the thesis at the appropriate
places.

It is further stated that no part of this dissertation has been submitted, either in part or
full for any other degree of Jamia Hamdard or any other University/ Institution.

Date:

Md. Faruque Ahmad

Deptt. of Food Technology
Faculty of Engineering and
Interdisciplinary Science
Jamia Hamdard (University)
New Delhi -11062
 ACKNOWLEDGEMENT
Foremost I like to thanks the Almighty Allah for his blessing in
the accomplishment of his work. All my success in my task can
come only from Him.

It gives me immense and fathomless pleasure to place on record my profound sense of appreciation
and indebtedness to my supervisor Dr. Bibhu Prasad Panda, Assistant Professor, Department of
Pharmaceutical Biotechnology who provided much needed constructive criticism, unending inspiration,
valuable and pains taking guidance, encouragement, untiring and constant help and above all her
positive attitude towards my abilities made the achievements of this goal, a challenging, enjoyable,
rewarding and stimulating experience.

I thankfully acknowledge to Dr. Z.R.Azaz Ahmad Azad, Assistant Professor, Department of

Food Technology for his guidance, supervision, providing resources and subjects, offering direction,
passing me many leads, discussing human factors, and helping me to set up the project in the first
place.

I deem it a proud privilege to express my heart-felt gratitude to Principal co-ordinator of Food

Technology Dr.Farhan Jaleess Ahmad , Associate Professor Department of pharmaceutics ,faculty of
pharmacy,for unending zeal and facile suggestions & for providing me all necessary facilities timely,
during the period of work.

I am grateful to Incharge Department of Food Technology Dr.Shakir Ali Professor Department of

biochemistry ,faculty of science ,Jamia Hamdard, New Delhi, for his support .

I thankfully acknowledge to Dr.Mohd Mujeeb (Assistant Professor ) and Dr.Aquil, (Assistant

Professor). his co-operation during my work and encouraged me to do the best.

I am grateful to the man with the mission who left us mourning forever (Late) Hkm. Abdul
Hameed Sb., a great visionary and founder of Jamia Hamdard, New Delhi, whose personality can
never vanish till we exist for providing us a congenital atmosphere and the necessary infrastructure.
 I could not have brought my work to a super-finish without the timely and precious guidance
extended to me by the staff of CIF, INSDOC, NML Library, and my faculty staff Mr, Naseem
and Waseem my heartfelt thanks to all of them.

I extend my warm thanks to my friends, Yunus,Imran,Kaify,Shoaib Akhlaq, Nazli,Jameel,

Nitin,Ilyas,Minhaj, Manower, Parisa,Sana, Afroz,Shahzad and myclassmates AmirAshraf
Anshul,Abhishek,Archana,Shamsh,Pranay,Santosh,Amit,Deepshikha, last but not the least for their
co-operation during my work and among whose company I spent my memorable two years.

My heartfelt thanks to my elder brother Mr.Fakhruddin Ali Ahmad who not only
encouraging me but gives me direction ,spirit and passion to do excellent honest work in every
moment of life, I can’t forget it till my death.

A very special thanks to Mr.Ejaz,and Mr.Ynus and Mr.Rizwan his co-operation during my
work and encouraged me to do the best.

I express my sincere thanks to seniors, Mr.Sarim,Mr.Israr,Mr.Tanveer,Mrs.Alkapuri,

Mr.Makhmur, Mr.Javed , and juniors Siraj, Mushariq,Noorulla,Sandeep,Jalal, Pankaj,Ashok,Rajeev
who helped me a lot in carrying out the work.

I don’t have words to express my emotional feelings towards my parents, Sisters and brothers
.Mr.Wasi, and Mr.Quamar and my niece and nephew Laiba,Shaqlain,Qousain, who showed their love
and affection on me, even though living miles apart from me and without whose blessing the aim could
never be achieved.
Lastly, I would like to thanks all those who in whatever degree contributed their bit in the
completion of this thesis
(Md Faruque Ahmad)
 Contents
Page No.

Preface
Acknowledgement
Abbereviation
List of tables
List of figures

1. Introduction and Objective 1-10

1.1 Mushroom 1
1.3 Nutraceutical 3
1.4. Effectiveness and Safety 6
1.5.1 Nutraceutical from Mushroom 7
1.5.2 Nutraceutical from Ganoderma 7
1.5.2 Nutraceutical from Ganoderma lucidum 8
1.6. Objective 10

2. Literature Review 11-40

2.1 Fermentation 11
2.2.1 Production of Ganoderic Acids 12
2.2.2 Analysis of Ganoderic Acids 17
2.2.3. Pharmacological Activity of Ganoderma lucidum 19
2.3 Nutraceuticals or Medicines 22
2.4 Ganoderma lucidum 30
2.5 Cultivation of Ganoderma lucidum 32
2.6 Phytochemicals 33
2.7 Medicinal Effects of Ganoderma lucidum 36
 2.8. Safety in the Different Age 38
2.9. Comparative Medicinal Value of Ganoderma lucidum 40
and Other Mushrooms

3. Materials and Methods 41-52

3.1 Microorganism 41
3.2 Equipments 41
3.3 Chemicals 42
3.4 Preparation of Sub Culture 43
3.5 Preparation of Seed Culture 43
3.6 Developmen of Elicitor 43
3.7 Optimization of Nutrients 44
3.8 Data Analysis of Plackett-Burman Design 46
3.9 Response Surface Methodology 47
3.10 Submerged Fermentation 50
3.11 Quantification of Biomass 51
3.12 Extraction of Ganoderic acid 51
3.13 Estimation of Ganoderic Acid 51
3.14 Extraction of Ganoderic Acids from Mycelium 51
Powder of Ganoderma lucidum
3.15 Estimation of Ganoderic Acids 52

4. Results and Discussion 53-88

4.1 Regression Analysis with Standard Plot of Ganoderic Acids 53

4.2 Plackett-Burman Design for Production of Ganoderic Acids 55
4.3 Concentration of Ganoderic Acids B and C2 56
4.4 Response Surface Methodology for Optimization of Ganoderic
Acids B 62
4.5 Response Surface Methodology for Optimization of Ganoderic
Acid C2 71
 4.6 Illustration 80
4.7 Discussion 86

5. Conclusion 89
6. Future Prospect 90-91
7. Bibliography 92-100
Appendices 101-111
 List of Tables

Table Tittle Page No.

Table 2.1 Comparative Medicinal Value of Ganoderma Lucidum 40

Other Mushrooms
Table 3.1 List of Equipments 41
Table 3.2 List of Chemicals 42
Table 3.3 Plackett-Burman Experimental Design of 12 Trials for Eleven
Variables 44
Table 3.4 Concentrations of Variables at Different Levels in Plackett- 45
Burman Design (Pbd) for Ganoderic Acids Production in
Submerged Culture
Table 3.5 Levels of Nutrient Parameters Used in Experiment 47
Table 3.6 Box-Behnken’s Response Surface Design for Three Parameter
for c2 48
Table 3.7 Levels of Nutrient Parameters Used in Experiment for C2 49
Table 3.8 Box-Behnken’s Response Surface Design for Three Parameters 49
.Table 4.1 Observed Biomass Production by Fermented Broth 55
Table 4.2 Plackett-Burman Experimental Design of 12 Trials for Eleven 58
Variables (9 nutrients + 2 dummy) Along with Observed
Concentration of Ganoderic Acid A and C2 in Fermented Broth
Table 4.3 Influence of Medium Variables on Ganoderic Acid A Production 59
Table 4.4 Influence of Medium Variables on Ganoderic Acid C2 Production 60
Table 4.5 Box-Banker Design with Result (Actual And Predicted) 62
Table 4.6 Analysis of Variance Table 64
Table 4.7 Statistical Data of Response Surface Model 65
Table 4.8 coefficient Estimation Data 65
Table 4.9 ANOVA Summary of Calculated Model For Ganoderic Acid B
production 66
Table 4.10 Influence of Medium Variables on Ganoderic Acid C2
Production 71
Table 4.10 Box-Behnken Design with Result C2 (Actual and Predicted) 73
Table 4.11 ANOVA for Model Terms of Response Surface Quadratic Model 73
Table 4.12 Statistical Data of Response Surface Model of C2 74
Table 4.13 Coefficient Estimation Data 74
Table 4.14 ANOVA Summary of Calculated Model for Ganoderic Acid
Production 75
 List of Figures
Page No
Fig 1.1 Nutraceutical was Coined from Nutrition and Pharmacuetical 4
Fig 2.1 Nutracuetical Inhabit Dark Area Beween Food and Drug 22
Fig 4.1 Calibration Curve In Mobile Phase of Methanol ,Water and
Trifluoroacetic Acid. 53
Fig 4.2 Chromatogram of Ganoderic acid B in Mobile Phase of
Methanol Water and Trifluoroacetic Acid. 53
Fig 4.3 Calibration Curve in Mobile Phase of Methanol ,Water and
Trifluoroacetic Acid 54
Fig 4.4 Chromatogram of Ganoderic acid C2 (standard) in Mobile Phase
of Methanol,Water and Trifluoroacetic Acid 54
Fig 4.5 Observed Biomass Production by Fermented Broth 56
Fig 4.6 Chromatogram of Ganoderic Acid B from Fermented Sample in
Mobile Phase 57
Fig 4.7 Chromatogram of Ganoderic Acid C2 from Fermented Sample in
Mobile Phase of Methanol ,Water and Trifluoroacetic Acid of
Methanol ,Water and Trifluoroacetic Acid 57
Fig 4.8 Graph Showing Percent Contribution and Effect of Factors Used
in Plackett- Burman Design. 60
Fig 4.9 Graph Showing Percent Contribution and Effect of Factors Used in
Plackett-Burman Design 61
Fig 4.10 The lines in Graph Represent Influence And Sensitivity of
Respective Factor Ganoderic Acid B Production 67
Figure 4.11 (a) and (b) Response Surface Graphs Showing Relatives
effect of Nacl and Urea on Ganoderic Acid B
Production Keeping Other Nutrients Constant 68
Fig 4.12 (a) and (b) Response Surface Graphs Showing Relative Effect of
Nacl and Soya Flour Ganoderic Acid B Production While
Keeping Other Constant. 69
Fig 4.13 (a) and (b) Response Surface Graphs Showing Relative Effect of
 Urea and Soya Flour on Ganoderic Acid B Production While
Keeping Other Nutrients Constant. 70
Fig 4.14 The lines in Graph Represent Influence and Sensitivity of
Respective Factor Ganoderic Acid C2 Production 76
Fig 4.15 (a) and (b) Response Surface Graphs Showing Relative Effect of
MnSO4.H2O and Maltose on Ganoderic Acid Production While
Other Nutrients Constant. 77
Fig 4.16 (a) and (b) Response Surface Graphs Showing Relative Effect of
MnSO4.H2O and NaCl on Ganoderic Acid C2 Production While
Keeping Other Nutrients Constant. 78
Fig 4.17 (a) and (b) Response Surface Graphs Showing Relative Effect of
Maltose And Nacl On Ganoderic Acid C2 Production While
Keeping Other Nutrients Constant. 79
Fig 4.18 Subculture of Penicillum citrinum 80
Fig 4.19 Subculture of Ganoderma lucidum 80
Fig 4.20 Subculture of Penicillum citrinum and Ganoderma
lucidum 81
Fig 4.21 and 4.22 Cmplex Media Used for Submerged Fermentation 82
Fig 4.23 Fermented Product Obtained by Submerged
Fermentation Process (Plackett – Burman Design) 83
Figure 4,24 and 4.25 Fermented Product Obtained by Submerged Fermentation
Process (PBD) 84
Figure 4,26 and 4.27 Fermented Product Obtained by Submerged Fermentation
Process(RSM) 85
Preface
Application of biotechnology in the field of food sciences rejuvenated this field in

the form of ‘Nutraceuticals’. This field has tremendous potential for research; important

is exploration of things correctly.

In 1964 Jackson S. Foster once said, “Never underestimate the power of the

microbes”. Microorganisms play vital role in preparation of food biotechnology afood

biotechnology afood biotechnology and nutraceutical active compounds. Commencement

of manufacturing these compounds starts with the help of fermentation technology. An

optimally balanced culture medium is necessary for maximal production of product

through fermentation processes. This gives greater emphasis on medium designing for

fermentation processes.

In the present study attempt has been made for optimization of nutrient medium

for the production of ganoderic acids by Ganoderma lucidum under submerged

fermentation. According to my knowledge this study has been carried out first time, no

any report was found regarding this work.

The thesis comprises seven chapters: Chapter 1 Introduction and objective,

Chapter 2 Literature Review, Chapter 3 Materials and Methods, Chapter 4 Results, and

Discussion Chapter 5 Conclusion Chapter 6 Future Prospects Chapter 7 References.

All the obtained results and things required for repetition of work have been duly

reported.

MD FARUQUE AHMAD