Production of Astaxanthin from Cultured Green Alga Haematococcus pluvialis and its Biological

342 Pages·2011·9.26 MB·English

Production of Astaxanthin from Cultured
Green Alga Haematococcus pluvialis and its
Biological Activities

A Thesis submitted to the
Department of Biotechnology of
University of Mysore
In fulfillment of the requirement for the degree of

Doctor of Philosophy
in
BIOTECHNOLOGY

By
Mr. Ranga Rao, A.,

M. Sc., PGDIT

Under the supervision of
Dr. G. A. RAVISHANKAR

PLANT CELL BIOTECHNOLOGY DEPARTMENT
CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE
(A constituent Laboratory of Council of Scientific & Industrial Research)
MYSORE - 570020, INDIA
January, 2011
DEDICATED TO,
MY DEAR FAMILY, TEACHERS &
BELOVED WIFE
Mr. RANGA RAO, A., M.Sc., PGDIT
ICMR-Senior Research Fellow
Plant Cell Biotechnology Department
Central Food Technological Research Institute
Mysore – 570020, Karnataka, INDIA

CERTIFICATE
I Mr. Ranga Rao, A., certify that this thesis is the result of research work done
by me under the supervision of Dr. G. A. Ravishankar, Scientist-G & Head at
Plant Cell Biotechnology Department, Central Food Technological Research
Institute, Mysore. I am submitting this thesis of possible award of Doctor of
Philosophy degree (Ph.D.) in Biotechnology of the University of Mysore.
I further certify that this thesis not been submitted by me for award of any
other degree/diploma of this or any other University.

Signature of Doctoral candidate
Signed by me on date:

Signature of Guide Date:

Date: Counter signed by

Signature of Chairperson/Head of Department/
Institution with name and official seal.

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Dr. G. A. RAVISHANKAR
Ph.D., FNAAS, FNASc, FAFST, FBS, FISAB, FAMI. FIAFoST, FIFST (UK)
Scientist –G (Deputy Director) & Head
Plant Cell Biotechnology Department

CERTIFICATE
This is to certify that the thesis entitled “Production of Astaxanthin from Cultured
Green Alga Haematococcus pluvialis and Its Biological Activities” submitted by
Mr. Ranga Rao, A., to the University of Mysore for the award of the degree of
Doctor of Philosophy in Biotechnology, is the result of work carried out by him in
Plant Cell Biotechnology Department, Central Food Technological Research
Institute, Mysore under my guidance during the period October 2007 to October 2010.

Date: G. A. RAVISHANKAR
Place: Mysore-570020 (Research Supervisor)

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ABSTRACT

Micro algal biotechnology has gained importance due to its potential to produce
bioactive molecules. Among the micro algae, Haematococcus pluvialis is a green
alga belonging to chlorophyceae, which produces astaxanthin & its esters in large
quantities under stress conditions. Carotenoids are being intensively investigated
regarding their potential to prevent diseases and vitamin A deficiency. The present
study is focused on the production of astaxanthin from Haematococcus pluvialis and
its biological activities. This thesis consists of effect of various stress conditions on
the production of astaxanthin in Haemtococcus pluvialis; isolation and
characterization of astaxanthin & its esters; evaluation of stability of astaxanthin in
various edible oils; safety evaluation of biomass; evaluation of its biological activity;
bioavailability and vitamin A conversion; anticancer properties in rat experimental
models.
Culture of Haematococcus was done using appropriate media & incubation
conditions in laboratory. In sodium nitrate (25 mM) treated culture, the maximum
biomass yield was 3.3 g/l, total carotenoid content was 2.9% and astaxanthin content
was 2.5%, whereas in a potassium chloride 16 mM supplemented culture the biomass
yield was 2.5 g/l, total carotenoid content was 2% and astaxanthin was 1.87%. Among
various carbon source studied, ammonium carbonate (3 mM) treated culture showed
the biomass yield of 2.9 g/l, total carotenoid was 2.6% and astaxanthin was 2.2%.
Among the various solvents used, ethylacetate, isopropyl alcohol: hexane (1:1) and
acetone were found to be efficient for extraction of the carotenoids from the
Haematococcus cells.
Carotenoids, astaxanthin & its esters were quantified and identified by
following techniques such as thin layer chromatography, high performance liquid
chromatography and liquid chromatography mass spectrum using APCI mode. The
major carotenoids were quantified in Haematococcus biomass and identified as
astaxanthin & its esters followed by neoxanthin, violaxanthin, astaxanthin, lutein,
zeaxanthin, β-cryptoxanthin and β-carotene. These were confirmed by their retention
times and the absorption spectra of the respective reference standards. Based on the
mass spectral data obtained from an astaxanthin mono-di esters such as ME C , ME
16:0
C , ME C , ME C , ME C , ME C , ME C , ME C DE C /C , DE
17:2 17:1 17:0 18:4 18:3 18:2 18:1, 16:0 16:0
C /C , DE C /C , DE C /C and DE C /C were identified in H.
16:0 18:2 18:1 18:3 18:1 18:2 18:1 18:1
pluvialis. Edible oils were also used as a vehicle of carotenoid delivery. To achieve
maximum extractability various oils were tried. The maximum extractability of
carotenoids was found in palm oil and olive oil. Astaxanthin & its esters were further
confirmed by recording unambiguous 13C NMR, 1H NMR, HSQC 2D, NOESY 2D
and COSY 2D NMR. Further, COSY and NOSEY spectrum confirmed the existence
of both cis and trans forms of astaxanthin & its esters in H. pluvialis.
The Haematococcus extracts exhibited 80% antioxidant activity in -carotene
linoleate model system (-CLAMS), 1, 1-diphenyl -2-picrylhydrazyl (DPPH) method,
and hydroxyl radical scavenging model systems. Antibacterial properties of H.
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pluvialis extracts were evaluated against selected bacteria. Among the different
solvent extracts of H. pluvialis, chloroform extract exhibited highest antibacterial
effect followed by ethyl acetate extract. Astaxanthin & its esters also showed
significant antioxidant activity and hepatoprotective ability in carbon tetrachloride
induced albino rats. Among the groups of experimental rats, the one which was
treated with astaxanthin esters at 250 µg/kg b.w. showed maximum hepatoprotective
activity i.e protection when compared to control treated group. However,
pretreatment of rats with 250 µg/kg b.w of astaxanthin esters preserved catalase,
peroxidase and SOD activities, when compared with control values in untreated
animals.
Feeding trials, for experimental rats were carried out to assess the safety and
efficacy of Haematococcus biomass as a source of astaxanthin. The study revealed
that Haematococcus biomass was safe in both single and repeated dose in
experimental rats. In vivo experiments demonstrated that astaxanthin from
Haematococcus was effective in retinol formation and its accumulation in serum and
liver. Time course study of carotenoids in rats after administration of Haemtococcus
biomass showed peak levels in plasma, liver and eyes at 2, 4 & 6 h respectively. In
the repeated dose study, the astaxanthin levels in plasma, liver and eyes of rat over 15
days after intubation of H. pluvialis biomass was recorded. In liver, astaxanthin levels
were 1.7 and 1.8 fold higher than in plasma and eyes. In plasma and liver, antioxidant
enzymes catalase, SOD, peroxidase activity was higher in astaxanthin treated rats
when compared to untreated control rats.
The anticancer properties of astaxanthin & its esters was also studied in human
glioma cell lines (LN-229, HNGC2) and liver hepatocellular carcinoma cell line
(HepG2). The apoptosis cells were observed under phase-contrast and confocal
microscope. Anticancer properties of astaxanthin & its esters were evaluated against
skin carcinogenesis, which is reported for the first time. Tyrosinase enzyme activity
was inhibited by using astaxanthin & its esters using in vitro models. Pre feeding of
rats with astaxanthin esters 250 μg/kg b.w. prior to treatment with carcinogen showed
3-4 fold reduction in tumor index. Hematological and histopathological studies were
examined which substantiate the protective role of astaxanthin esters. The plasma
astaxanthin and retinol content were estimated in serum and liver homogenate.
Biochemical changes like lipid peroxidation, catalase, superoxide dismutase,
glutathione reductase activities were estimated in control, cancer induced animal
groups. Astaxanthin metabolites isolated from plasma, liver from in vivo studies were
characterized by HPLC and LC-MS (APCI) techniques to elucidate their structure.
Epoxy carotenoids were tentatively identified in liver and plasma. This study
emphasizes the influence of astaxanthin and its beneficial effects on the metabolism in
experimental animals. Based on animal experiments and bioavailability studies, it was
evident that astaxanthin & its esters could possibly be safely used as nutritional,
antioxidant, anticancer agent.

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ACKNOWLEDGEMENT

I would like to express my profound gratitude and sincere thanks to my mentor
and guide, Dr. Gokare Aswathnarayana Ravishankar, Scientist-G & Head, Plant
Cell Biotechnology Department, CFTRI, for suggesting the research problem,
inspiring guidance, and constant encouragement throughout the course of
investigation. His ever encouraging and highly positive approach has influenced
me a lot and I am truly indebted to him for his helpful attitude and for the
freedom to pursue my specific goals in his laboratory.
I wish to express my heartfelt thanks to Dr. V. Prakash, Director, CFTRI,
Mysore, for giving me the opportunity to utilize the excellent facilities available
at CFTRI.
I am ever grateful to Dr. Ravi Sarada, Senior Scientist, Plant Cell Biotechnology
Department, who gave me the cultures to start my research work and her constant
encouragement, support and helping hand during the pursuit of my research work
by way of scientific discussions and I am also thankful to Dr. B. Panduranga
Narasimha Rao, Deputy Director, IGNOU, New Delhi.
I would like to extended my gratitude to the members of my doctoral committee
Dr. B. R. Lokesh, Dr. K. K. Bhat, Dr. H. P. Ramesh, Dr. T. R. Shamala, Dr. V.
Baskaran, Dr. Muralidhara, for their constructive criticism and valuable
suggestions which helped me make this thesis look better.
I thanks to Dr. K. Udaya Sankar, Senior Scientist, Food Engineering Department
for his help in NMR interpretation of the data and structural elucidation of
astaxanthin metabolites.
I would like to express my heartfelt thanks to Dr. Shylaja. M. Darmesh, Dr. V.
Baskaran, Senior Scientists, Biochemistry & Nutrition Department for their
excellent guidance on bioavailability and bioactivity studies.
My sincere thanks to Dr. Anjali Shiras, Senior Scientist and Dr. E. Maheswara
Reddy, National Centre for Cell Science, Pune for their constant help,
suggestions and instrument facility during cell culture studies.
Thanks to Dr. S. M. Aradhya, Senior Scientist and Mr. A. Harshavardhan
Reddy, CSIR-SRF for their help in antibacterial activity studies.
I thank Dr. P. Srinivas, Senior Scientist, Head PPSFT and Mr. J.R. Manjunatha,
ICMR-SRF, PPSFT Department for their help during NMR experiments.
I thanks to Dr. P. Vijayananda & family, Senior Scientist, Fruit & Vegetable
Technology department for their support and encouragement during my research
investigation.
My special thanks to Dr. C. V. Subrahmanyam, Associate Professor, R.V.R & J C
College, Guntur and also Mr. Ch. Sambasiva Rao, Head, Oils & Fats
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Department, V.R.S & Y.R.N College, Chirala, Andhrapradesh for their co-
operation and encouragement during my research work.
My heartfelt thanks to the staff of PCBT Dr. T. Rajasekaran, Dr. M.S. Narayan,
Dr. Bhagyalakshmi Neelwarne, Dr. P. Giridhar, Dr. Nandini Prasad Shetty,
Mrs. Karuna, Mr. Srinivas Yella and Mr. Shivanna who have always been so
helpful.
I am grateful to all the scientists of CFTRI who have directly or indirectly helped
me whenever required.
I acknowledge the timely help and co-operation of staff of supporting
departments, B&N, HRD, CIFS, FPT, FE Pilot plant, FOSTIS, I&P, Animal
house, Health centre, Computer center, Stores & purchase and all the
administrative departments at CFTRI during this investigation.
My special thanks to Mr. V. Lokesh, CSIR-SRF for his kind support.
All my dear Friends, Seniors, Juniors and Colleagues in the PCBT department
and other departments who have been kind enough to extend a helping hand and
support me at all times. Therefore I place on record my heartfelt thanks to all
these people who made my stay at CFTRI a memorable one.
Many thanks to my Guruji`s Shri Pamulapati Ankineyudu, Ponnur, and also
Late Shri. G. Subba Rao & family, Chirala, for their astrology and suggestions
during my career.
The delicious breakfast at the Canteen cannot go unmentioned, for it made my
life much simpler, sparing me from that extra time in the kitchen.
To all others, who had helped me directly or indirectly wherever they are goes my
thanks and with them the assurance that their assistance will not be forgotten.
My deepest sense of gratitude goes to my family. Thanks to my parents Mr. A.
Venkateswarlu & Mrs. A. Tulasidevi, Brothers, Sisters, Uncle, Aunt, Brother-
in-law and Sister-in-law whose support, sacrifice and patience were instrumental
in accomplishing this task.
My genuine and very special thanks to my dear wife Mrs. G. Deepika for her
unflagging love and care during the preparation of this thesis.
My special thanks to Dr. (Mrs) Sandhya Diwakar, Deputy Director General,
ICMR who helps me every time to release my fellowship grants during the
fellowship period.
The award of Senior Research Fellowship by Indian Council of Medical Research
(ICMR), Government of India, New Delhi is gratefully acknowledged
Last but not the least, thanks to God for all that I am today and for the test of life
which have made me emerge much stronger and more confident.

(Ranga Rao, A.,)
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TABLE OF CONTENTS
Sl. No. Details Page No
1.0. General introduction of algae 1
1.1 Microalgae as source of food 1
1.2 Microalgae as source of pharmaceuticals 2
1.3 Microalgae as source of high value metabolites 3
1.3.1 Pigments and carotenoids 3
1.3.2 Stable isotope biochemicals 5
1.3.3 Antioxidants 6
1.3.4 Polyunsaturated fatty acids (PUFA) 7
1.3.5 Lipids, oils, sterols and fatty acids 8
1.3.6 Protein & amino acids 9
1.3.7 Minerals and vitamins 9
1.3.8 Nutraceuticals 9
1.4 Bioactive compounds from microalgae 10
1.5 Pigments 14
1.5.1 Natural colors 14
1.5.2 Carotenoids 15
1.5.3 Carotenoids chemistry 17
1.5.4 Carotenoids as natural food colorants 19
1.5.5 Carotenoids pharmacology 20
1.5.6 Carotenoids absorption 20
1.5.7 Carotenoids health benefits 22
1.5.8 Carotenoids metabolism 23
1.5.9 Carotenoids storage 23
1.5.10 Transport and tissue distribution of carotenoids 24
1.5.11 Carotenoids biological activity 25
1.5.11.1 Carotenoids bioavailability 25
1.5.11.2 Methods to determine bioavailability of carotenoids 26
1.5.11.3 Plasma/serum response after caroteniods ingestion 26
1.5.11.4 Safety and toxicity 27
1.6. Haematococcus species 27
1.6.1 Taxonomic classification 27
1.6.2 Introduction 28
1.6.3 Origin and occurance of astaxanthin 28
1.6.4 Chemistry of astaxanthin 29
1.6.5 Production of astaxanthin 32
1.7 Applications of astaxanthin 33
1.7.1 Toxicity and human clinical trials of astaxanthin 33
1.7.2 Bioavailability of astaxanthin 33
1.7.3 Human clinical studies of astaxanthin 34
1.7.4 Astaxanthin as anti chronic and acute agent 35
1.7.5 Astaxanthin as source of medical and nutraceutical applications 36
1.7.6 Astaxanthin conversion into retinol 37
1.7.7 Astaxanthin as general biological antioxidant 40
1.7.8 Astaxanthin prevention for neurodegenerative disease 40
1.7.9 Astaxanthin protect from UV light 41
1.7.10 Astaxanthin for prevention of cancer 41
1.7.11 Astaxanthin support for the immune system 42
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1.7.12 Astaxanthin for treatment of infections 43
1.7.13 Astaxanthin for prevention of arteriosclerosis and related disease 43
1.7.14 Astaxanthin for prevention of inflammation 43
1.7.15 Astaxanthin prevention cardiovascular disease 44
1.7.16 Astaxanthin as antiskin carcinogensis agent 45
1.7.17 Astaxanthin as anti age macular degeneration (AMD) agent 45
1.7.18 Astaxanthin in cellular health 46
1.7.19 Astaxanthin prevention of ulcers, gastric injury, stomach cancer 47
1.7.20 Can astaxanthin help in diabetics? 47
1.7.21 Antihypertensive effects of astaxanthin 48
1.7.22 Astaxanthin for salmon and trout feeds 50
1.8 Growth and carotenogensis of microalgae 51
1.8.1 Current market status of astaxanthin 51
1.8.2 Astaxanthin as nutraceutical 53
1.9 Objectives of the present study 54
Chapter-1: Production of astaxanthin from various culture conditions in H. pluvialis
2.0. Background 55
2.1 Materials and methods 56
2.1.1 H. pluvialis 56
2.1.2 Glasswares 56
2.1.3 Stock culture maintenance 56
2.1.4 Normal growth condition 57
2.1.5 Growth in carbon source enriched condition 58
2.1.6 Growth measurement 58
2.1.6.1 Dry weight 58
2.1.6.2 Chlorophyll content 58
2.1.6.3 Total carotenoid and astaxanthin content 58
2.2 Influence of different stress conditions (carbon, salt, nitrogen) on 59
the production of astaxanthin
2.2.1 Effect of different nitrogen concentration on growth and astaxanthin 59
production
2.2.2 Effect of different carbon source on growth and astaxanthin 59
production
2.2.3 Effect of different salt concentration on growth and astaxanthin 60
production
2.3 Downstream processing of carotenoids from H. pluvialis 60
2.3.1 Haematococcus biomass extraction in different solvents 60
2.3.2 Haematococcus extraction in different edible oils 60
2.4 Results and discussion 61
2.4.1 Maintenance of H. pluvialis stock culture 61
2.5 Influence of different nutrient conditions (nitrogen, carbon, salt) on 61
the production of biomass, total carotenoid and astaxanthin
2.5.1 Influence of nitrogen source on growth of H. pluvialis culture 61
2.5.2 Effect of different nitrogen sources on biomass yield, total 62
carotenoid and astaxanthin production in H. pluvialis
2.5.3 Effect of nitrogen sources on carotenoid composition by HPLC 63
2.6. Influence of carbon sources on growth of H. pluvialis culture 64
2.6.1 Effect of carbon source on biomass yield and carotenoid 64
composition in H. pluvialis
2.7. Effect of salt stress on growth of H. pluvialis culture 66
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