STUDIES ON A NEW STRAIN OF Rhodotorula rubra ISOLATED FROM YOGURT
BY
©RAVI NDER PAL KAURHARI SANGHA(B.Sc . M.Sc.)
A thesis submitted tothe school of Graduate studiesin partial fulfilmentof the requirementsfor thedegree of
Doctor of Philosophy
Department of Biology Me mo ri al university of Newfoundland
Ma r c h , 1994
St. Jo h n' s Ne wf o u n d l a n d
ABSTRACT
A newstrainof re d yeast wa s isolatedfromyo gu rtand identifi e das Rhodotorula.l.:Y.!:lnTP1. Studies weru conducted on it s pigment production, morphology, se xualit y, growth kineticsand possibleus e in aquaculture.
Studies on the sexuality indicated a basidiomycetous affi nityof the isolateandhenc e the first rep o r t of a sexual stagein Rhodotorula,~.
The is ol at e showedgood growth on different substrates.
It grew re a di l y on a molasses and wo r t medi um. Pe a t hydrolysate alsosupported asatisfactorygr owth ofthe yeast.
The pHprofileswe r e also studiedand growthwas fo un dwit hi n a broad pH zone of 3 to10.
Studi es on the genetics of the isolate included mut.aqeneaLa withnitrosoguan idine. One of the two mutants obtained utilized additional subst ra tes for growth and pigmentation. This mutant also showed a highe r value for productivity,yie ld coeff icientand economic coefficient.
The kine t i c s of growth and pigmen t formation of the is ola t e sho wed pigmen t atio n in the exponential pha s e, lik e
f.hill.iA ~ .The isolate wa s grown in a 1500Lbatch
fermente r us i ng molasses andwort as thegrowthme dium and the inf luenc e of vario us kin e t i c pa rame te r s on the yi eld coefficient and specific growth rate wa s ob s e rve d . The
i i
fermenter cul t ure showe d a hi g he l' va lue fo r the specific growt h rate and product ivity th an that of the shake flask cul tu r es.
Stud ies on aqua c ul ture invo lve d a feed ing trial fo r 9 wee ks usi ng rai nbow trout. Th e newyeas t was fo u nd to be a good source of pigm entsaswel l as nutri e nt sfo rthefi sh when fed as int a c t cells. Though the commercia l astaxanthin cont a i n ing di e t induced better pigm entat ion than tha t containing the test yeast,the to talcolor dif ferencebetween the fish fed with thetwo dietsdecl inedto almo s tone-third atthe end ofthe feeding trial.The test yeastfe dfis h had high e r growth ra t e s than thos e fed with the commercial pigment . The prox im ate ana l y sis of the fishwas done wherei n the test ye as t-fed fish showed an incre ase in th e protein amoun t atcb e end of the feedin g tr ia l wh ilethe synthe tic astaxant hin -fed fishsho wed a decrease.The lipidlevelsof bot h groupsdecre a s ed at the end of theexpe riment.
iii
ACKNOWLEDGEMENTS
I wi s h to express my sincere gratitude to my supervisor Dr. T. R. Patel for his valuable guidance and sustained in t e r e s t during the course of my studies. I would like to tha nk theot he r members of my supervisory commi ttee (Dr . P.
Dabinett and Dr. A.M. Martin) for their constant encouragement.
The fundingprov i de d by the School of Graduate <:;tudiesas aMemoria l UniversityFellowship is kind l y appreciated . I am also grateful forthe financialheLpprovidedby theSeabright Co r po r a t i o n Ltd. as well as theca na d d.e n Centrefor Fisheries Inno vation .
I appreciatethe useof facilities at theDe pa r t me n t s of Che mis t ry and Biochemistry at the Memorial University of Newfoundlandand the Northwest Atlant i cFisheries Centre .The assistance provided by Dr. R. Taylor, Dr. J. Bonoub, Dr.
Jablonskyand Mr. Kenneth Kean is much appreciated.
I acknowledge the assistance providedby the following people and institutions:
MemorialUniversity of Newfoundland:
Mr.D.Hall and Ms.S.Banfield,The Amino-AcidAnalysisUnit Mr. Roy Ficken, PhotographyServices
Me.Carolyn Emerson, EM Technologist
iv
Food Research and Development Centre, at . Hyacinthe:
Mr. Claude Gagnon
SalmonHatcheryat Bay d'Espoir:
Mr. De nni s Rose
MarineInstituteof M.U .N .:
Dr. Steve Goddard, Facultymember Mr. Keith Rideout,stude nt
Mytha n k s are due tothe cheerfuloffice staff at the Departmentof Biology as well as all my friends an d we ll - wi s he r s . I am especially thankful to Ruth Roy and A. B.M Siddique.
Finally, l owe it to my familyto bring out the be stin me. Iapprecia tethe affection and inspir a tionprovidedbymy loving parents, brother Maninder and sisterHarinder. I am gratefUl to my beloved husba nd Dr. Aj mel Sangha fo r his pre c i o u s lov e andmo r a l support duri ng dif fi c ul t times.
TABLE OF CONTENTS
Page Abstract... .... .. .. .. . ... ii Ac knowledgements.. ... . .. ... iv
Tab l eof Co nte nt s vi
Lis t of Tabl es x
List of Fi gu r J!s xii
List of2bbre'l~iations xv
Lis t of manu s c r ipt s. .. .... . .. .... ... ... . ... . ... xvi Chapte r 1:Reviewofthe1itl;lt""ature••.••.. .•• •• • • .••• . 1
1.1 General introduction 1
1.1.1 Chemistryof pigment . ... ... ... .. 1 1.1. 2 Types of pigments... .... . . ... . 2
1.1.2 .1 Tetrapyrroles 2
1.1.2.2 Indol i c bi o chromes. ... ... 3 1.1.2.3 N-Heterocyclicbiochromes 3 1.1.2 .4 oxyq e noue heterocycl ic
cdc cbrcmee-cneflavono ids 4
1.1.2.5 Quinones 4
1. 1.2.6 Carotenoids . . . .. ... . ... . .... .... 5
1.2 yeast s 8
1.2.1 Taxono my 8
1.2.2 Cultivat ion of yeast 8
1.2.3 Yeastge ne t ics 8
1.3 Redye a sts 11
1.3. 1 Caroteno ids of redyeasts... .. .. 12 1.3. 2 Analysisand identification
of ca r o t enoids 12
1.3.3 Yeast as a sourceofca r ote no i d and their nutrit ivevalue 17
1.4 The genusRhodotorula 18
1.4.1 Description of Cryptococcaceae 18 1.4.2 Description of genuaRhodotorula 18
1.4.3 sexualityin Rhodotorula 19
1.4 ." Commercial importanceof Rhodotorula 19
1. 5 The redyeast ~ ~""" " " " " " " 20 1.5 .1 Descriptionof Rhodotorula~" " ' " 20 1.5.2 Sources... . ... . .. .. ... .... 21 1.5.3 Gr owt h andpigme n t a t i o n
inE.~" "" " """""" "" "" 21
1.5.4 Enzymologyof caro tenogenes is .. ... ...24 1.5 .5 Commercial si gn if icance
ofB,.
mn
251.6 Research objectives 39
Chapter21Investiga t ionof the new iso late•. .••••.. .• 41
2.1 Introduction 41
2.2 Materialsand methods , 42
vi
2.3 .4 2.2.6 2.2.7. 2.2.1 2.2.2 2.2. 3 2. 2.4 2.2.5
Orga nism 42
Cul tu r e conditions 42
rdentLfIca t.Lcn of the isolate 42
Growt hon different media 43
Nutritiona l andgr owt h
character istics 43
Morphology and ultrastructure
of theis ola t e .... . ... . . .. .. .. . . .. ...43 Studieson pigme n t productio n
by the is o l at e 44
2.3 ~~lts 47
2.3 .1 Assi milationof carbohydrates 47 2.3.2 Growt hondif fe ren t media.. .. . . 47 2.3 .3 Nutritionaland growth
characteristics 48
Morphology and ultrast ructure
of theisolate 48
2.3.5 Studies on pigment production 49
2.4 Discussion 70
Chapter 3: Studies on the sexuality
in R. ~TP 1•. .•••••••.•••. .••..•.••... •75
3.1 Introd uction 75
3.2 Materialsandmethods 76
3.2 .1 Org anisms. .. . .. ... .... . ... . .... . .. ... 76
3.2.2 Sporulationmedia 76
3.2.3 Microscopic examination... ... ... . 77 3.2.4 Ul trast r u ctureof thecellwal l 77
3.2.5 Urease test 77
3.2 .6 oiazoniumblue Btes t 78
3.3 Results 78
3.3.1 Microscopicmethods ,78
3.3.2 Ultrastruc tureof the cellwal l 79
3.3.3 Urease te s t 79
3.3.4 DiazoniumblueBtest. ... . 79
3.4 Discussion B4
Chapter4I Growthkineticsof Rhodotorula~ TP1on different substrates••.•.••..•• B8 4. 1 In t r o du c t i on . . . .. . .. . ... .... . . .. ... ... . .. B8 4.2 Ma t e ri a l s andMeth o d s. ... . .. ... ... .. 91 4.2.1 Organism...•.. .. .. ... ... • . ... . 91 4.2.2 Subst rat es... ... .... . ... . . . ... . . .. 91
4.2.3 CUlt ureconditions 91
4.2.4 Biom assproduction. ... ... 93 4.2.5 Proximateanalysis. ... . . . ....•.. . .... 93
4. 3 Results 94
4.3. 1 Growthon molassesand pea t
hydrolys a t e ... . .. •... ... ..94 vii
4.3.2 Growthonmolasses andwort... . . ... 95 4.3.3 Growthon sulfite wa ste liquor 95 4.3.4 Effect ofpH... .... . .. ... . ... 95 4. 4
~i;~~ssion~~~~~~~~~
.~~~:~~~~:
: : : ::::::: :::. ......~g6
Chapte r 5: Nitrosoguani dinemutage nesisin a
new strainof~lJ: ~
TP 1 IIsolati o nand characterization ofmu t a n t s•... ... .. ... . ....
5. 1 Int r od uc t i o n .
5.2 Ma t e r i a l s andMet hods .
5.2.1 Microorg a ni sms .
5.2.2 Nitroaoguanidinemutagenesis .
5 2.3 Charact erizationof mutan t strains .
5.2.4 Growth kinetics .
5.2.5 Mutagenicity of NTG ..
5. 3 Results .
5.3.1 Is o l a t ionof mutants .
5 . 3.2 colonymorphology .
5.3.3 Cell morpholo gy .
5.3. 4 Nutritional and biochemical
charact.er-Iet.Lcs .
5.3 .5 Growth kinetics .
S.4
~i;~~ssion~~~~:~~~~~~:. ~:
.~~ :
::: :: : :: : ::: :::::::Ch a p te r 6: Gr ow t h and pigmentatio nin Rhodot orularubraTP 1
and Phaffi a~ .
6.1 Introduction.... . .. .. ... ..•. . .. ... ... . ...
6.2 MaterialsandMethods .
6.2.1 Organi s ms.. .•.. . . .... ... .. .. ...
6.2.2 Culture condi tions .
6. 2. 3 Drywe i ght .
6.2.4 Mecha ni cal ruptureof cells by
Fre nc h press .
6.2.5 Pigment extraction .
6. 2.6 Enzymatictreatment
for cell rupt ure .
6.3 Res u l ts .
6.3 .1 pig ment rele a s e d by enzymat icmetho d
inE..~ .
6.3.2 Compari son of enzyma tic and Fren c hpr e s suremethod s in
£. _ .
6.3.3 Pigme nt rele a sedby mechanica l rup tureinE. X~.··..• · •••·•··••.•• • 6.3.4 The enzyma t i c allyrele ased pig ment
vii i
112 112 113 113 113 11.
11' 115 116 116 116 116 117 117 116 129
132 132 133 133 133 133 133 13' 134 13 5 135
13 5 136
inR.~..... .... ... 136 6.3.5 Comparisonof pigment formationby
f.~anll&. ..D.I..b.n ..•. . ....•• . .136
6.4 Discussion 14 2
Chapter 7:
14' 160 14 6 146 14 7
7.3.2
7.4.2 7.2.2
~~~~its ... G~~~th' ki~~ti~~' i~ '~ . ~h~k~ fi~~k""' "
and termenter 148
Comparison of growthinashake
flask and fermenter .
Discussion .
7. 4 .1 Measureme ntof maintenance
coeffi cient 160
Comparisonof growthina
shake flask and termenter.... ... . ... 161 Shake flaskandfermenter cul ture studies of Rhodotorula~TP 1 ••••• •
Introduction .
Materialsand Methods .
7. 2 . 1 Conditions of growth
in a sh ake flask... ... .... ... . . .. .... 14 7 Conditionsat growthin the termenter.. 147 148 7.1
7.2
7.3
7.4
ChapterB: Rhodotorularubra TP1as a pigment and nutrientsource forra i nbow trout (Onchorvnohus.D1ykissl•.•. .••. • ... 162
8.1 In tro d u cti on 162
8.2 Materia lsand Methods 163
8.2.1 Rearing and sampling of the fish 16 3
8.2 .2 Color parame ters 16 5
8.2.3 Growthrate 16 6
8.2.4 Specificgrowt h rate 166
8.2.5 Proxim a t e analys is 166
8.3 Resu lts 166
B.3.1 Visual inspectionof the fiah 166
8.3.2 Measurementof differentcolor
parameters... . . . ... 167
8.3.3 Relations hip betwe en duration ofpi g me n t at i o n and
the di f feren t color parame ters.... . .... 16 8
8.3.4 Growthrate 169
8.3 .5 Specificgrowthrate ... .. .. .. ... 169
8.3.6 Proximate analysis 17 0
8.4 Di s cu s s i o n 187
Chapter 9: References
General discussionand conclusions••... 19 0 ..•••.•.• •.. .•..••.•••••••• • •.•• ••••••• 198
ix
Table 1.1 Table 1.2 Table1. 3 Table 1.4 Table1.5 Table 2.1
Table 2.2
Table 2.3 Table 2.4
Table 4.1 Table 4.2 Table 4.3 Ta b l e 4.4 Table 4.5
Table 5. 1 Table 5.2
Table 5.3
Table 5.4 Table5.5
Table 6.1
Table 7.1 Table 7.2
LIST OF 'fABLE S
Page ca ro c enofds used as food additives... 27 Worldwide carotenoid markets.. ... ... . 28 Taxonomyof yeasts... . . 29 Sub s t r ate s for yeast
biomass production ... .. ... . . ... 30 Differentiationof red yeasts.. ... ... 31 Analytical ProfileIn d e x 20C
(Ar t ) test for identification
ofth e isolate... .. . .. . .. . . ... . 52 Nutritionaland biochemical
properties of the new isolate
compared withg.rubra ATCC 9449 53 Growth characteristicsof
the two red yeasts... ... .... . .. ... 54 Thin layer chromatography
of thepu r if i e d pigment. .. ... ... ... 55 Compositionof cane and beet molasses. . 97 Compositionof brewer's wort 98 Composition of the peat extract 99 Compositionof sulfitewaste liquor 100 Proximate composition ofB. ~
andf:. ~ 101
Yeast strai ns us e d inth e study .. . .. . . . 119 Nutritional and biochemical
cna r e ce e r de e f.c e of R..ntt!nTP 1 and the two mutants A andB... .. . . .. 12 0 Nutritional and biochemi cal
characteristics of~ ~
ATCC 24202, RhQdQtQrulaI:l.l..Qu
ATCC 9449 and ahcdot.cru'ta~... 121 Growth kineticsof th e yeasts.... .... .. 122 Effect cf nitrosoguanidine
concentration onpercentas··kill... .... 123 The pigment yields of2,. ~ andR.~using the tWQ methods of cell rupture. . .. . .. ... .. . ... .. 138 Compositionof medium for the growth of R.rubra in a fermenter 15 0 Growthco nd i t ionu of g.!J.llliin fermenters.... . . ... . . ... .. 151
Table 7.3 Table 7.4 Ta b l e 7.5 Tabl e 8.1
Ta ble8. 2 Tab le8.3 Tab l e8.4 Ta b l e8.5 Table8.6 Table 8.7 Tab le 8.8 Table 8.9 Tabl e 8.10
Ta ble9.1
Dimens ions of vari o u s .lt ru c:t ura l compon e ntsofth e ferme nt ers .... . .... .. 152 Growth parametersobserved
ina ferm enterculture 153
Growth data in shake flaskcult ures 154 Compositionof expe rimentaldiets for trout pigmenta t i on; study
con duc t e d at Bay d' Espoir... .. ... 171 Relation of fee d i n g timewith
the colorparameters , a- and b' 17 2 Relation of feeding time with
the colorparame ters, a'lb ' andL'... ..17 3 Relation of feedingtime wi ththe color pa r a me t ers , hue and chroma... .. 171 Tot a l color di fferences between two feeding periods... . . .. .. . . .. 175 To tal col o r dif f e r e nc e
betweentwo die ts... . ... ... .. ... . . 176 The growthra tes of rainbow
troutfedwiththe three diet s 17 7 The specificgrowthratesof rainbow tro ut fed with the three die t s 178 The proximate composition of
fishfedwiththe threediets . .. .. . . . .. 179 The amino-ac idlevels of
fishfedwi t hdiets2 and3
at the end of feedingtri a L .. .. ... . . .. 180 The maintenance ene r g y requirements fo r differentor g anisms... .. . ... 197
xi
LIST OF FIGURES
Page Fig. l.l. Cyclic tetrapyrrole. . . .. . .. ... 32 Fig. l.2. Line ar tet r a p y r ro l e ... . ... .. . . ... . . .. 32
Fig.1.3. Indolicbiochrome 32
Fig. 1.4 . N-Heterocyclic bi o chromes 33
Fig. 1.5 . Flavonoids 34
Fig.1.6. Quinones 34
Fig, 1.7 .Carotenoids 35
Fig . 1.8a.Conversion of acetyland acetoacetyl CoAto geranylgeranylpyrophosphate ... 36 Fig. 1. 8b .Conversionof geranylgeranyl
pyrophosphate to cis-phytoene 37
Fig. 1.ac. Co n v e r s i on of cis-ph ytoeneto
to r u len e and torularhodin... .. ... .... 37 Fig. l.Sd.Conversion of phytoeneto astaxanthin 38 Fig . 2.1. Schemeof pigment extraction. . . ... ... . .. 56 Fig . :2.2.Ascanningelectron micrograph of
E•.r.!.Y2nTP 1 showing a circular
cell shape 57
Fig.2.3.Ascanning electron micrograph of E.~showingan ellipsoidal
cell shape 57
Fig. 2.4. Atransmission elec t r on mi c r ograph of
£. ~.• • ... • ••.••... ••. ...• •.. .•.. SS Fig.2. S. Atr an s mi s s i o n electron micr o g rap h of
E.~TP1 58
Fig. 2. 6 . Abso r p t i o n spectrum in acetone. .... . . .. ... S9 Fig.2. 7. Absorption epectrumin methanol 60 Fig. 2.8. The mass spectrumof the purifiedpigment
from E. ~TP 1 61
Fig. 2.9 . Thema s s spectrumof astaxanthin . . .... ...61 Fi g. 2.1 0.Chromatographicbehaviour
us ingthe binarysolvent system 62 Fi g.2.11. Di o d e array analysisof peak 2 fromth e
HPLe analys is (us ingone solvent ) ofthe pigment extract from R.D!£llTP 1. 63 Fi g. 2.12.Dio de array analysis of peak 3... .... .63 Fig. :2.13.Diode array analysisof peak 4.. ... . .. ..64 Fig. 2.14.Absorbancespectrumof fractio n#3
of the pigment extract from CF-l1
cellulosecolumn 64
Fig. 2.15.HPLCanalysisof fraction#3of the pigment extract. ... ... ... ... 6S Fig. :2.16.Diode array analysisof fraction
#3of the pigment extract... . ... . ... 65 xii
Fi g. 2.17. Absorbancespectrum of fr ac t ion
#I5 of th epi gment ex tra c t from
CF-llcellulosecol umn. .... . 66
Fig. 2. 1 8 .HPLC analysis of frac t i on #I5of the pigme nt ext r a c t... . . ... . ... 66
Fig. 2.19.Diodearrayanalysisof frac t i on #I5 of the pigmentextract... . ... ... .... 67
Fig. 2.2 0.Total ion current (A), mas s spectrum (Blan dext rac ted io n ch romatogramatm/ z 596 (C) of thepigmen t from,e. ~... . . .......... 68
Fig.2.21.To tal ioncurrent (Al,mass spectrum (Blandextractedionchromatogramat m/z 596 (C) ofthe pigment fr om R.~TP1 69 Fig. 3.1 (A&B).Fusion of opposite mating types ofB,.~TP 1 80 Fig. 3.2. Zygote formationin R.rub raTP1. .. . .. . .... BO Fig. 3.3 (A&BI. Sac-shapedascus of B,.~TP 1 withfourspores B1 Fig. 3.4. Mul ti -sporedascus of R.
nmu
TP 1. B1 Fig. 3.5. cylindr i cal ascusof g. ru b ra TP1 wi t h four spores... .. . ... . ... ... B2 Fig. 3.6 (A&ai,As c o s pore s of B,.~ TP 1 stained wi t hDAPI B2 Fig. 3.7. ,a.~stainedwith DAP I showi n g a singlenucl eus.. ...B3 Fig. 3.8 . Atransmiss ionelectron micrographof,S..~••••••. .•••. .•.•.83Fig. 4.1. Effect of pre t r e at me n t of molassesand peatex tra cton the growt h ofyeast ...10 2 Fi g . 4.2 . Gr owt h on molass e s and peat ext r a ct using dif fe r en t ino r g a ni c nitrog ensources... . . .102 Fig. 4.3 .Growthon molasses andwort.. ... . . ... ... .... 103
Fi g. 4.4.Growt honsulf ite was t e liquo r.. . . . ... . ... 10'
Fig. 4.5. Eff ect ofpH ongrowth... . . .. . .... . .. . . 105
Fig. 4.6. Ef f e ct of differe ntbuffers on growt h ...105
Fig. 5.1. Co l o nymorph o l ogy ofmut antA••• • • • • ••••• • • •12. Fig. 5.2 . Colonymorpho logyof mu tan t B.... ...12'
Fig. 5. 3 . Ce ll morpho l o gy ofB.. Dll>uTP1. . . ... ...12.
Fig. 5. 4.Cell morp hology ofmu tan t A.. . . .. . ... .12 5 Fig. 5.5.Cell mo rpho l ogy of mutantB... . ... .. ..125 Fig. 5.6. Cell mo rpholo gy ofR·~ATCC 94 49.... ... 125
Fig. 5.7 Cell morpho logyof E. glytlnll.... .. . . .. . . .. 126 Fig . S.B.Cellmorphologyof£.~ATCC 24'202.. 12 6 Fi g. 5.9. Growthcu rve sof pare nt
xiii
andmutant yea s t ~ 12 7 Fi g.S.lO.Gr o wt hcurve sof threeyeast specie s 12 7 Fi g . 5.11 . The su rvival ratiosof ye asts
tr e a ted with ni t r o s ogua n idine 128 Fig. 6.1. Pig me n t releasedby enzymatictreatment
of ~ ~ce l l s 139
Fig.6. 2.Efficacyof enzymaticand mechanicalrupture in the
rele as e of pigmentof .E.~•..••..••• 140 Fig.6.3 . pigme nt relea se dfrom Rhodotorula
XYl2nby mechanical ruptu r e 141 Fig. 7.1.Effectof spec ificgrowthrate
on the specific rateof sub st r a te
consumption 15 5
Fig. 7.2.Yiel dcoe f f ici e nt asa funct i o n of
sp e c ific growth rate 156
Fig. 7.3.Yiel dcoe ff ici e nt asa funct i on of ti me
of cultivat i on 156
Fig. 7.4.Growth cur ve of the yeast grownin a
fermente r 157
Fig . 7.S .Diss olvedoxygen statusof the fermenter 15 7 Fig . 7.6 . Effec t of consumedsub s tra t e
co nc e nt r a t i o n on the biomass 158 Fig. 7.7.Spe c i f i c growth rateofthe ferme nt er
cult ur e asa fun ctionof time of
cultiv a t i o n...•... . . . .. 159 Fig. 7.8. Spe c if i cgro wt hra t e of the
sha ke flask culturesas a function
of time of cultiva tion 159
Fig. 8.1.
Fig. 8.2;
Fig.8.3 . Fig.8.4. Fig.8.5.
Fig.8.6.
Fig.8.7. Fig. 8.8.
Pi gme nta tio n of fish (On c ho rync husI!!Ykin) at the beginningofthe experiment . Pigmentati onof fish (Onc hor ync hus ~)
after6weeks of feeding .
Pigmentation of fish (Onchorynchus~)
after 9 weeks of feeding .
Re g r e s sio n of a'sco r e s of the fish
flesh onduration of feeding .
Regression of b' sc oresofth e fish fle s h onduratio n of fe e d i ng . Regressionof hue of the fish
fleshon durationof feeding .
Regressionof chroma of the fish
flesh on duration of feeding .
Regressionof a'lb·ratio of the
fish fleshondurationof feeding .
xiv
1B1 1B1 1B1 1B2 1B3 1B4 1BS 1B6
LIST OF ABBREVIATIONS
4, 6-Diamidino~2-phenyl-indole Diazonium Blue B
Yeast extract/malt extract medium Sabouraud'sdextrose agar
Potato dextrose agar Trypt ic soy agar Total ion current sulfite waste liquor
N~methyl-N'-nitro-N-nitrosoguanidine Maintenance coefficient
High performance liquidchromatography- mass spectrometry
Scanning elect ronmicroscopy Transmission electronmicroscopy
CAPI
DBB YM
SDA
PDA TSA TIC SWL NTG
SEM TEM
LI:STOF MANUSCRIPTS
This the s i s wa s prepared in the form of manuscripts submitted or to be submittedfor publication. Eachchapter denotes work already pub l i s he d or in the process of publication .
Followingis the list of manuscripts:
Har i, R.P . K., T.R. Pa t e l andA.M. Mar tin (1 992). Anew strain of~ ~isolatedfro m yogu rt. J.Industrial Mi c ro b i ol. 11:43-51.
Hari , R.P. K., T.R. Patel and A.M. M~r t. in (1993). An ove r v i e w of pigment production in biol o g i c al systems: Functions, biosynthesis and app l ications in fo o d industry. Food Reviews Inter national. 10 (1): 49-70.
Har i, R.P.K. , T.R. Pat el and A.M. Mar tin (1993). Growt h kinetics of Rhodotorula .ntl2U on diffe re n t substrates. J.Basi cMicrobiol. 33: 379-388.
Har i, R.P .K. , T.R. Patel and 1.M.Ma r t in (1 9 93 ) . Growth and pigmen ta tionin Rhod ot o r u l aXl.ll2uand~
~. J.BasicMi c rob i o l. (Submitted ) . Har i, R.P .K.. T.R. Pate l and A.M. Martin (1 9 9 3).
Nitrosoguan idine mutagenesis in a new str ain of Rhgdo tgDl l a D!t!.u: Is o l a tion and characterization of mutants. JBasicMi crobio l . (s ubmi tt e d) .
xvi
CHAPTER 1 REVIEW OF LI TERATURE
1.1 General introduc t ionsince times immemorial,colors in the livingwor l d have always fascinated and amazed mankindle a v i ngitwonde rs truc k;
the present study haa alsobeen inspiredbythe i r provocative and conspicuousnature.
Th e visibleperceptionof coloris causedbythe pigments or biochromes, the chemical compounds absorbing specific wavelengths of visiblelight.
1.1.1 Chemistry of pigmen t
The term • pigment' is applied to a materia l of known or unknown physical state or to an unanalysed and unkno wn colour e d material.However.a more ap pro p ri at e and scientific te rm is 'hiochr ome' wh i chis def inedas a specificche mi c a l sub stance with a colored molecule, synthesized by living organis ms (Ne e dham, 1974a) .
The structural feature of a bioc hro me which ie re s po ns i b l e for the absorption of light is called the chromophore , forexample,in theca r o t eno i ds , the chromophore is the conjugateddouble-l·'':od system. Otherfunctionalgroups or substituentsin the moleculewhichare ab le to mod ifythe abso r p t ion maximum are called euxcchrcmee (Br i tton, 1983).
The absorptionof vis iblelight bya molecu l eresul tsin theexc i t a t i onof anelectronin theou t e ror b i t a l to a higher
orbital. These transitions are chara c te r i s ti c of most biological materialsbut are par t i cu l a r ly pronounced inthe biochromes.This is becausetheenergy increment is minimized by a number of facto r.sli ke: la r gemc keculer- ::i::J,-;;e , conj uga ted double bonding, polar st ructure and hi g h dipole moment
(Needha m, 197 4b ) . 1. 1. 2 Types of pigmente
The six ma j o r groupsofpigmentsfound in the biological systems include caro tenoids, tetrapyrro les , indolic baccnrceee , N-heterocyclic bioc hromes (ot he r than tetrapyrroles ), oxyg e nou s heterocyclic biochromes (the flavonoids ) and quinones . The most important group, carotenoids, will be discussedlaterin the chapter.
1.1.2.1 Tetrapyrro1es
Thetetrapyrroles co ns i s t ofthe N-heterocycl icpyrrole , a very stab leheteroa romaticsystem.These are of twotypes, cyclic tetrapyrroles having the basic structure of porphin with four pyrrole residues linked toget her (Fi g. 1.1) and line a r tetrapyrroles, also called bilins (Fi g. 1. 2 l . Tetrapyr roles play importan t roles in plants and animals.
Cyclic tetrapyrroles like chlorophyll ha ve function in photosynthesisin green plantswhilehaem and haem proteins such as haemoglobin,myoglobin andleghaemoglobinare oxygen carriers .Cytochromesare essentialin the electrontransport chain and the two haemoprotein enzymes, catalase and
peroxidase, co ntri b u t e totheredoxreaction s (Br i tton ,19831. 1. 1.2.2 Xndolic biochromes
The indol ic biochromes containth e indolenucleusas in trypt op ha n .A commonexa mp leis melanin ,a polymer ofindole- 5, 6 quinone (Fig. 1.3).
The role of melanins is that of providing
photoprotection, captur ing stray lig h t An d pr o t e c tio n in general. Eume l an i n s affordde fe n s e tocuttle fi s h ,allow the capture of straylightof all wave lengths inthe back ofeye and affordphotoprotect ion in animals.Al l mel ani ns commonto plants form the protectivecoating of many ripe seeds and serveas directionalguidesfo r polli nati ng insects(Britton, 19 8 3 ).
1. 1.2.3 N-Beterocyclicbiochromel!l (o t h e r tha."\tetra pyrr o le . ) This group of biochromes is representedby eompoun dawith very complex structure s fou ndin purines, pterins, flavins, phenazines, phenoxaz inesand betalains (Fig. 1.4).'rhe purines ad eni ne and guanine are found in nucleic ac ids and nucle otides.Th e twopt"rin derivat i ves, biopterinand folic acid,are importantinth e redox.reactionsand ","8an es s e n t i a l vi tamin , re s pe c t i vely . Riboflavin (belo ng ing to th e flav i n groupof bioch romes)occursuniv e rsallyin livingorgan isms as a component of two coen zymes FMN an d FAD. Iodin!n and pyccyan in are examples of phe n az i n e s, synthes ized by Ps e u domo n a s spp. and havi ng bacteriostatic prop erties .
Actinomycin , an example of phenoxazine and produced by St r e p t omyc e s , has antibiotic propert ies. Two groups of
?e t a l ains , beta c yan i n s {e. g. be t an i d i n} and betaxanth ins (i nd ica xa nt hinl, help in se e d dispersal and pokLi.nat.Lon (Britto n, 1983).
1.1.2.4 Oxy g e n ou B heterocyclicbiochromes - the flavonoids ; Theyhav e thebas icstruc ture offlavon eandfl ava n(Fi g.
1.S) .Thesehavebeenfoundin allplant tissues- leaves,wood , roo t s, fruits , seedsand allpa rt s of the flower, especially petals . They occur as antho cyanins , chalkones, aurone s and flavan derivatives and are important in color a tion , pollinat ion , p-ceecefc n and di sease re sis t ance. (Bri tton, H83).
Applica t i ons of flavonoids in food indu s t ry :
In the USA, onlytwo anthocyani nprepar.ationshave be en legalisedas food colorants; one is enocyani n fro m sk i ns of wine grapes and theothe r is fromlees in thebot t om of ta nks of grape juice (Fr a c i s , 1989).
1.1.2.5 Quinones
The basic quinone structure is that of an unsaturated cycli c diketone dez-Lved from a monocyclic or polycyclic aromati c hydrocarbon. Example s include be n z oq ui no ne s, na p ht ha qu i non e s andanthraquinones (Fi g. 1. 6) .Colorationsby qui nones are ob s e rved in the animal kingdom, co nt ri buto r s
bei ng spino c hr ome s and ech i no c hr ome s foun d in echinoderms (Tho mson, 1971; Thomson , 1976 ). Qui no ne s have found appl.i cat i on s in industry as dye s , food colorants and medic inals.
1.1. 2•6 Carotenoids
Caro tenoidpigments belong tothe class of polyenes and are probably the most widely dis t ri bu t e d. Almost all carotenoi ds eithe r are,or are der ived fr om, tetraterpenes ,C- 40 compound s with aca r bon skele tonbuilt up from eightc-s isoprene unit s (F ig. 1.7) , e.g . carotenes, xanthophylls, re tro- ca rotenoids,aeco-andapo-carotenoids, nor-car oteno ids andhigherorhomo-c aro te noi ds (Bri tto n, 1983) .
Functionsof car oteno ids·
The ca r ot e no i ds play a role in photoreception (v i s i o n ) , photosynthesis, photoprotection , phototaxis, sporangiophore formation and integumental colur s (Mos s and Weedon, 1976;
Britton, 1979) .
Applicationsof caTgtenoi ds'
1. ~ : Thecolor of fishis an impo r t a n t f:l.ctorin consumeracceptanceof aquacultured fish.Hence the use of carotenoid pigment in the diets of farmed aalmonids has increaseddramattcally in the pastfe w years. 2. Food indu s try : Xanthophyllsof the algae~have
been used for chickenegg yolk pigmentation (Ande r s on~
al.1991).Pigmentsfrom~ ~have similarly beenusedinpoultryfeeds tocolor egg yolks.Table 1.1 summarizes the so urce s of pigments used as food addit i v e s .
3. Pharmaceut"ica l ana cosmetic products: Carotenoid formulations for cosmeticsandpharmaceuticalshave been produced by Hoffman La Roc he, e9, tablet coatings, suppos i t ories,gela tincapsules, fat-based ointments and crea ms , vitamin emulsions, lipsticks and toothpastes
(Tayl o r, pe r s on al communi cat ion ). 4. Medi calapplications:
Caroteno ids recommended for erythropoietic protoporphyria and congenitalporphyria (Taylor ,personal co mmun i c at i o n).
b. Retinoidshave been demonstrated to be chemopreventive age ntsforexperimentalca rcinog enes i s ofma mma ry gland, urinarybladder, lung s, skin, l iv e r , pancreas, colonand es ophagus (Moon ,198 9 ) .
B~Carotenehas been showntobe protective againstthe development of lung cancer (Zi e g l e r, 1989) as well as enhance immune function (Bendi c h, 1989a) .
d. Ce rta i n carotenoidswith ant ioxidantactivitieshave been shown to act as anti-mutagens and anti-carcinogens, protect~gainstradiation dama g e and block the damaging
eff e ctsof pho t o s en s itizers (Ben d ich, 1989b ) .
Business implicat ions and valueQf the market i. Thecarotenoidmar ket, whic h generated world-wide sa les
of $100 mi ll i on in 1989 , is exp e c t e d to expand mo r e beca use of the proposed link between ca r-o t.e n o fd a and can cerpreve nt ion (Tab le 1.2 ).
i i. Na t uralB-ca rote ne nowcons tituting lOt of the tota l B- caro tene mar ke t , is expected to ca pture25t of th emarke t because of incre a s i ng demand (Taylo r , pe rso n a l communicat i o n) .
pis t ri butio n of caro tenoida·
Carotenoids ar e wel l spread amongst bi olog i c a l systems in c ludi ngthe following:
A. Higher pl a n t s andalgae: The pigme nt sof highe r plant s and green algae include ,B-carote ne, lutei n and vi o laxant h in(Br i t ton, 198 3 )whileother clas s e s ofalgae produce acety lenic carotenoids, e.g, fucox a n t h i n (sue warc , 19741.
B. Bacteria: The no n-pbotosyn tbec ac ba c t e r i a produ ce gl ycos i d e s of C- 30, C-40 andCOSO carote no f d a while the ph o t osy nthe t i c bac teriasyn t hesi ze acycli c,aromati c and glycos idic car o tenoids (B:t:"itton, 198 3 ) .
C. Ani mal s1Mo s t ani ma ls ar e characteri ze dbyoxyc a ro te no i ds
as well as carotenes (Britton, 19 83 ).
Fung i:Mo s t carotenogenic fu n gi accumulate ca r ote nes like (3-caroteneand "'(-carotene. The yeastswhichaccumulate car.. 'ienc Ld e belong to fami l ies Deuteromycetes and Basidiomycetes (Britton, 1983) .
Th e presentpiece of work is devoted to the pattern of caro t e nog e ne s i s inth e yeast Rhgdg t gru1a.Ji:l.l..Qnaswell asit s gra..rthkinetics,n'OEPhologyandsexuality, arrong other characteristics.
1.2. Yeasts:
A ye as t is defined as a uni c el lula r fungus which reproduces by bUddingor fist?ion (Kr e g e r-v a n Rij, 1.984).
1..2•1 Taxonomy
Yeasts are classified in th e division Eumycota and Ln c Ltrde Ascomycetes,Basidiomycet esandDeute romyce tes(Table 1.. 3) (Kr e g er-v a n Rij, 1984) .
1.2.2 Cu l t i v a t i o n of yeast
SubBtratesfor yeast biomass production:
Di f f e r e n t sub stra t es ha v e been used to growyeast fo r biomasspr o d u c t i o n (Table)..4) . The substratechosen should have a few desirable ch a r a c te r i s t i c s , e.g., i t sh o uld give maximum yield of pro d uc t and mi n i mum yi e l d of undesirable products;it shouldbein expe nsi v e , of cons iste ntquali ty and readily availablethro u gh o u t theyear ;it shouldcausemi n imum
problems in aspects of production process like aerati on, agitat ionand wastetr ea tment (St a n b u ry and Whitaker, 1984). 1.2.3 Yeast genetic s
Winge 11935 ) demonstratedthe haploid and diploid phases in the lifecycle of Saccha r omycesellipsoideu8 Hansen while Mendelian segregat ion was first observed in Sacchargmyces
~ (Mort imerand Haw tho rn e, 19 6 9).
Yeasts have been found to present many advantages for gene t i c studies,e.g., rapid growth, easeof clon ing,handling and storage, ad a pta b i l i t y replica plating, mi croman ipulat ion an d an array of biochemical pro cedures
(Mort im e r and Hawthorne, 196 9 ).
Methods for genetic manipulationl 1. Mlltag e n esi s l
A. Mutagenesisthrough radiation·
a) Ultra-viol et radlationl Sh o r t wavelength ultra-v i olet rays be twe en 200-300 nm have been found to be effective for mutagenesis,withan optimumat 254 nrn, absorption maximumof DNA. The productsof UV ac t ion are dimers be tween adjacent pyr i midines or pyrimidinesof complementarystrands.
Long wa v e l e n gt h 1N radiationbet.ween 30 0 and 400 nm is less le th a l ; howeve r, in the presence of variousdyes which interact wit hDNA, i t induces increased mutation frequency. b) Ionizing radiation:Ionizingra d i a t i o nswh i c hinc l u d e x- ray s , B-rays and 'Y-rays,are seldom used for mutagenesis in
10
industrialstrain development (Crueger and Crueger, 1989).
B. Mutagenesi s with chemical a g e n t s ·
al Mutagens affecting non-replicating DNA: These include nitrous acid, hydroxylamine and al ky lating agents. Nitrous acid deaminates adenine to hypoxanthine andcytosineto uracil while hyd r o xy l a mi ne reacts with pyrimidines. The alkylating agentshave been found tobeone of the most potentmutagenic systems. These include ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS) , diethylsulfate (DES) , ddepoxybut.ane (DEB ) •N- me t hy ! - N'-nitro-N-nitrosoguanidine(NTG) , N-methyl -N - nltrosa- ureaandmus t ard gas.
bJ Base analogs:Ba s e analogs such as 5-bromouracil(BU) and 2-a mi no puri ne (AP) are incorporated into replicating DNA because of structural similarity. However, these not important for practical application.
c) Frameshitt mutagens: These intercalate into the DNA moleculecausing errorsand resultingin an al te ra tionofthe reading frame and thus lead to thefo r ma t i o n offaulty pr o t e i n or no pr o t ein at all. Some examples of auch mutagens are acridine orange, proflavineand acriflavine.Though us e ful in research, these are not sui table for routine purposes in strain development (Cruegerand Crueger,1969 ).
2. Protoplaat fusion;Thisinvolvea cell fus i o nfollowe dby nuclear fusion occurringbetweenprotoplastsof strains wh i c h nor md lly do not fuse. Thus, protoplast fu s i on is used to
11
re c o mbi na tion barriers. Protop last fus i on has be e n shown in Streptgm yces spp. (Ho pwoo d l l l l . 1977). filamentous fungi (Fere ncz y and Zsol t , 1974) and yeasts (Si pic z k i and Fe r en c z y, 1977). Intergeneric fusion of B. !:Ybu with §..
~has been successful (Evans and Conrad, 1987).
3. Recombinant DNA techniques : In these techniques. a plasmid from a yeast carrying the des i re d gen e to be introduced into the recipient strain is used to transform Es c he r i c h i a ~andthe.plasmid DNAis amplified.DNA from the bacterial strains is ampli fied, reisolatedand used to tr an s for m there c i p i e ntye ast . The transformantsareselected andthentestedfor the pveeenceof the desiredgene(spe nc e r tt li. 1988).
4. Pulsed field electrophgresis: Thi s tec hni q u e permits separation of intactchromoaomes andinformationabout their size. Here, the yeast cells, embedded in agar osegel blocks and lysed enzymatically , are subjected to el ectrophor es is wherein.pulses ofcurre nt of unequal durationare applied and then reversed at intervals of several seconds producing homogeneous electricfieldsandgivingsharper separationsof the bands. Thistechniquehas alsobeen us e d for karyatyp i ng of a number of yeast species (Spenc er ~Bl.1988).
12 1.3 Red yeasts
The car o t e nog enic yea st s fall into seven genera , Sp o r o b o l o rny c e s , Sporidioho lus, Rhodospgr idi1!ID, Rbodotorula, PhaffiC,'. cryptococcus (Phaff tt ll. H78) and ~ (Ko mag ata.tl.iU,. 19B7 ).
The s e be differentiated biochemically and morpho logically (Ta ble 1.5) .
1.3.1 Carotenoidsof redyeas t s:
~is theon l y yeast studied todate that contains as taxanthinasitsmaj o r pi gmen t (Fig. 1.7 ) .Otherca rote no ids synthe sized B-carote ne , ueurosporene, v-ceroeene, lycopene, echinenone, 3 ·hydroxyechinenone, 3-hydroxy-3-4 - di dehydro -B-caro t en-4- oneand phoe ni c o xa n thi n (And r e we s II .2.1.1976).
The yeasts of gener"? belo nging to Cryptococcus, Rho d o t Qr u l a, RhodQsporidium ,sporidioboluB and soorobolomyces ma i n l y synthesize &-carotene, y-carotene , torulene and torularhodin (Fi g.1.7) (Goo dwi n, 1972; stmpeonej;ill.19 71). The ge n e r a Rhodotorulaand Rhodosporidiummay alsoproduce
t-
caro t e n e , phytoene, phyto flueneand E-zeacarotene (Hayma nII li. 1974). plectaniaxanth in has also been found in a fe w Rhodotorula spp (Ra t l e dg e and Evans , 19 8 7) as well as crypto c o c c u s ~ (Ba e t tl i. 19 71). A species of B,.
~ ha s been found to produce 2-hy d rox y
13
plectaniaxanthi n (Liu ilt. .e.l. 1973) . The carot enoid a of
~areyetunkn own.
1.3.2 Analys is andiden ti f icati o nof ca ro teno id.:
A. Extraction of carotenoida:
1) Mechanica l met h o d s:Haar d (1988)gro u n dth e freez e -dried .E.~cel ls wi t h fine sandand 60% met ha no l inw,tt~ l'l Joh nsonandLewis(1 979)mixedPha f fi a cellswi t h91a 8')1~;;ld..
followed by vibra t ion in a Braun ho mo g e ni z e r I Th e French pressurecell ha s been used as a method for cel l rupture by Ha r iI Ill. (1992).
2) Chemicalmethods:Ok a g b ue and Lewi s (1984 al treatedthe yeastcell pe llet wi t h 2NHC! fo llowed by mildheat; treatment in a boilingwa t e r betr, for:-2eunuc ae andthenrap i d cooli ng and extraction; Okag bueandLewi s (1984b l used autol ysis, of
£.~,in di st i lled wa t erand O. 02Mcitr atebuffer wi th or wi tho utdi thio t hreit ol as ame thod toex t r a c t astax a n t h i n from the yea s t cell s; Bonnerll.21. (1 94 6 ) shoo k thecell pellet withbenzene and 20% KOH in methanol.
3) Enzym a ti c me t ho d s; Joh ns on gt, 2.1. (1978) ind uc e d the lyt i c enzymesof~ ~WL-12 bythe growthof th e org anismon heat-kil l ed
e.
~ce lls.Okag bue an d Lewis (1 985) used a mixed culture of ,g. ~WL-12 and E.rh o d o zyma to render the yea s t pigmen t extrac t a b l e by the bacterialenzyme comp l e x . Gentles andHaard (1991) treatedth e E.~cells.,litha commercial enz yme fr oma mutantof
1.
Trichoderma~ (funcelase enzyme) for the extractionof the pigment .
B. Separation of carotenoids:
1) Column chromatography: The adsorp t ionof pigments onto packed columns of powdered solids was first appl ied to carotenoids by Tswett (1906). The adso r bentsmost commonl y used for carotenoids are calciumcarbonate,magnesiumoxide, calciumhydroxide and aluminum oxide.
2) Thin-layerchromatography (TLC\: For the TLC, ma g n e sium oxide(Sa do ws kiand Wojcik,19B3), silica (Singh l l l l . 1973) and kieselgel (Liaaen-Jensen and Andrewes, 19 85 ) are the commonly used materials and different developing systems are used dependingon the polarity of the pigment.
c. Identification andcharacteriza t ionofcarotenoids:
1) Ultra-viQ le t vi e i b l e spectroscgpy: The characterist ic max ima l absorption peaks in visible spectrum giveo valuable information about the kindof carotenoid. The characte rist ic absorptionspectrumis definedbyth e numberof doublebonds, various additionalstructural featuresandthe type of solvent used (Vetter~2.1..19'71) .
2) Inf ra -red spectroscopy lIBl: Th istechnique is not used extensively in the carotenoid identification.It is used for theas s i g n me n t of different functional groups and different carbonyl functions, an d alIeneearereadi lyrevealedbythis technique (Bellamy , 197 5 ) .
15
3) Maag spectroscopy (MS): Th e MSspec t rum re veal s thre e kindsofinforma t ion , na mely , molecularweight andel e me nt a r y co mpo s ition of a compound isob taine d,struc tu r a l fea turesare de d ucib l e fr o mthe fragmentat ionpatte rn and theproof ofthe ident ity of diff eren t sample s is pos sib l e (Ve t t e r g.t. gl.
1971 ).
4) Hig h-p e rf o rm a nce l iqu id chromatog r a phy (HPLe): In the carotenoid field,theuseof this techniquebegan in 1971when St ewart and Whe a t o n reporte d the se p a r a tion of comp lex mi xtures on pre c i pitated zinc carbonate and magnesium ox i de usingsteel columns .Some of the advantagesof HPLCare ra p idanalys i s time , high se n a itIv dt.'~, high res olving power , high reco very, non - de structive con d ition s (Ta y l o r II ill.
1990 ) , sele ctiv i t yandef f ic i e ncy(Ste wart andWheaton ,1971 ). Sili c a and bond ed nit r ile columns are co mmonly us ed for normal- pha s e HPLC. In re c e nt years rever s e-ph a s e HPLC is usual ly th e me tho dof choi ce; al umi n a co lumns ar e commonly used , typi cal col umns being 15-30 cm in len g t h with an inte rnaldiamete r of3-8 mm. Guardcolumn s whar-hare 20\' of the le ngth ofanalyt icalcolumnare als o employe dtoprotect the life of theco l umn (Ta yl o r.e.t.ill. 1990 ).
5) ~: In this technique , HPLC separation and mass spectral ampli f icationis achievedin one procedure .However, it entails high cost of instrumentation and maintenance
(Ta ylor .c.!;.~!. 1990 ) .
"
6) ~:Thistechniqueis usuallyemployed wh e n high amounts of contaminants are pre s e n t in the carotenoid mixt ures. Here, mass spectrometer provides two sequential stages of mass separat ion and hence chemical analysis, separation and identificat ion are obtainedinone technique.
TheHPL C-M S-M S hasbeen used for the Ldent LfLcatton ofa-and a-cerccene from the algaI2.J.m..ili!;.ln.J..iDA (Taylor IIli.
1990).
7) ReponanceRaman (RR) spectrgscopy:This techniqueisused forinvesti ga t i ng carotenoid-proteininterac t ions (Salareag,t.
Al. 1977) .RRspectroscopyrevealsthe specific vibrational mo de s of the chromophoreeven whe nit is presentin a complex biologicalmedium at a low concentration (Merlin, 19 85) . RR spectroscopyhas many advantagesoverIR spectroscopy,namel y, spectra are obtained in aqueous solutionsaswate r exhibits veryweak Raman li n e s; time resolution reduces the analysis time; analysisof very small amounts of materialinclude d in a heterogeneous mediumis possible ; spectracan be obtained fromsingle liv i n g cells (Merlin, 1985).
8) Circular dichroism (CD): The CD spectra are us u all y employed for carotenoid·protei n complexes (Za g a lsky ~ll.
1983)andre v e al the geometrical configuration ofa carotenoid (Goodwinand Brit t on, 1988).
9) Nu c l ea r ma g n etic re s o n a nc e (NMR1:Thisis a powerfu l techn i quefor carotenoidstructureelucida tion.The ca rotenoid
17
end group as s i gnme nt isposs i blewiththe1H NMR (Goo d win and Britton,1988).Howe v er, locat i o n ofcis-bo n dsis poss i ble by
»cNMR(Liaaen- Jense n and Andrewes, 138 5 ) . Anumber of oneand two dimensiona l NMRte chniqueshave be e n desc ri be d byEnglert (1 9 9 1 ) •
1.3 .3 Yeastas • source of carotenoidsand i t . nutritive value:
The pink to red colorofthe flesh of salmo nids is of economic importance due to consumer preference for colored fish .SalmonidaquaCUlture has increas edoverthe years and so has the use of carotenoid pigments in the fish feed. The dominant pigment source used in aquac ulture is synthe t i c astaxanthinor canthaxa n thinwhi c harecommerc ially pr odu c ed by Hoffman LaRo c he (Ba s l e , Swit zerland )and mar ketedunde r th e tr a d e names of 'Carophyl l pink' and 'Ca rophyl l red' re e p e c t fve Ly (To r r issen II a1,. 1989).Crust aceanby- p roducts ha v e also been used ae an al te rnat ive pigme n t sourc ealtho ugh be c a u s e ofth e irlow astaxanthin contentandhigher mine r a l conten t, they havea limitedpotential (Johnsonl ll l.1980). The gr e e n algaehi gh in astaxanthin havebeenused to co lor salmon. However, the i r high concentration of asta xa n t h in estersnecessitatesthedeve lop mentof a suitab l e hydrolys is pr o c e s sto inc re a sethe amou ntof fr e eastaxan t h i n (To r r i s s en n a1,. 19 8 9). In re c e n t year s yeas ts have been tried as a
,.
pigment source for fish and poultry . Lai ne and GyUenberg (1969) us e d Rhod ot.orula sanneii tofeed rainbow trout an d found thatthe yeastyiel d ed poor weigh t develo pme nt in the fis hbut the color ofthe fish was enha n ce d . Savolainen and Gyllenberg (1970) fed RhodQtorula .u.nn.a.il pre p arati on s to ra inbo w trout. However. they did not re po r t any increase in the fishcolor.JohnsonAt.Al. (1 977 &;19 8 0 ) us e dly o p h il iz ed and fr ee ze-driedE. ~to feed ra i nbowtrou t and the asta xa nt hinJe vefwa s found to increasefrom 5mg/ KgtolOrng/Kg bo d yweight. Johnsonitt.g l. (1 9 8 0)also used the abo v e re d yeastto rtgment eggyol ks of la yinghens and Japanesequail and reportedthat astaxanthin frombroken yeast or prepa red yeastoil but notfrom int a c t yeast cells was deposited in the eggyolk s.
1.4 The genus Rbodotorula:
The genus ~ bel o ngs to the family
cryptococcaceae (Kreger Van-Rij, 1984) and sub-family Rhodotoruloideae (Lodder and KregerVanRi j . 1954) . 1.".1 Descriptionof czyptococcac e a s:
Budding yeast celle are always pr esent ; however, pe e udomyce lium, true myceliumandarthrospo res maybefor med. Cellsare hya line, or re d , ora ng e oryellowdue to ca rotenoid pigments. very seldom brown or black. The break-down is
19
strictly oxid a t ive or oxidative and fermentative (Kre g e r van- Rij, 198 4 ).
1.4 .2 Descripti on of genusRhodotorul&:
As describedbyHarrison (1928 ) , me mbe r sof the genus show no fermentati on, nitrate is sometimes assimilated. inositol is not assimilated, starch-like compounds are not pr oducedand urease is produced.
Kreger Va nRij (1984) describedthe membersof the qenue as cons i s t i ng of spheroidal, ovoida l or elongate cella. Reproduct i on isby multilateralbudding and strains of some species form pseudo- or true mycelium . Ascospores or bal1istospores are no t forme d . Red or yellow ca r o t e noid pigmentsare synthesi zedin malt agarcu l t ure s. Many strains havea mucou sappearance due to capsule formationbut others are pasty, dry and wrinkled.
1.4.3 Sexua l it y in Rhodotorulal
The members of the genus do not form aaccspo res or ball istospores and reproduce vegetatively. Rhodotorula has beenrepo r t e d to have a haploid lifecycle (Fowell, 196 9 ).
Kreger Va n-Ri j (1 969 ) reported tha t the above genus may represent life-cycle stages of basidiomycetes. Fell (1970) provided biochemical evidencethatcert a Ln species of~ , Rhodotorula and Sporobolomvces related to the heterobasidiomycetes rather than to the ascomycet es. The basidiomycetousorigin has alsobeen shown by HamamotoU ll.
20
(1987) . The perfect forms of a number of atrains of R.
~ have been found and trasferred to Rhgdosporidium
~ (Ba nn o , 1967). 'rbeee strains had oppositemating types, dikaryotic mycelium with clamp connections and ch'lamydcapoxea ,
1.4.4 COWD.ercialimportanceof Rho dotorula;
1. Some species of the genushave beenfound to metabolize aromatic compounds while others produce glycolipide co n t a i n i ng polyhydroxyalcohols (Spencertt !!l.1988).
2. A Rhodotqrula sp p. isolated from so il hadthe capacity to degrade aliphatic and aromatic hydrocarbons and hence could be used to tre a t oil sludge (Sh a ilub h ai §.t.Dl.
1,984)•
3. Milkclot t i ng enzymeshave beeninvestigated from strains of Cryptococcus and Rhodotgpl]a. The milk cl o t t i ng activitytogetherwith proteolytic ability could be used in cheese manufacture (Fe de r i c i , 198 2).
4. Some strains of E. ID.inl.lll were found to pxoduce isobutene, the starting ma t e r i a l in petrochemical industry (FujiiIIU.1987) .
5. Lipases have been reported in BhodgtOIUJiii, app, by Zvyag!ntseva and Pitryuk (2975) as wel las Zvyagintseva (l972)•
21 1.5 The red yeast ~..nLlADlbI:I.
1.5.1 De8cription ofRh odotorula
.ma,
As described by Lodd er (19 3 4) . R. .D.!..t!uassimilate s eucxose, tr e halose , raffinos e,D-xylos e , ribitoland succinic ac id wh ile ga l actose, maltose, cellobiose, L-a r a b inos e, 0- ribose,L-rha mnose, D·mann i t oland citric aci d are assi milated by some strainsof th eep e cie s (Kr e ge r va nRij. 1984).TheG+C contentis59.0-61.2molet (Ha ma mo t o J:.t.Al.1986; Nakaseand Komagata, 1971) . The ubiqu inone system is eo01D and the require'llents for bi o t in as well as p amino b e n zo i c acd.dhas be en shown to be negative (Yamada and Ko n d o , 197 3). The carbohydratepat t ern s of wholecell hydrolysatesarefoundto co nt a i n fuc o seandmannoseas thedomi na nt sug a r s whi l e ot her components inc lude manni t o l and arab initol (We i j ma n and RodriguesDe Miranda , 1988 1.
1.5.2 Sou rcesI
Rhodotorul a st r a ins hav e be e n isola t e d fro m leave s , flowers, atmo s phere, soilandma rine sources (KregerVa n Rij, 1969 )•
1.5.3 Growthandpigmenta tioninB,DW.n: Effect of cul tUral cgnditions on pigmentatig n:
11 Carbonsources:Different carbonsources ha vebe en sho wn to be effecti v e in promoting pigm enta t i on . In .8.. LWi2n. gl y cerol is ef f e c t i vein promoting ca r o t en og e n e s i s (Fr o me g e o t
22
and Tchang, 1938). However, fo r Rhod9torllJa sp. no. 10 0, glycerol was less effective tha n glucose while phenol, resorc inolandko jLc acid stimula ted&-caroteneproductionin the above strain (S i mp s o nn.5!l.197 1 ). Foranotherstrainof g. ~, th e best yields of to rularhodi n were obtainedon glycer-:>l withasparagineas the nitrogensource while sucrose gave the highest yieldsof ca r oteno i ds torulene, B-carotene and v-cerot.ene (Wit t ma n n , 1957). A strain of Rhodotorula
~was found to produce the highest pigment on carro t juice (Ve cher .e..t. s j,1965).Speciesof Rhodotorulahave also beengro wn on petroleumhydrocarbons (Ni kol a evtt li. 1966; vaskivnyuk and Kvasnikov , 1968) .Strains of R. ~, R.
~and R.mucilaginQ 9a (nowB,.~) have also been isol ate d from oil- containingsoils .
2) Nitrogen sources: One stra i n of R. ~ gave the hi g h e s t pigment yieldon leucineand glutamicacid,when used as the sale nitrogen source (Veche r ~.91. 196 7) while an another strain ofthe same yeastsho we d maximum pigmenta tion on ammoni umnitrate(S im p s on U l l .1971).Fora Rh-lOOstrain of Rh ..,d o t o r ula , the bes t yieldsof carotenoidswere obtained with valine, le u cin e and asparagine . For an isolate of g.
~, among a series of in o r ga n i c and organic nitrogen sources. the highest yield of total carotenoids per unit dry weight of cells was obtained on ammoniumnitrate (Wittmann, 1957). However, th ecel lyield was onlymoderate. The highest
23
concentration of torularhodin was obtained with histidine whilemaximum yieldsof torulene and B-carotenewe r e obtained on ammoniumnitra te while y-carotene production was the hi g hest on asparagine.
3) Light:Light has beenshowntoenhance ca rote nog e ne s i s in Jl..D!Wand
z.
~(Simp s o n IIe,l.1971).Maxwell§.t.li.(1966) showed that caro tenoids ing.~strain48-23T afforded protectionfr o mpho todynamicdeath by monochromatic light at 632 nm butnot between 300 and40 0 nm.
4) Temp e ra t u r e: With a ch a n g e in the cultivation temperature, most yeaat.e showa dec rease in the carotenoid level thoughthe rati oof differentpigments re ma i ns the same (Fr o ma ge o t and Tc han g , 1938; NakayamattAl,. 1954, Bobkova, 196 5a ) . However , two strains of 8.. ~ (4 8-23 Tand48- 23W)were shown to be pink at25 ° Cand yellowat5°C (Pha f f~
li. 19 52). A Rhod otorula rubra st r a i n also showed color change s at sim il a r tempera tures (Simp s o n gt. li. 19 7 1). cha ngeinthe cultivation te mpe r a t u r e of g.~ (str.48·
23T), from 5 to 25°C resultedin a decrease in the level of0;-
and B-carotenewhilet.he amounts of torularhodfnand torulene increased (Na k a y a ma§.t.i!l. 1954).
5) Time of growt!1: While carotenogenesis in ~
~occursintheexponentialgrowth phase (J o hns on and Lewis,1979) , pigme ntproduction in R..D.!..bu (Good wi n , 1959, 1972) as wel l as othe r caro t e nog e n i c organisms like 8..
24
~ (Vecher and KuBkova, 1968), Sporobo lomycee~ (Bo b k o v a , 1965bl, ~ bJakesleeaPlls (Leenheer and Nelia, 1991) arid~ ~ (Go o d wi n , 1959, 1972) has been shown to occur in the stationary phase.
6) Other growth requirements: Agar in a concentrationof 2.5\along withye a s t autolysate (lOt,v/v) and glucose (s t ) has been shown to enhance pigmentation in B,. .D.l..Q.u, B,.
~and many strains of crvocococcue.The liquid medium of the same composition gave good growth of the yeast but lower yieldsof the carotenoidswereob t a i ne d (Nakayama.e.t.li.
19 54 ) •
7) Effect of inhibitors: 15-Iooooe in a concentration of 250mg/Linhibited the formation of carotenoidsin R.~by destroying a-carotene andto r u l a r ho d i n when added to mature cultures (S i mp s o n II g . 1971). Other compounds found to inhib i t carotenogenesis in R.IDudlaginoRa (B.nlW)were the diphenyl derivatives (Villout reix, 1960 ).
1.5 .4 Enzymolo~yofca ro ten o gene s is:
The pathways for carotenoid synthesis in Rhodotorula spp.
have been worked out as shown (Fi g. l.8a, b, c and d). The effect of light on carotenogenic enzymes has been particu larly studied in this yeast. In g. minuta the stimulation of carotenoidbiosynthes isby li g ht was found to occur in two phases: the first phase involves a photochemical reaction independent of temperature (light reaction) and the second
25
involves biochemica l reactions independent of light (d a r k re a c tio n) as shown by Tada&Shiraishi (196 2a) . The workers also reported the followi ng aspects of photoregulative caroteno g ene sis in the above organism (Ta d a & Shiraishi , 1982b):
Thequ anti t yof ca rotenoidproduc e d as well asthe rate of carotenogenesis were regulatedby li g ht dose.
b. The photochemical product , postulated to ae rve as an in d u ce r of carotenoid synthesis, was st a b le , not me tabolized in vivo and decreased as the ca r o t en o i d syn thesisprogressed.
The s e resear ch ers alao showed that the photoregulat.ion of carotenogenes!sin R·.llIi..ml..!aresulted from the photoregula tionof HMG·Co A reductase synthesis while the enaymeerequiredfor the conversion of phytoene toca r o t e no i d pigmentswere not induced by light(Ta d a&
Shiraishi. 1982c).
1.5 . 5 Commer cia l sign ific a n c eof R.~:
1. Two R . .J.:!.!l:!.n st r a i ns were found tode g r a de 4-hydroxy be nzoateand therefore coul d be exploited for oilsludge tr e a t me n t (Wright& Ratledge, 1991) .
2. Ast r a i n ofthe red yeast has been reported to produce extracellularmannan whichwas found to have a moderate in h i bit o ry actionon the infec tiousnessof tobacco mosaic virus and hence could be us e d to control the plant
26
infections caused by this virus (Elinov.!:..t.ai. 1980). 3. A st.rain of g. rubra MGU 691 was found to produce
extracellularcapsular material that could be used in food, pharmaceutical. textile, paper, oil refi ning,paint and varnish Lnduat r Les fGolubev~21. 198 0 ) . 4. Productionof extracel lularproteases was reported from
a strain of B. ~. It WAS speculated that these proteases could be used in degradation of proteins remainingin beer and wine that form ha z e s during storage (Ogrydziak, 1993 ) .
27
Table 1.1. Carotenoidsused asfoodaddit ive s Food products
A. Bakery productsI Cinnamonrolls Frozen yeast dough Yeastbun s Kaiser rolls Shortbreadcookies Wafers
Doughnut Softcookies
pigme nt Amountused
a-eeeeeene g/100Ib 12.5 32.51 11. 9 12.2 12.6 21.26 0.08 1.0
S~c aro t en e 30t
a-eeeeeeee 3.6\
B. Beveragssl Orange flavoreddrinks, fr u i t ju i c e blends C. Fat and oi l productsI
Margarine, process cheese, winter butter, pop p i n goil
D.Dairy products: a-ceeeeene
Imi tation milk, whipped topp i n g s, fluid&dry coffee whdtieriez-s, sourcr e a m, frozen desserts, eg, custard, water ice,fruit sherbert&:ic e milk E.Frozen&:dried egg So-carotene
yolk products: Frozen yolk, frozen whole egg&driedyo l k
F.Popcorn S-carotene
G.Tomatobased products: Canthaxanthin Toma to soup, vegetablecocktail
spaghetti, barbecue&:pizzasauce
H. Dry spice&:breading mixes Apo-caroten.l I.confectioneryproductsI
Candy&:fruit jel l y S-carotene
Apo~carotenal
Ca n thax anth i n Gordon (1982)
0.1-1.0ppm
110ppn
40ppm
.,
0.0005%
O.OOlt 0. 0 04%
28 Table 1.2. Worldwide carotenoid markets Carotenoid
rate
$ million 19 8 9 19 9 5 ·
Annual growth (>'
s-carceene Other carotenoids Total
60 45 105
100 95 195
8- 10 10-15 10-1 2
projectedin 1989 dollars Taylor (Personal communication)
Table1.3.Ta x o nomyof yeasts.
29
Su b·d i vi s i o n As comycotina
8asidiomycotina
Deuteromycotina (Form su b-division)
(Kreger -vanRij, 1984) Class Hemias c omyc etes
Blastomyc etes Ord e r Endo myce t ale s
Ustilaginales
Tremel l ales
Family Spe rmo p h t ho r aceae Sacch aromyc e taceae
Fi l ob a s idi aceae Telios po r e forming yea sts Sirob asi d iaceae Tremel laceae
Cryp toco c c a ceae Spo r o bo l omyc et a ceae
- -
>· t
.~
~~~
i~
•3 ~I~•
H
J::~~~~~ 'gil]:SSg
~~::~~
i~ l~
Ii ~~ !~
*
e'i
g]
i l~
~~~~,5o!lJ I
~j ; [~ a
31
Table 1.5. Diff ere n t iatio n of re dyeas t s (Davenport, 198 1 ; Komag atafl.t.U.19B7).
Organisl" I F N M SS T B Principal habit a t Su bdivision
.Ellil.f.lJl + Tre e exuda t e s and Deute romyc ot in a
cactuspl an t s (R)
Cryptococcu s + ± Various (e) Deu t eromycot ina
Rbod g t orula ± + Ubiquitou s Deuteromycot i na
gbodo s cor-Idf u m- ± + + + Anta r c t i c sea Basidi o myco tina
wa t e r orplant surf a c e s (R)
SDorobo l o~± ± + Pla ntsurfaces/ Dcute rom ycot i na
air (e)
Spo ri di obol us ± + + + + + Pla n tsu r f aces (R) Basidiom yc otina
~ + soil Deu t eromycot i na
I- assimilatio n ofino sitol; F. fe rme n ta tionof glucose;N. as simila t ionof nit rate ; M- myc eliumand/or ps e d omy c el ium formed; 5S- sexualstates; T. te l iospore s ; B. ballistospore s; R. ra re (usuall y ne e d specialcul turalconditions ; c-common;±_...&.-strains; ~• al l st rains neg a t ive ; + • al l stra i nspos iti v e
32
Pig. 1.1. Cyclict.etrapyrrole
A , A r , , , A '
Pig. 1.2. Lin••r htrapY%'role
Pig. 1.3. Indolic biochrom.
J3
IH~~;•.
JJ,..
~II~~
•:',,..~y~~:
JM~~~
• " f
"'~
~" ". I .
" 11NO liN' II.
"
Uft•••••~...I.,
I'
t •
'ex'~~,
' .
~,l'f~h
- -
... ..-..-
"
: 6c: . .. b :
.... -- ... -
. .
.Q<:c..
1 , ',
II I.
1tOOC...~11
I••
• . r
~.~(lI••••COClIlI; .f••1I)
rig. 1.4. N.let:e rocyclicbiocJtto.ea
~
.; ,I
b ~ Is'
'4 1 ' "
.LV--, . . .
J34
co·
.... 1vO
¢ .
' , -
,1Q. 1. 5. Flavonoid.
e¢ .
1. 4M. . . .
Pig. 1. 6.QuiDon••
~ ~
--
35
AlTAXANTtnH
~
e.uau lo'l.lo. CAIIlO'l'OII,~
p _~ .~
. .
1OllVID'
'00...-.
36
Fig. 1. 8a. Conversi.onofaClftyl a..nd acetoacetyl CoA to geranylgeranyl pyrophosphate (Simpson ~ !!!l..
1971; Britton, 1983).
Acetyl-eoA+Acetoacetyl-CoA
S-Hydroxy-S-methylglutaryl-CoA (HHG-CoA)
Mevalonic acid
Mevalonic acid 5-phosphate
Mevalonic acid S-pyrophosphate
:Isopentenyl pyrophosphate
'Y-'Y-Dimethylallyl pyrophosphate
~ +Isopentenyl pyrophosphate Gsranyl pyrophospha te
-It
+Isopentenyl pyrophosphate Farnesyl pyrophosphate" +Parnesyl pyrophosphate Geranylgeranylpyrophosphate
37
Fig.. 1. 8b.. Conver s i onofgeranylgereyl pyrophospb.teto cis-phytoene 2 X Geranylgeranyl pyrophosphate
~ (Albnan!!1y . 1972; Qureshi!!1Ai. 1972) Prephytoene pyrophosphate
~ (AltmanISy. 1972,Qureshi.!!..t..!.l.1972) Cycle propylcarbinyl cation
~ (Cama ra.atl l. 1980,1982) Cis-Phytoene
Fig.. 1. 8e. Conv ersion of cis-phytoene to
torulene lin dtorularhodin (SimpsonttIA. 1971) Phytoene
~ Phytofluene
~ r- Ca r o t e n e
~ Neurosporene
" 'It
Spirilloxanthin4- Lycopene 'It
S-Zeacarotene
'V- Ca r o t e n e '" a-Carotene
Plectaniaxanthin'"Torulene
~
17'-Hy d r oxyt o r u l e ne
~ 17'-Oxotorulene
~ Torularhodin
e.-Carotene
3B
Fig. I..8d.conversIon at phytofJ.afJ to astax4Dth1n (Andrewe.g .I.!.I 1916; JaMsoD aDd An, 1991).
Torulene
.
Phytofluene t
1
-caroteneI
Phytoene
Neurosporene
! "<,
Lycopene £-Zeacarotene
I -., - _....
./ < ,
B-carotene
Echlnenone 4-Keto-torulene
3-Hydroxy- HDCO
=~~_'M'~/~CD\
"I ~: • ...I
1fTrans-Asta~anthin cis-Astaxanthin
39
RESEARCH OBJECTIVES
Dueto therisi ngcost ofthe syntheticpigments ad de dto aquaouLt.uz-ediets, an escalating concern among the ge neral pubkdc regar di ngtheir safetyinfoods, and more stringentFDA regulations,morestudiesarebeingconducted that reportthe us e of natural sou rcesof the s e pigments. One of the ar eas whe r e the pr e s e nt research was focused wa s to examine the possibility of using the new Rhodoto rula ru bra is o l at e reported in this thesis, as a carotenoid source for aquac u l turedfish, andto compare it ' s eff icacy with syn thetic pigments.
The objectivesof my research we r e :
1. Id enti f i c a t i on of the unknown yeast strai n that isolatedfromyog u r t.
2. Studies on the morphology and ult rastructure of the is o l a t e.
3. Ide ntif ication of th e major pigments produce d by the isolate.
4. Studieson the sexualityof the yeastisolate.
S. Studies on the growth pa r a mete r s onche a pe r substrates including industrial by -products.
6. Genetic studi es invo lving mut age n e s i s with nitrosoguanidi n e.
7. Studieson the kine tic s ofgrowth and pigme nta tion.
