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“Bioactive Glucan and Melanin from Marine Yeasts: Characterization and Evaluation


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by LSY WILS Reg. No: 418



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To the Lord Almighty...


Professor Cochin University of Science and Technology Fine Arts Avenue, Kochi-16


This is to certify that the thesis entitled “Bioactive Glucan and Melanin from Marine Yeasts: Characterization and Evaluation” is an authentic record of the research work carried out by Ms. Wilsy Wilson under my supervision and guidance in the Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and Technology, in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology under the Faculty of Marine Sciences of Cochin University of Science and Technology, and no part thereof has been presented for the award of any other degree, diploma or associateship in any university. All the relevant corrections and modifications suggested by the audience during the pre-synopsis seminar and recommended by the Doctoral committee have been incorporated in the thesis.

Dr. Rosamma Philip (Supervising Guide) Kochi-16

December 2016



I hereby declare that the thesis entitled “Bioactive Glucan and Melanin from Marine Yeasts: Characterization and Evaluation” is a genuine record

of research work done by me under the supervision and guidance of Dr. Rosamma Philip in the Department of Marine Biology, Microbiology and

Biochemistry, Cochin University of Science and Technology. The work presented in this thesis has not been submitted for any other degree or diploma earlier.

Wilsy Wilson Kochi-16

December 2016


It is generous su completion. M

I was Professor and express my de sincere gratit enough kind accessibility, appreciable.

student, rese During my c driving force been possibl

I gra of Marine Bi providing me

I wis coordinator, especially for express my h needed.

I am encourageme Dr. Aneykutty valuable advi

I than the departme of Marine Scie I tak Technology a and Senior R

my great pr upport durin My cherished s immensely b d Head of the eep sense of g tude and love dness, affecti

, coupled wi Mam, you ex earcher and s course of res e. Without yo

e. I am enoug atefully ackno

ology, Microb e all the help, sh to express National Cen r the shrimp heartfelt grati

very much th ent. I thank y Joseph, Dr. S ice, suggestion

nkfully ackno ent for their h ences, CUSAT ke this oppo

and the Univ Research Fello

ivilege to exp ng the cours

d ambition…

blessed with d e Department gratefulness to e towards you

on, motivatio th meticulou xceptionally i so on. You we search, at the

our support gh fortunate t

owledge Dr.

biology and B , support and s my deepest ntre for Aqua bioassays, th itude to the g

hankful to all Dr. Babu Ph S. Bijoy Nanda n and constan owledge all th help and assis T, for extendin ortunity to versity Grant


press sincere se of the pr

divine mercy w t as my resear o you mam. On u mam, for y on and bless us way of ap inspired a lot ere a real mod e dawn of ea and encoura to be called a C.K. Radhakr Biochemistry d encouragem gratitude and tic Animal he hroughout my genuine effor

the teachers hilip, Dr. A.V.

an, Dr. Sajeeva nt support.

he office staff, stance. I would ng the necessa

acknowledge Commission

e gratitude to resent thesis

who directed m rch guide. Th n this occasion your valuable

sings. Your p pproach to so t and enriche del to me and ach day you agement, this as your studen

rishnan, Profe y, for the eno ment.

d sincere than ealth for prov y work perio rt and timely

of the depart Saramma, D an T.P and Dr.

technical sta d like to than ary facilities.

e Cochin Un n for the fello

o each one o work for i

me to Dr. Ros e words are n n, I would like

guidance, en pleasant natu

olve a probl ed my overal d I learnt a lot were my ins s work woul


essor (Retd).

ough kindnes

nks to Dr. I.S.

viding all the od. Sir, I have help provide

tment for thei Dr. A. A. Moh

. Valsamma Jo

ff and non-tea k the library

niversity of owship awar

of you for the its successfu

samma Philip not enough to to express my ndless support ure, patience em is deeply ll growth as a t from you….

spiration and d never have

., Departmen ss offered and

. Bright Singh e lab facilities e no words to ed whenever

ir support and hamed Hatha oseph for thei

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from Arabian Sea and Bay of Bengal. The marine yeast isolates used for my study were isolated by Dr. Reema.

Words are not enough to express my sincere gratitude to Dr. Swapna P. Antony, for teaching me the basics of molecular biology and taking care of me as a student of you.

Your valuable suggestions, encouragement, timeless help throughout my research and thorough revision of manuscript is deeply appreciable. Thanks a lot for your invaluable help and friendship….

I am much obliged to Dr. Jayesh Puthumana for his heartfelt support and timeless help all the way through my research and always being a helping hand whenever I needed.

My immense thanks to Ms. Sruthy K.S, for the invaluable help received for the molecular works and also for her concern, encouragement and support during various stages of research.

I am much thankful to Ms. Divya T. Babu for the help and assistance received in setting the aquarium tanks and the help rendered on all occasions.

My whole hearted thanks to Ms. Aiswarya Nair, Ms. Manomi S and Mr. Jayanath G for their timely help and support.

I sincerely acknowledge the assistance received from Dr. Douglas Lawman for NMR analysis of glucan and melanin samples for the accomplishment of research.

Heartfelt thanks to Dr.Sudhir N S for the timely help in WSSV inoculum preparation for bioassays.

I would like to express my sincere thanks to my seniors, Ms. Chaithanya E.R, Ms. Jimly C. Jacob, Ms. Ramya K.D, Dr. Deepthi Augustine, Dr. Naveen Satyan, Dr. Afsal V.V and Mr. Anil Kumar P.R. for their help and support.

I cherish the friendship of my fellow colleague Ms. Neema Job, and a cheerful group of juniors, Ms. Archana K., Ms. Bhavya Kachiprath, Ms. Dhanya Kesavan, Mr. Solly Solomon, Ms. Sephy Rose and Ms. Anju M V., for your timely help, support and joyful time.

My special thanks to all fellow colleagues and research fellows of Marine Biology, especially, Ms. Aneesa, Ms. Emilda Rosmine, Ms. Jesmi Youssaf, Ms. Deborah Alexander, Ms Reshma S, Ms. Susan, Dr. Nifty John, Dr. Divya P.S, Dr. Neethu C.S, Dr. Mujeeb Rahman, Ms. Ally, Ms. Bini, Ms. Thasnim, Ms. Melji, Ms. Soumya Krishnankutty, Mr. Rithin, Mr. Jabir T, Mr. Ajin and too many…. for your friendliness, help and support. I also would like to thank the research scholars of NCAAH for their help during my research.


blessing that helped me in fulfilling this effort. I humbly offer you with prayers, my thankfulness and admiration.

I am so much grateful to my parents-in-law, Mr. C.V Jose and Mrs. Rosily Jose for their concern, prayers and encouragement. I express my sincere thanks and deep sense of gratitude to you for the genuine interest, initiative and effort taken to continue my studies. My special thanks to my sister-in-law, brother-in-law and their family for the concern and help whenever I needed.

I would like to thank my uncles, aunts, cousins and family friends for their encouragement, prayers, help and support.

The abundant prayers and immense blessings of my parents Mr. T.D Wilson and Mrs. Alphonsa Wilson was my strength throughout my life. Your invaluable sacrifice and hard work is behind all the achievements of my life. I take this opportunity to express my deepest gratitude and immense thanks to them. I also thank my sisters for their love, prayers and support.

Finally, I must tribute to my husband Mr. Munna Chirayath, who has always been my pillar of support, my joy, my strength. Thank you so much for your love, care and constant encouragement. My research endeavor would not have fulfilled without your support and sacrifice.... I deeply appreciate the immense affection and sacrifice of my son Akhil and my daughter Ann mariya who have adjusted well in my strain.

I thank all the experimental animals (Penaeus monodon) who sacrificed their lives for my research work.

The Lord is my strength and my shield (Psalm 28:7). I bow my head and surrender my life to the God Almighty for granting me the strength, health and wisdom to overcome the hardships of research and empowering me for the fulfillment of this endeavor. I do believe that with God all things are possible.

Thank you so much one and all…….

Wilsy Wilson


Chapter 1 General introduction --- 1

Chapter 2 Screening of marine yeast glucans as immunostimulant in P. monodon post larvae --- 17-78 2.1 Introduction --- 17

2.1.1 Shrimp aquaculture --- 18

2.1.2 Diseases in shrimp aquaculture --- 19

2.1.3 Crustacean immune system --- 21 Haemocytes --- 23 Cellular immune responses --- 23 Phagocytosis --- 23 Nodule formation --- 24 Encapsulation --- 24 Humoral immune responses --- 25 The clotting system --- 25 Prophenoloxidase system (proPO system) --- 26 Antimicrobial peptides --- 27 Reactive oxygen compounds --- 28 Pattern recognition proteins (PRPs) --- 28 Lectins/Agglutinins --- 29 Peroxinectin --- 30

2.1.4 Disease control strategies in shrimp aquaculture --- 30 Use of chemicals and antibiotics --- 31 Vaccination --- 31 Probiotics --- 31 Use of immunostimulants --- 32 Bacterial cell wall components as immunostimulant --- 33 Peptidoglycan --- 33 Lipopolysaccharides --- 33 Glucan as an immunostimulant in aquaculture --- 34


2.2 Materials and Methods --- 37

2.2.1 Microorganisms used for glucan extraction --- 37

2.2.2 Glucan extraction --- 38

2.2.3 Structural characterization of the cell wall glucans --- 40

2.2.4 Determination of biocompatibility --- 40

2.2.5 Preparation of glucan incorporated diet --- 41

2.2.6 Experimental animals and rearing conditions --- 42

2.2.7 Experimental design and WSSV challenge --- 42

2.2.8 Gene expression analysis --- 43 Total RNA extraction --- 43 Reverse transcription --- 44 Semi-quantitative RT-PCR analysis --- 44 Agarose gel electrophoresis --- 44 Expression analysis of target genes --- 45

2.2.9 Statistical analysis --- 45

2.3 Results --- 47

2.3.1 NMR spectra and Glucan yield of different marine yeast isolates --- 47

2.3.2 Biocompatibility of yeast glucans --- 51

2.3.3 Estimation of the immunostimulant potential of yeast glucans --- 51

2.3.4 Expression profile of control genes in P. monodon post larvae in response to various marine yeast glucan and WSSV challenge --- 53

2.3.5 Expression profile of AMP genes in P. monodon post larvae in response to various marine yeast glucan and WSSV challenge --- 55

2.3.6 Expression profile of immune genes in P. monodon post larvae in response to various marine yeast glucans and WSSV challenge --- 59


post larvae in response to various marine yeast

glucans on challenge with WSSV --- 63

2.4 Discussion --- 68

Chapter 3 Characterization and evaluation of selected glucans as immunostimulants in adult P. monodon against WSSV challenge --- 79-116 3.1 Introduction --- 79

3.2 Materials and Methods --- 85

3.2.1 Microorganisms used as source of glucans --- 85

3.2.2 Glucan extraction --- 86

3.2.3 Structural characterization of the cell wall glucan --- 86

3.2.4 Preparation of glucan incorporated feed --- 87

3.2.5 Experimental animals and rearing conditions --- 87

3.2.6 Virus inoculum preparation --- 88

3.2.7 Determination of WSSV titer --- 89

3.2.8 Experimental design and WSSV challenge --- 89

3.2.9 Hemolymph collection --- 90

3.2.10 Gene expression analysis --- 90 Total RNA isolation and reverse transcription --- 90 Quantitative Real Time PCR analysis of gene expression --- 91

3.2.11 Statistical analysis --- 91

3.3 Results --- 92

3.3.1 Glucans from marine yeast isolates --- 92

3.3.2 Comparison of the immunostimulatory efficacy of the yeast glucans --- 97

3.3.3 Expression of immune genes in haemocytes of P. monodon in response to marine yeast glucan and WSSV challenge --- 98

3.4 Discussion --- 105


from a marine black yeast, Hortaea werneckii --- 117-165

4.1 Introduction --- 118

4.1.1 Sources of melanin --- 120

4.1.2 Fungus as a source of melanin --- 122

4.1.3 Enzymes for microbial melanin synthesis --- 124

4.1.4 Types of melanin --- 126

4.1.5 Characterization of melanin --- 126

4.1.6 Functions of melanin --- 127 Melanin as a radioprotectant --- 127 Melanin as a factor of virulence --- 129 Melanin as antioxidants --- 129

4.2 Materials and Methods --- 132

4.2.1 Microorganism used for melanin production --- 132

4.2.2 Extraction of melanin --- 132

4.2.3 Characterization of black yeast melanin --- 133 Solubility --- 133 UV-VIS spectra of black yeast melanin --- 133 Nuclear Magnetic Resonance (NMR) Spectroscopy --- 133 Fourier Transform Infrared Spectroscopy (FTIR) --- 134 Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES) --- 134 Scanning Electron Microscope - Energy Dispersive Spectrometer analysis (SEM- EDS) --- 135 Elemental analysis of R23 melanin (CHNS analysis) --- 135

4.2.4 Assays for antioxidant activity --- 135 ABTS radical scavenging assay --- 136 DPPH radical scavenging assay --- 136


4.2.5 Biocompatibility and cytotoxicity of R23 melanin --- 135 2, 3-Bis [2-Methoxy -4-Nitro-5-Sulfophenyl]- 2H-Tetrazolium-5- Carboxanilide (XTT) assay --- 138 Sulforhodamine B (SRB) assay --- 139

4.2.6 Phototoxicity assay of R23 melanin --- 140

4.2.7 Statistical analysis --- 141

4.3 Results --- 141

4.3.1 Production and extraction of melanin from black yeast ---141

4.3.2 Physicochemical characterization of black yeast melanin ---142 Solubility of black yeast melanin --- 142 UV-VIS spectra of R23 melanin --- 142 Fourier Transform Infrared Spectroscopy (FTIR) --- 143 Nuclear Magnetic Resonance (NMR) Spectra of melanin --- 144 Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES) --- 145 Elemental analysis of melanin (CHNS analysis) --- 146 SEM-EDS analysis of melanin --- 146

4.3.3 Antioxidant activity --- 148 ABTS radical scavenging assay --- 148 DPPH radical scavenging assay --- 149 Lipid peroxidation inhibition assay --- 150

4.3.4 Biocompatibility and cytotoxicity of R23 melanin --- 151

4.3.5 Phototoxicity assay of R23 melanin --- 152

4.4 Discussion --- 154

Chapter 5 Evaluation of marine yeast glucan and melanin as anticancer agents by in vitro gene expression analysis --- 167-206 5.1 Introduction --- 167

5.1.1 Anticancer agents from plant sources --- 169


5.1.3 Anticancer agents from marine sources --- 172

5.1.4 Genes in cancer regulation --- 175

5.2 Materials and methods --- 180

5.2.1 Microorganisms used for the study --- 180

5.2.2 Preparation of marine yeast glucan compound --- 180

5.2.3 Preparation of marine yeast melanin compound --- 181

5.2.4 Cell lines and testing anticancer activity in vitro --- 182

5.2.5 Gene expression analysis --- 182 Total RNA isolation and reverse transcription --- 182 Real-time quantitative PCR analysis of target gene expression --- 184

5.3 Results --- 186

5.3.1 Effect of marine yeast β-D-glucan on the expression of cancer-related genes in lung cancer cells ---186

5.3.2 Effect of marine yeast melanin on the expression of cancer-related genes in lung cancer cells --- 189

5.4 Discussion --- 192

Chapter 6 Summary and Conclusion --- 207-212 References --- 213-263 Appendix I - GenBank Submissions --- 265

Appendix II - List of Publications --- 266-277


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isolated fr ed using sea s were isolat

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from diffe vertebrates, arine yeasts habitat whe are able to g meyer, 1979

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and YPD ation of mar 005; Masud ons, compon ch as chlo

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applied to ular biology

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methods ha

yeasts and ently marine sediments s etc. Yeasts ate solely in inated from environmen

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identificatio identificatio imparts mor strains (Rec

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on and ch on methods re powerful cek et al., 20 previous st nus or grou ch as Cand la, Saccharo ts (Bhat and

nho et al., 1 anho and S ishi et al., 2 yces and Rh

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presence of 963) from ff San Die dium pullula oast (Zalar e

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aracterizatio , genetic ch l means of s 002).

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2010; Galk hodotorula Debaryomyc

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marine yeas ndian Ocean

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f black yea en reported

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f traditiona r techniques tion amongs

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rine organis enormous n 4; Lordan e to explore t ing. The e e, salinity, p nisms comp es. The biom ogical poten

reported tha such as am ns with pot cal industri (Chi et al., 2 e terrestrial s been exten ce ancient tim

of alcohol tillation and ch is highly sible utiliza yeasts to pr otease, acid sively studi 002; Gupta 2007; Bhadr in food,

sms have number of b et al., 2011;

the bioactive extreme env pH and man

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tential appli ies as well 2009; Sarkar lly isolated nded to the m mes, yeasts

and carbon d baking ind substantial ation as feed

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considerabl biologically Chen et al.

e potential o vironmental ny other fact arine yeasts produced by

s use as cos yeasts are glucans, glu ication in th l as for m r et al., 2010

bioactive marine envi were used in

dioxide, w dustries. Yea

in aquacultu d for anima erent extrac ellulase, phy ziak, 1993;

3; Vakhlu an 008;). Most armaceutical

le importan y active com

, 2016). Ext of marine m l conditions

tors have be s to produ these organ smeceutical

able to pr utathione, to he food, ph marine cultu 0). Due to th

compounds ironment.

n many indu hich are im ast, also use ure owing t als or in aqu cellular enzy ytase, lipase,

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s such as een fortified uce potenti nisms have g

s and nutra roduce man oxins, enzym harmaceutica ure and en

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cell protein ional quality imilarly, the as amylase and inulinase

Buzzini and et al., 2006 ind potentia fermentation

g d e s n e e at It e e c al s, d

s g, n y e e, e d 6;

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industries. F as thiamin pyridoxine, one of the b been notice Ergosterol, important p main materi extracted fr et al., 2011) Sprenger et acts as an an most of its Pohanka, 20 Kille yeast strain Cryptococc Metschniko Zygosaccha producing k 2004; Oro, yeast strain Metschniko pathogenic expansum, vitro and in

Furthermore ne, pantothe choline, glu biomolecule ed in Debary

another be harmaceutic ial to produc om several ). Productio t al. (1999).

ntioxidant. T effect sugg 011; Rodrigu er toxins are ns which cus, Debary owia, Pichia aromyces a killer toxins 2014; Meh ns belonging owia pulch

fungi such P. digitatum n vivo wound

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of the ide and Yagiu, t al., 2014).

coccus albi d Aureoba tis sp., Asp nilia fructico

o, 2014). Ye

ources of a n vitamin B d p-amino be ny of the cell 2002; Reye with varying sor of vitam cortisone an 003; He et a t S. cerevisia lator of circ of melatonin

monal activ 2).

le compoun tural bioco ora, Hanse tilago, Toru ntified yea

1978; Staib Furthermo idus, Wicker asidium pu pergillus ca ola were al easts or its m

number of vi B6, and vi

enzoic acid.

lular functio es-Becerril e g functions min D2 and c

nd progestero al., 2007; G ae has been cadian rhyth have been r vity (Jacob e

nds produced ntrol agent enula, Kluy ulopsis, Wi ast genera b, 1999; Pfe re, antifung rhamomyces ullulans ag arbonarius, lso reported metabolites

itamins such itamin B12

Polyamines ons also have

et al., 2011) such as an cortisone, the one was also omez-Lopez n reported by hms and also reported and et al., 2002

d by severa t. Candida yveromyces illiopsis and

capable o feiffer et al.

gal effects o s anomalus gainst some

Penicillium d through in can be used h 2, s, e ).

n e o z y o d 2;

al a, s, d f ., f s, e m n d


as biocontro pre- and po

The bioactive d made up of The cell w extraction.

health indu modifiers (B of the imm infections.

antigenotox application β-Gl wall (~50-6 in animals.

The first r (Chihara et including an et al., 2005 the immun against pat inflammato Geastrum s vitro cytoto cancer cells extracted fr

ol agents an st-harvest d cell wall p due to the d

f glucans, m wall polysac

These poly ustries. The BRMs) due mune system Kogan et xic activitie in anticanc lucans are h 60 %) and a

A variety reported ma

al., 1970).

ntifungal, an ) and chole ity towards thogens tha ory, antioxid

saccatum m oxic study o s exhibited s rom various

nd it will be decay of man polysacchari

diverse func mannans, ma ccharides ca ysaccharides ese polysac e to their ab m against tu al. (2008) s of the yea cer preventio homopolysa are potent st of biologic ajor functio

Many other ntiinfection sterol reduc s various di at are alre dant and cyt mushroom ha

of β-glucan strong antic s mushroom

a valid alte ny fruits.

ides of yeas ctions/appli annoprotein an be separ

s have vario ccharides ar bility to trigg umor cells, i

reported th ast polysacc on/therapy.

accharides o timulators o cal functions

on of β-glu r biological

(Onderdon ction (Wolev

iseases (Ho eady active

totoxic activ ave also be

from Maita cancer effect ms exhibited

ernative to fu

st also have ications. In ns and mino rated from

ous applicat re known a ger a usuall inflammatio he antioxida charides and

of glucose, p of nonspecif

s of β-gluca ucans was activities h nk et al., 199 ver et al., 20 ofer and Po

in human vities of β-g en reported ake mushro t (Fullerton d a positive

fungicides in

e been dem yeast, the or compone each other tions in foo as biologic ly non-speci

ons, viral a ant, antimu d suggest th

present in th fic defense m

an have be its antitum have also be

92), radiopro 011). Yeast ospisil, 2011

ns and anim glucan-rich e d (Dore et a

oom in hum et al., 2000 impact aga

n controlling

onstrated as cell wall is ent of chitin

by alkaline od, feed and cal response ific reaction nd bacteria utagenic and

eir potentia

he yeast cel mechanisms en reported mor activity een reported

otective (Gu ts strengthen 1) and figh

mals. Anti extract from al., 2007). In man prostatic 0). β-glucans ainst a range g

s s n.

e d e n al d al

ll s d.

y d;

u n ht - m

n c s e


of cancers Due to its e can prevent screens, oil (1993) repo in vaccines In re have attract For instanc multiple b immunostim special atten threat by va increases t responses, b has been rep enhance d immunostim

Aqu various viru shellfish ag immunomo been studi immunostim 2011c). Va (THC), ph phosphatase

(Okamura e emulsion-sta t skin injurie s and gels ( orted that ye against furu ecent years,

ted much a ce, polysacc biological mulating, an

ntion in aqu arious patho the resistan

but by enha ported that disease resi


uaculture is uses and ba gainst bacter odulator (Ch ied by pre mulant (Saje arious imm henoloxidase

e activity an

et al., 1986 abilizing eff es caused by (Mason, 200 east glucan a unculosis in , bioactive p attention in charides or activities ntioxidant uaculture sec

ogens. Subs nce of the

ancing nons polysacchar istance in

facing sev acteria. Gluc rial or viral hang et al., 2

evious rese eevan et al., munological

e (PO) ac nd acid pho

6; Kimura e ffects and an

y solar radia 01; Michiko also showed n Atlantic sa

polysacchar the field of their glyco

including etc. Simila ctor as an im stances used

host, not specific defe rides from y shrimp, a

vere threat can enhance l infection a 2003). Marin

earchers an , 2009; Suk

parameters ctivity, res osphatase ac

et al., 1994;

ntioxidant a ation and th o and Yutak d an adjuvan almon (Salm

rides isolate f biochemis conjugates

anticarcino arly, polysa

mmunostim d for immun

by enhanc fense mecha

yeasts also c a process

due to the es the defen and thus act ne yeast der nd proved kumaran et a s such as t spiratory bu ctivity have

Kodama e activity, yea herefore are ka, 2007). R nt effect wh mo salar L.).

ed from natu stry and pha were show ogenic, an accharides h mulant agains nostimulatio cing specif anisms (Sak confer prote often refe

e continuou nse potential ts as immun rived glucan d its poten al., 2010; A

total haemo urst activit

been used t

et al., 2002) ast β-glucans

used in sun Rorstad et al hen included

ural sources armacology wn to exhibi nticoagulant have gained

st the severe on therefore fic memory kai, 1999). I

ection and/or erred to as

us attack o l of fish and nostimulant ns have also ntial as an Antony et al.

ocyte coun ty, alkaline to detect the ).

s - l.


s y.

it t, d e e y It r s

f d t/

o n ., nt e e


immunolog aquaculture significantly serious risk S169, Cand of the mar aquaculture Subramania of attentio chemothera In ad the yeast ce important r D-mannose glucose, gal supplement immune sy polysacchar gut health ( Huu and Jo growth of b also used as have been p such as imm and binding

In th not only du

gical effect e. Earlier

y enhance t k to aquacu dida tropica rine yeasts e (Sajeevan

an and Phili on as a apeutics in th

ddition to gl ell wall. Yea role in vari , but some lactose and s and provi ystem of th rides in feed

Ponton et al ones, 2014).

beneficial ba s adjuncts fo proposed to p munomodul g of toxins an

he same wa ue to the abil

of glucan studies rep the survival ulture indus alis S186, C used for et al., 2009;

ip, 2013). T valuable a he fight aga lucans; mann ast derived m ious biologi

etimes cont xylose. Man ide a range he animal,

d helps to m l., 2001; Gri

Mannan c acteria in the or fish and a promote ani lation, oxida nd pathogen ay, natural p lity to incre

n against port that g l of shrimps try. Debary C. haemulon the study

; Sukumaran he use of im alternate to ainst infectio

nans and ma mannans/ma

ical activity tain minor nnans have b of benefici growth and maintain a h

isdale-Hella can also fun e gut. These animal feed imal growth ative status, ns (Kurtzman

pigments ha ase the mar

bacterial o glucan inco s against W yomyces ha nii S27 and of glucan n et al., 201 mmunostimu

o the use ous diseases annoprotein annan-oligos y. These are

saccharide been added ial effects. I d survival.

healthy gut f and et al., 20 nction as pre e yeast cell w ds. In genera h and health b

, interaction n et al., 201 ave also bec rketability o

or viral in orporated f WSSV attack ansenii S8, C. sake S16 and its ap 0; Antony e ulants have e of antib s in aquacult ns represent ~

saccharides e composed e componen in many of It helps to Incorporatio flora and th 008; Refstie ebiotic by fa wall polysac al, these poly

by various m ns with gut


come a cho f products;

nfections in feeds could k, one of the D. hanseni 65 are some pplication in et al., 2011c

gained a lo biotics and ture sector.

~ 30-40% o (MOS) have d mainly o nts such as f animal feed improve the on of these hus promotes et al., 2010 avouring the ccharides are ysaccharides mechanisms


ice of study but also due n d e ii e n c;

ot d

f e

f s

d e e s 0;

e e s s, s

y e


to advantag Melanins a preparation for the prod industries.

Yea property; p point of vi yellow to b common pi the pigmen frequently a function ag and extrem an importa preparations 2009), free r effects (El- melanin.

Mela microorgan Pathogenic immune sys brasiliensis pathogenic produce me source of m

geous biolo are frequen ns. Earlier st

duction of v

sts are able igmented y iew. It prod bright orang igments pro nted yeasts b

associated w gainst damag e climate co ant candid s. Photoprot radical-scav -Obeid et a

anin is an isms. Melan yeasts pro stem. Crypt and Wang yeasts (da elanin and a melanin prod

ogical activ ntly used i tudies revea various pigm

e to produc yeasts are an

duces differ ge, red, pin duced by ye belong to a with the cell ges caused onditions. T date in ma tection (Par venging (Roz al., 2006) a

n ubiquitou nin producin duce melan tococcus neo giella derm

Silva et al., apart from te

ducing yeas

vities as an in medicine aled that, yea ments and it

ce varying h n interesting rent colours nk and dark easts compr ascomycota wall of yea by ultravio These protec any of th ramonov et zanowska et are some of

us pigment ng yeasts are nin as a pr

oformans, C atitidis are , 2009). Ter errestrial ha st and most

ntioxidants e, pharmac asts are also t may find p

hues of pig g group fro s of pigme k brown or

rise melanin and basidi asts, which c olet (UV) li ctive effects e pharmac al., 2002), a t al., 1999) a f the biolog

t found in e generally k rotective me Candida alb

some of t rrestrial fun abitat, marin t of them as

and antican cology, and o a promisin potential app

gments and om the biote ents ranging even black n and carote omycota. M can provide

ght, oxidan of melanin cology and antioxidatio and immuno gical effects

n plants, a known as ‘b easure again

icans, Para the melanin ngus has bee ne environm

ssociated w

ncer agents d cosmetics ng candidate plications in

due to this echnologica g from ligh k. The mos ene. Most o Melanins are

a protective nts, radiation n make them d cosmetics n (Tu et al.

omodulatory s of natura

animals and black yeasts’

nst the hos coccidioides n producing en shown to ment is also a ith high sal


s e n

s al ht st f e e n m s ., y al



st s g o a lt


concentratio triangularis halophilic f marine blac species in h Cimerman a Caro colours of f colour of th nutritional of function antioxidant have antic aquaculture their antiox stressors (C 2011). Rec AY-01 with (Yoo et al., been isolate Rhodotorula al., 2001).

capacity to biological a Cont is a relevan pathogens a

on. The asc s, Trimmat fungi that i ck yeasts, H hypersaline and Plemeni otenoid is

fruits, flowe his pigmen supplement s, especially ts. In vitro cancer prop e animal he xidant defen Chien et al., cently, caro h significant , 2016). Car ed and mo a, Rhodospo Former stud increase the activities suc

tinuous outb ant problem are various

comycetous tostroma s inhabit hyp H. wernecki waters on itas, 2006).

a pigment ers, birds, in nt make it a

ts. Apart fro y in relation cell cultur perties (Fr ealth, carot nse ability a

, 2003; Pan otenoid pigm

t antioxidant rotenoid pro ostly they b oridium, Spo dies have re e marketabi ch as antioxi breaks of dis m in aquacu viruses and

black yeast salinum an persaline wa

ii has been three contin

responsible nsects and m a colourant om this, car

n to human re experime raser and

enoids play and resistan n et al., 200 ment from t and antiba oducing yea belong to b

oridiobolus eported that lity of produ idant and ant

eases and th ulture. Main d bacteria.

ts, Hortaea nd Aureoba ater of sola identified a nents (Koge

e for most marine inve

in many o otenoids als

health and ents have s Bramley, y an impor nce to disea 03; Amar et yeast Rhod cterial activ asts from ma basidiomyce

and Sporob natural pigm ducts, but als

ticancer pro he constant u nstream dise

Bacteria su

werneckii, asidium pu ar salterns.

as the dom ej et al., 20

of the ye ertebrates. T of the foods so have exte their role a hown that 2004). Si rtant role in ases and env t al., 2004;

dotorula m vities have b arine enviro etous classe bolomyces (N

ments not on so exhibit a operties.

use of chemo ease causin uch as Vibri

Phaeotheca ullulans are Among the inant funga 004; Gunde

llow to red The pleasan

s, feeds and ensive array as biologica

carotenoids imilarly, in n enhancing vironmenta Yang et al.

mucilaginosa een reported onment have es, including Nagahama e

nly have the dvantageous

otherapeutics ng infectious io vulnificus a e e al -

d nt d y al s n g al , a d e g et e s

s s s


(Sung et al alginolyticu harveyi (Am (WSSV) (C Taura synd hematopoie myonecrosi causing age most alarm worldwide Different str have been Administrat been consid use of immu use of antib farming (Su The paved the w cancer. Eve hallmark is in increased through apo involved in cancer effec concern of Espinosa-A

l., 1994), V.

us (Wang an mparyup et Chang et al.

drome virus etic necrosi

is virus (IM ents in aquac ming shrimp shrimp cult rategies suc proposed fo tion of imm dered as a p unostimulan biotics and v ubasinghe, 1 e emergence way for the t en though m

the loss of b d cell repli optosis (Han n the apopto cts of extrac f many of t Aguirre, 2004

V. campbellii nd Chen, 20 al., 2012) a ., 2003; Saj (TSV) (So s virus (IH MNV) (Nune

culture. Amo pathogens, ture industry ch as the use

or the contr munostimula potential mo nts has gaine

vaccines in 997).

e of new bio treatment of many factors balance betw cation and nahan and W osis, an acti

cts from va the research 4; Park et

i, V. proteo 005), Aerom and viruses

jeevan et al ong et al., HHNV) (Y es, 2010) ar ong the dive , and it has y (Takahash e of probioti rol and prev ants especial ode of protec

ed lot of atte the fight ag

oactive com f many of the s contribute

ween cell pr the failure Weinberg, 20 ive form of arious natura

h study (Le al., 2006; B

lyticus (Ma monas veron such as wh l., 2009; Su

2003), infe Yeh et al.,

re some of t erse pathoge s caused sev hi et al., 199 ics, vaccines vention of d

lly glucan i ction agains ention as a v gainst infect

mpounds fro e life threate

to the deve roliferation

of damage 011). Netwo f cell suicid al sources h eone et al., Bachmeier e

arques et al nii (Lin et a ite spot syn ukumaran e ectious hypo

2009) and the problem ens, WSSV

vere devast 94; Wang e s and immun diseases in in shrimp cu st WSSV in valuable sub tious disease

om natural s ening diseas elopment of and cell dea ed cells to orks of gene de (Wong, 2 have been em

, 2003; Ab et al., 2008;

l., 2006), V al., 2011), V ndrome virus et al., 2010)

odermal and d infectious matic disease is one of the tation to the et al., 1995)

nostimulants aquaculture ultures have nfection. The bstitute to the es in shrimp

sources have ses including f cancer, one ath, resulting be removed es have been 2011). Anti merged as a bdullaev and Zikri et al.



s ), d s e e e ).

s e.

e e e p

e g e g d n - a d .,


2009; Leon various nat (Giovannuc Beca have been c compounds longa L., m signaling pa survival pa pathway (ca death recept pathway (JN extracts of t with signifi apoptosis in Recently, an exhibited st viz., MCF-7 and DU-145 Duri metabolites have been i Amador et become imp Rece culture extr

nardi et al., 2 tural source cci et al., 199

ause of the i considered a for their an modulates g

athways inc athway (Bc aspase-8, ca tor pathway NK, Akt, an the dark fun cant change n human pro

n in vitro stu trong antica 7, MDAMB 5 by MTT a ing the pas

with divers isolated from

al., 2003).

portant sourc ently Sekar racts of mari

2010; Kim es have also

95; Narisaw intricacy of as a tool for nticancer eff growth of tu cluding cell cl-2, Bcl-x, aspase-3, cas y (DR4, DR nd AMPK) (R ngus Ganode es in the exp ostate cancer udy of the re ancer potenti B231, T47D

assay (Athav st 20 years se biologica m various m

Subsequent ces of new b et al. (2015 ine yeast, C

et al., 2011 o been mad wa et al., 1996 this disease r the better u fects. Curcum umor cells t

proliferation cFLIP, X spase-9), tum

5), mitochon Ravindran e erma lucidu pression of g r cell lines P ed pigment e ial on a pan

and prostate vale et al., 20 s, thousand al activities r

marine sourc tly, marine- bioactive mo

) reported th Candida albi

). Similarly de an area 6; Nahum et , gene expre understandin min, major through regu

n pathway ( XIAP, c-IAP mor suppres

ndrial pathw et al., 2009).

um have exh genes associ PC3 and DU

extracted fro nel of three e cancer cell 013).

s of novel ranging from ces (Garcia- -derived mic olecules (La hat selected icans can ac

y, pigment e of anticanc t al., 2001).

ession based ng of the ne component ulation of m (cyclin D1, P1), caspas ssor pathway ways, and pr . Ethanolic a hibited antica iated with ce U145 (Kao e om Serratia

breast canc l lines viz., P

compound m antiviral t -Fernandez croorganism am, 2007).

compounds ct as an antic

extracts from cer research

d approaches ewly isolated of Curcuma multiple cel

c-myc), cel e activation y (p53, p21) rotein kinase and spore oi ancer effects ell cycle and et al., 2014) a marcescens

cer cell lines PC3, LNCaP

ds and thei to anticancer

et al., 2002 ms have also

s from crude cancer agen

m h

s d a ll ll n ), e il s d ).

s s P

r r 2;


e nt


by the prot subjected fo fucoxanthin pathways in several othe metastasis (

The Candida al from coast carcinoma Green Mon Kathiresan, cancer cell three yeast normal Ver extract ind cancer act environmen

Sear expanding o Henceforth provide the perspective yeasts as Saccharomy immunostim branching a

ein ligand d or anticance n exerts its ncluding the er molecules (Kumar et al

cytotoxic a lbicans, Kur

al mangrov cells (MCF nkey kidney , 2015). Al

s (MCF-7, t strains, S ro cell line dicate more

tivity. Auth nt will be pr rching for n

our ability t the marin e novel bio

, the presen a source o yces cerevi mulant. Eff as well as th

docking stud er studies in

antiprolifera e Bcl-2 prot

s that are in l., 2013a).

activity of raishia caps ve ecosyste F7), human y cell lines ll extracts w

HepG2) an S. cerevisiae es and at th

efficiency hors sugge romising fo natural prod to find nove ne environm oactive com nt study was

of bioactive isiae has al ficacy of y he number

dy. A marin various cel ative activit teins, MAPK nvolved in e

three differ sulate and S em have be

hepatocarc (VERO) u were able t nd the norm e showed he same tim

against M est that th r further de ducts in rel el, potent an ment, the l

mpounds w s undertaken e compoun lready been yeast glucan

of glucose

ne caroteno ll lines, has ty through d K, NFκB, C either cell cy

rent extract Saccharomy een studied inoma cells using MTT to inhibit th mal Vero c

more than me the IC50

MCF7 cells his yeast evelopment latively unt nd selective argely unex with immen

n to explore nds. Glucan n proved to ns depends molecules i

id pigment shown that different mo Caspases, GA ycle arrest,

ts of the m yces cerevis

against hu s (HepG2)

assay (Sen he prolifera cell viability 80% cell value of S suggesting species fr as an antica tapped sour e bioactive xplored res nse possibil the potenti n from Bak o be a good s on the fr

in the β- 1,

fucoxanthin the pigmen olecules and ADD45, and apoptosis o

arine yeasts siae isolated uman breas

and African nthilraja and ation of the y. Amongs

viability in S. cerevisiae g anti-breas

om coasta ancer drug.

rces will be compounds servoir may lity. In this al of marine ker’s yeast d source o requency o 6 branching

n nt d d r

s d t n d e t n e t al

e s.

y s e t, f f g


points. Earl property an mortality da glucans fro shrimps and in aquacultu Prev melanins. In since the in and functio undertaken Obje 9 Extr yeas 9 Test imm 9 Fun

expl The topic is gi primary scr against WS characteriza adult P. mo of melanin melanin an

lier studies nd the survi ata. The pre om various d selection o ure.

vious studi n this study nformation onal charact

to understan ectives of th raction and st isolates ting the b munostimula nctional cha loring its bi Thesis is c iven in Ch reening of g SSV infect ation and e onodon agai n from mar nd evaluatio

have show ival in shrim esent study

marine yea of the poten

ies on mel , marine bla on this me terization o nd its prope he study are d characteri

bioactive p ant in Penae aracterizatio ioactive pot comprised o

apter1. Ext glucan as an ion is pres evaluation inst WSSV rine black on of its bio

wn yeast-wis mps estima

was undert asts in term ntial strains a

lanins are ack yeast, H elanin is ver of the marin erty for poss

as follows:

ization of g

potential o eus monodo on of marin tential of six chapt

traction of n immunost sented in C

of selected infection. C

yeast, phy oactivity. Ch

se variation ated by imm

taken to und ms of immu as source of

mainly foc Hortaea wern ry much lim ne black y sible comme

glucan and

of marine on

ne yeast gl

ters. A gene glucan fro timulant in P Chapter 2.

d glucans a Chapter 4 il ysicochemic hapter 5 de

n in immuno munological

derstand the une gene ex f glucans for

cused on p neckii has b mited. Phys east melani ercial applic

melanin fr

yeast glu

lucan and m

eral introdu om marine

P. monodon Chapter 3 as immunos

llustrates th cal characte

als with the

ostimulation l assays and e efficacy o xpression in r application

plant/anima een selected sicochemica

in has been cations.

from marine

ucan as an

melanin for

uction to the yeasts and n postlarvae deals with stimulant in he extraction erization o e evaluation

n d f n n

al d al n




e d e h n n f n


of glucan analysis. A followed by

and melani A summary y references

in as an an and conclu s and appen

nticancer ag usion of the ndices.


gent by in e study is p

vitro gene presented in

e expression n Chapter 6

n 6,


2.1 Introd

Aqu plays a crit developing food and nu source of in acquired by single comm value of int shrimp prod as a result America. A prevention

Dise causing hug developmen


uaculture is tical role in and develo utritional se ncome and y FAO in modity in v ternationall duction volu

of disease- As the shellf and control ease outbrea ge economi ntal stages

one of the n global foo oped countr ecurity, man livelihood i 2014 indica value terms, ly traded fis umes decre -related pro fish aquacul l increases c ak has been ic loss durin

of the anim

fastest grow od security a

ries. In addi ny millions in the aquac ates that sh , accounting shery produ

ased in 201 oblems in s lture industr concurrently a major thr ng the last mals are be

wing food and nutritio ition to its

of people a culture sect hrimp conti g for about ucts in 2012 2 and partic some count

ry grows, th y.

reat to the s few decade elieved to b

sectors in th onal needs o growing im around the w tor. Recent inues to be 15 percent 2. Globally, cularly in 2 tries in Asi he necessity

hrimp farm es. Generall be more su

he world. I of people in mportance in world find a information e the larges

t of the tota , the farmed

013, mainly a and Latin y for disease

ming industry ly, the early usceptible to It

n n a n t al d y n e

y y o


pathogens t Penaeus m developing White spot problems a reaching 10 immunostim shrimp farm and protec components immunostim Burgents et al., 2010; Su 2005); mann (Boonyaratp et al., 2000) Leung et al.

rendering hi 2.1.1 Shri

Shri aquaculture the world m more than cultured shr the top pro producing c and now re farming ind

than adults monodon is countries in syndrome v and is usua 00% within mulants as ming help to ction from

s are used a mulants of al., 2004; R ubramanian noproteins ( palin et al., 1 ) known as ., 2006) hav igher surviv mp aquac mp culture e crops worl market. Mos 50 countri rimps come oducers. In country. Shr epresents a dustry produ

(Lightner the most n Asia and i virus (WSSV ally associa n 3-10 days prophylact o control infe

drug resis as immunost

biological o Rodriguez et n and Philip, (Tizard et al 995; Zhang biological r ve been prov val rate and

ulture has grown ldwide. All st coastal co

ies practice e from Asia

the Wester rimp aquacu multi-billio uced an esti

et al., 1983 farmed cru is under per V) infection ated with h of infectio tic agents r fections and stant patho timulants in origin such t al., 2007; S , 2013;Mast ., 1989;Abu g et al., 2014) response mo ved to enhan

resistance to

n into one o kinds of sh ountries have

shrimp aq with Thaila rn hemisphe ulture expan

n dollar ind imated 1.6 m

3a; Momoy ustacean sp rsistent threa n is consider high inciden on (Liu et a rather than

therefore le ogens. Yeas n aquaculture h as β-gluca Sajeevan et

tan, 2015); c u-Elala et al

) and lipopol odifiers (BR nce immune

o infections.

of the large hrimps are h e a harvest i quaculture.

and, China, ere, Ecuado nded signific dustry. In 2

million met

yama and S pecies in m at from vira red as one o nce of larv al., 2009a).

n chemother ead to reduc

st cell and e over the y an (Chang e

al., 2009; Su chitin (Wan ., 2013); pep lysaccharide RMs) (Yan responses in .

est and mos highly desir

industry for Around 80 Indonesia a or is the m

cantly durin 2002, the gl

tric tons of

Sano, 1989) many of the

al infections f the serious al mortality The use o rapeutics in ced mortality d cell wal years. Use o et al., 2003 ukumaran e ng and Chen ptidoglycans es (Takahash

et al., 2005 n organisms

st importan rable now in r shrimp and 0 percent o

and India as major shrimp ng the 1980s lobal shrimp shrimp, and ).

e s.

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