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CHARACTERIZATION OF Rkodororula rubra TPI MUTANTS

by

Subhashini Mauidi, B.Se.

Thesis submitted to the School ofGraduate Studies

in partial fulfillment of the requirements

for the degree of Msder of Science

Department of Biology

Memorial University of Newfoundisnd.

April 2003

SL John's Newfoundland & Lnbrsdor

ABSTRACT

Carotenoid pigments exist in nature and are w~dely d~stributed as colourants

throughout the btologtcal systems, such as microalgae, insects, blrds, fish and

orusmeans. They are rqonslble for tnterestiog colours seen in various pans of these

organums, which play a great role in the biological functions like phatoreceptlon and

photosynthesis. Carotenaids are malnly used as ptgments for colouration of food

products and phameuticals. They also function as antiaxldants and help m minimizing

membrane-damage, and in controlling human dlsesses such as cancer, cataract and

atherosclerosa.

Astaxanthin 1s a red orange camtenaid pmduced by aquatic organisms such as

algae and is also found in yeasts hke Pha@ rhodorynn and Rhodotomla mbra. It a

used as a plgment m feed for salmon and shellfish and also enhances nnmune response of

fish and shnmp. Among yeasts. R mbro TPI is a good source of red ptgment and whole

cells induce pigmentation in fish. It has been shown in earlier work that R.mbra han

faster gmwh rate, shotter mcubation-time and ytelds more biomass than Prhodowma

Further, previous fcedmg-trial experiments carrisd out using rainbow trout have b-

successful and therefore R. mbrn TPL has economic potential.

In the current work the mutants MI, M2 and M3 of R mbro TPI were

characterized and their propertics compared with those of the wlld type yeasts. The

optimal pignent production was defemlned by growing the mutants and wild type yeasts

under different growth eandltions, such as different substates, temperamres, initial pH

and light. The maximum plgment recovery was achieved by using diffprent ertractlon

methods whlch include French Press method, Frccm- dried cells, soni~atlon and

enzymatic cell breakage. The spcch.ophotomoter graph and Thin Layer Chromatogmpy

(TLC) techniques were used to estimate thc total carotenold can~entration and to analyse

the pigment in each sample.

The experimental results showed that light enhances pigment production. Yeast

malt broth with peat exmct as a nitmgenous source showed more biomass yield. Bacto

craper dox bmth was found to be inhibitmy to growth of the mutants of R. mbro TP1.

The cells gave mon pigment at 25 k in the initial pH range of 5.0 to 7.0. The French

press method was found to be more enlcient to extract the optmum pigment for MI, M2

and M3 with values 250.6, 254.4 and 193.2 &gig, respectively. Mutant 2 alone gave

higher reoovery of tbe pigment with Freeze- dried method. Sonication method gave less

pigment recovery. The enzymattc method with a pH of 7.0 for all mutants gave recovery

values of 184.4, 164 2 and 1294 pgig for MI, M2 and M3, respcctlvely. The pigment

analysis eonflmed that all the mutants contarn Pcamtene, torulene and tomlarhodin

carotenoids in their pigments.

TABLE OF CONTENTS

Abstract Table of Contents List of Tables List of Figures List of Abbreviations Acknowledgements

CHAPTERI. INTRODUCTION

1.4 Appl~cat~onr of the catotenoids 1.5 Taxonomy of Rhodoloruld mbba 1 6 Muiagenesis 1.7 Astaxanthin 1.8 Significance of caratennndd 1.9 Camtenogic yeasts as sources ofcamtenoids 1 10 The genus Rhodotomln 1.1 1 Commerz~al Importance of Rhodolorulo species 1.12 The Redyeast,Rhodaroruln mbro TPI 1.13 Description ofR, mbra TPL 1 14 Potential commercial applieauans R. mbro TPI 1.15 Research objectives

CHAPTER 2. MATERIALS AND METHODS

2.1 Chemicals 2.2 Sources of mlcrwrganisms 2.3 Peat extract and cane molasses 2.4 Lyslng enzymes 2.6 1 Prepmatlo" of media and ~noculum 2.6.2 Gmwlh culhlres and harvesting ofyeast -11s 2.6.4 Gmwh on molasses and peat sub-ate$ 2.6.5 G m h measurement and aeneratlon tlmes

- . ? 6 J 2 Pi~ment crlr~;t~an. ulmg Free,e,lnlng 2 6 ') 3 t \lr8ctlon by ron8camn m:lhJJ 2 6 Y 4 P~ymcntricrauan by ul lu; rn,ynr< 2 11 9 J I h:e,c and Thrv mcthuJ 2 h Y I 2 cncit 01 II,,UI aq p8sn,eot moV:O 2 6 1, Ali~ruwmcnl oip.@!rr~r pnwcJuw 2 6 I I I hln l avc. Cl~rotnal,pr~ph\ 2 6 1: SlladsJ cnor ~n ll8e r l i ln

CHAPTER 3. RESULTS AND DISCUSSION

3.1.1 Growth on cans molasses and peat extract 3.1.2 Effects ofthe medm on biomass yield 3.1.3 Effects oftemperamre 3.1.4 Effects of initial pH 3.1.5 Effect8 ofLight 3.2 Effect of extraction methods on pigment recovery 3.2.1 French Press 3.2.2 Freezedrying 3.2.3 Sonlcation 3.2.4 Extracoon usmg enzymes (i) Effect oflytic nvymes on pigment release (ii) Effect of Buffers (lil) Effect of reducing reagonts (1") Effect of freezing and thawing

CONCLUSlONS REFERENCES

Table 1. Biomass yleld (&) in wild type and mutant yeasts at vanous temperatures 39 Table 2. Rf values of Carotenoids fmm various yeasts 47 Table 3. Effect of inilial pH on b~omass yields in yeasts. 50 Table 4. Absomuon soectra ofthe veast samoles. 54 . . . . Table 5. Effects of lys~ng enzyme from different sourees.0" pigment recovery 68 Table 6. Recovew of carotenoids by lytic enzyme (R. soloni) m Tns-HCI buffer 69 Table 7. Recovety of caratenads by lytic enzyme (R. solonr) m citrate phosphate

Buffer. 70 Table 8. Recovery of caratenotds by lyfic enzyme (R. solanil in DiUliothreitol (Dm. 72 Table 9. Recovery of carotenaids by lytlc enzyme (R solanil m Beta mercapto ethanol

(BME). 73 Table 10. Recovery of carornoids by LyUc enzyme (R, solonfi by freeze and thaw

method. 74 Table 11. Recovery of camenoids by enzymatic breakage and French press method. 79

Fig. I. Chermcal srmetnms of camteno!ds Rg. 2. lsoprenoid pathway Rg. 3 Chemical sbuctuie of Polyene chat" with a vanatton in the end groups Fie. 4. French nresr nracedure for olmnent extractton ~D - r ~X . " Fie. 5 . Freeze drvlne orocedum for exWaction . -. Fig. 6 Enymatlc cell breakage procedure for pigment extraction Fig. 7 Genemuon tlmes of yeasts Wg. 8. Growth of yeasts on YM-hmlh at IS 'C Fig. 9. Growth of d l f f e m yeas* on YM-bmU1 at 25 OC Fjg. 10. Tlme needed for achieving stationary phase for yeasts in llquid cultures Fig. I I Effect of 1 %peat m YM-broth on biomass yield Fig 12. Effect of 2 %peat extract m YM-broth on biomass yield Fig 13. Effect of3 %peat emact on biomass yield k ~ g I I I fi:l .>f;arhon WLICC on b#u1113,, )#<1.1 F y I5 k!lc:l ~r .~ghc un p#(rmcnl prod ~;llnn Fig In F!icrl n! Jar'<cs.% ~n plgtncnt proda:l\on b ~ g 17 Cllrir o f rwnrh PC:,, .,n ppmenl re:o%cr). ~n ~.ulrlcr Ihg lh I N<.t .,f I rei,c.dnlng on plgmmc rr:u\ :ly 8s 111 the ~rolarc- t ~g I9 Lnect 01 runl.at . n mnh.3 l I I p#g,l#cal rec.ncq .n all ~ h r ~ s ~ l n c , I.#$ I L I n;.s~~fcxlart~.~nnlclh~lJ~unpl~ma~lrc.d\en 60n> IPI I:#$ ? I P . : r u~ r~ ~?COL.LT\ imnl h lu l~nl I U ~ J C I (I Nrw!ll e\cncllon n l c l h ~ b I.#$ 22 I'.snlear rrcovrq f r ~ m hlucrcc 2 unJ:r (I>lPrctnr c\trdcl#nn mrlh <LI Fir 23 P.anc?! reco\cr\ from blrranr I unJ:r JdTcrml r&lmclton nrrhod; Fie. 24 Pigment recovery from Rm yeast, under vanous extractton methods - . h e 25 P t!meni w:.n :ly (rom KI yclrl under \anl>L,r\lnalun ntcll~vds t 19 20 Iv!ot o f r ~ h ~ l r r c r .'.,ncr.18rclon \errus opt~.'ll d c n r . ~ fdr ~ruhlc* ry 27 PI.., or a w ) m e eorlccntn~~.ln v r r ~ u \ ~ p c ~ ~ 11 dcr8-11) for ~<olali.

ABBREVIATIONS

Yeast Malt Broth Bacto Crapen Don Broth GramJLitre Micro gram Thin Layer Chmmatograph) Nanometer Beta mercapto ethanol Dtthlothreioml Degree centigrade

YMB BCDB pn rgig TLC nm BME DTT Oc

ACKNOWLEDGEMENTS

I express my sincere gratimde to my supervisor Dr. T. R Patel far the supervision

of the res-h projed and for his advice, encouragement and financial support. I dso

thank the other members of my supervisory committee, Drs. A.M Mart," and Jyotl Patel

for their interest in my research project and for making useful suggestions.

I am gratefd to the Schwl of Gniduate sludles, Memorial University of

Newfoundland, for their financial support

I acknowledge the technical assistance provided by Peter Earle, Gany

Colms, Kevin Snow, Kathy Anthle and Willlam Brown of the Biology Depament. The

adminishative coopeman provtded by Sham Quintan, Patncta Squires, Shirley Kenny

and Christine Everson is also appreciated

Special thanks are extended to my past and present colleagues in Dr. Patel's Laboratoly

and several of my friends whom dtverse ways made my stay m St John's enjoyable.

I appreciate and acknowledge the affection, encouragement and inspiration

provided by my loving parents, aunt, uncle and other members of my extended family.

INTRODUCTION

The quality of food, aside from the microb~olog~eal aspects, is generally based on

its colour, flavour, textore, and nuvittve value. Depending an the panicular food, these

factors may be weighted differently in assessing overall quallry. However, one of the

most important sensaly quahty attributes of food is colour, because no matter haw

nuumous, flavourful, or well terhlred a fwd is, it is unlikely to bc accepted unless it has

the appropriate colaur. The acceptability of food a remforced by economic worth since

in many cases raw food materials are judged on the basis of their colaur.

Pignenta an chemical compounds which reflect only certain wavelengths of

visible hght, mslang them a p a r ''colourful". Flowers, orals, and even antma1 skin

contain pigments, whtch give them thew colors The abihty of pigments to absorb light

of certain wavelengths is more important than reflection by them.

The term 'ptgment" is used to refer to a material of known or unLnown physical

state or to an onanalyzed coloured material (Sangha, 1994). Colours of various

carotenolds are related to the number of alternating carbon-earban double-bond pain in

the long palyene chain of the mobcule, known as the chromophore (Fig. I).

Speeitieally, list energy ir absorbed by the carotenoid polyene system between 4W -

700 nm, and is converted into vibrational energy and heat. Each cuotenold has a unique

resonance in this regard (for, 1976) through the lsoprenosd pathway Fig. 2) and they

produce dtverse compounds such as essential fatty acids, steroids, sterols, and vitamins

A, D, E, and K.

Fig. I Chemical structure ofCamtenoids (Hari efol, 1992)

4 CS lsopentyl pyrophosphate

+ CIO Oeanyl pymphosphate

CZO Geranylgeranyl pyrophosphate - Phylol

'ig. 2. lsoprenoid pathway (Tan&a.,1995)

3

Withln the varlaus classes af natural pigments, the carotenoids we the most wide spread

and slnrchlrally dtverse pigmenting agents They are responsible, m combination wlth

pmterns, for many of the brilliant yellow to red colors m plants and the wide range of

blue, green, purple, bmwn and reddish colors of fish and crustaceans. The general

distribution and metabolic pathways of carotenoids have been extensively detailed

(Goodwin 1984) Camtenoids are wtdespread thmughout b~ologeal systems They are

found in the plants, algae, baetena, animals and fungi (Goodwin, 1980). Several species

of yeasts produce earotenoids and are grouped as the 'red yeasts'. These carotenogenle

aseomyeetes, basidlamyeetes and deuteromycctes all tend to accumulate predominantly

hydrocarbon eamtenoids, such as beta-eamtene and gamma-eamtene (Goodwin, 1980).

1.1 Manifestation of calour:

Colour is displayed by organisms in rwo ways, namely, (I) physically, by

colourless panicles or ultramicmscopic stlvchlres called "schemochmes", and (2)

chemically, by naturally occumng chemtcal substances possessing a coloured molecule,

called "biwhmmes" (Fox, 1979).

Schemachromes are exhibited by both ealourless, randomly scattered, hghl-

diffracting submicmropic bodiss. These givs lise to the TyndaIl blues of scattering and

various striations or ulvathin successive tilms or layers which resalve incidcnt light lnto

its components producing interference colors (Fox, 1979).

Biachromes absorb wavelength, while reflecting andlor transmitting other

wavelcngthr of vlslble light (Fox, 1979). The shuchlral fsaturc of a biochmme

responsible far the absorption of light a the chromophore. For example, in carotenoids

the chmmophore a the conjugated chn-carbon double bond systcm.

Other functional groups or substihlents m a biochmme, which possess the ability

to modify the absorption maximum of the molecule are termed aunochromes. Vision in

humans and animals is a complex chemical phenomenon. The human eye, for example

is roughly spherical with an opening to admit light, which falls on a rear surface lined

wtth millions of cells. The molecules responsible for vision are altached to the cells.

Discnminatton behveen colours is possible because cone cells occur in three groups:

thasc receptive to blue light, those receptive to green light and those receptive to yello-

red light. Each type can absorb light in a range around its primary color. When an object

absorbs these wavelengths (visible range 400 and 750 nm), certain molecules within he

object become excited. A molecule is excited when one of ~ t a out= orbital clecvons is

raised to a higher orbital. These eicctmn transitions ere charactenstic of most biological

materials but are psrticulariy pronounced in biochromes (Needham, 1974a).

1.2 Major Pigment Types.

There are sin major groups ofpigments occurring in bioiogial systems. These are

carotenoids , tetrapymies, indolic blochromes, N-heterncyclie biochromes (other than

tctrapyrroles), oxygenous heterncyclic biodvomes (the flavmoids) and quinones

Carotenoids are nature's mast widespread pigments, with the earth's annual

biomass production esttmated at 100 million tons (Fennoma, 1996). In n a h m over 560

~arotenoid shuchlres have been identified and compiled. They denve their names from

the fact that they eonstihlte the q o r pigment in the carrot root, Do- C Y I O I ~ , one of

the first foods observed to possess this class of pigments (Klaui el ol., 1981).

1.2.1 Funcflons:

Most of the functlans of carotenoids are a consequence of thar ability to absorb

vtslble Ilght. It has been established that carolenoids play a role in photoreception

(vaton), photosyntheis, photoprotection, phototanis and ~ntegumental colors (Bumen,

1965; Needham, 1974; Goodurn, 1980; Sangha, 1994; Brillon et n l , 1995). Thc

luminous carotenoid eolours of topical fish are not only keys for species ~denttfication

and mating signals they have significant physiological roles as well. The spasonal

astaxanthtn levels in the carapace have shown that the egg, parallel with the exposure to

sunlight, indieatlng that the carotenotds serve to protect enema1 proteins and eggs from

u l m v ~ ~ l e t exposure. Beta-carotene is convened to vitamin A, which is required for the

biachnnlcal processes rnvolved in vision (Goodwin, 1980). Furthermore, vitamln A

plays an important role in the growth, development, and integrity of mucous surfaces.

However, the majority af research concerning askanthin and 0 t h ~ carolenoids has

been aimed at irs mle in photoprotection and as an antioxidant in quenchzng of oxygen

radicals.

Carotenoids owe thetr wlor to tbc absorption of hght by the feahlre of their

molecular structure ! a w n as the 'chramophore'. In most carotenoids the chromophorc

consists entirely of a conjugated system of carbon-carbon douhle bands, referred to aq

the 'polyeneehain' (Fig. 3). It ia possiblc to have up to I5 conjugated double bonds m

the chmmophore of a C40 camtenoid, although srmchlns with 7 U, 1 I such bonds a n

Fig. 3. Chermcal struchlre ofpolyene chain with a variation in the end groups. (Weedonet al., 1995)

more common. Other fearures of carolenold molecules that may constitute part of the

ehmmophore an mple bonds, temlnal allene gmups, substituted pholyl end groups, and

earban-oxygen double bonds (Weedan er 01.. 1995).

1.3 Appl i~st lon~ of Carotmoids:

Carolenoids have commrrclal appllcallan m varlour industries such as

aquaculm, food indusky, phmacculical, cosmeuc, and medicme (Baucrnfemd and

Klaui, 1981; Munzel, 1981: Sangha, 1994). The use of carotenoids as pigments in

aquaculture is well documented. It appears that their broader functions include a role as

an antioxidant and provitamin A activity as well as enhancing immune response,

reproduction, gmwth, mahlration and phatopmtection. An extensive body of data

stresses the vital role of carotenoids in the physiology and overall health. It concludes

that carotcnoids are csscntial nulnenls that should be included in all aquatic diets at a

minimum level of 5 -10 ppm pornissen, 1989).

1.4 Taxonomy of Rhodororula rubm.

Yeast is defined as a unicellular fungus that reproduces by budding ar fission

(Kregcr van-Rij, 1984) Yeasts are Lvronomically divene and classified in the divtson

Eumycota, which lncludes the classes Ascomycotina, Basldiomy~otina and

Deuteromycotina (Krcger van-Rij, 1984). The ascomycetcs are recognized as

unplgmented yeasts possessing asci witb ascospons, and reproduce by holoblastic

buddtng (Kratochvilova, 1990)

The system of taxonomy used today is the result of the development and

integration of various avenues of approach to the problem of yeast idenltfication and

classification &odder, 1970). Morphological and repmduetlve atuibutes are utilized lo

d u d e the matn faxonomy- to designate htgher m a whtle physiological evidence is used

to differentiate lower ma, and in particular, species classfication. Most of these

chmtenstlcs are defined based on a particular test, such as femcntatian and

assimilation (Kreger-van-Rij, 1984). The isolation of the mutants of Rhodolourla m b m

TPI used in this work has been reponed (Acheampang, 2000).

The uimtenoid-producing yeasfs include genera such as C v m c c w ,

Rhodoromlo. Rhodosporidiun, Spor~diobolus. Sporobolomyces, Phoflo (Johnson and

Lewis. 1979) and Sailoella (Komagata el a/., 1987). Yeasfs belonging to the genera

@plooccus. Rhodotomlo, Rhodosporrdiurn, Spotidtobolus, and Sporobolomyces

typically contain 0-carotene, ycamtene, tomlcne and tamlarhodin as major carotenoids

(Simpson el 01.. 1971). The generaRhodospridium and Rhodolomla may also produce

carotene, phytoene, and phytatluene, Z-hydronyplec!manlaxanthin have been found in a

strain of Rhodororulv ourantiaco (Lui er ol., 1973). Some species of Rhodolonrla dso

synthesize O-tarotenc, pzcacarotene and plectaniaxanthin, which are also found in

Cryploplococcus lourenrii (Lui el a1.,1973).

The yeast Phaffia produces aslaxanthin a s ilz most abundant carotenaid. Other

characterized carotenoids are Bcacotene, -+carotene, neumspame, lycopene,

echinenone, 3-hydmnyechinsnone, 3-hydmxy-3'. 4'-didehydm- 0-carotene - 4 - 1 and

phoeniooianthin (Andmues el ol., 1976).

1.6 Mutagenesis:

Several methods are available for genetic manipulations of biological cells

Newer techniques include protoplast fusion, pulsed field electrophoresis and

recombinant DNA techniques. However, dlfflculty anses in applying these methods

when genetic lnfonnation of a species is lacking. More fundamental approaches For

strain improvement involve genetic mutattons (Crueger and Crueger, 1989).

To enhance the potential of a microorganem, the genome can be manipulated by

inducing mutations m the gmomc. Common mutagensc agents include ulmmolet and

ionizing radiations and chemical agents mese affect "on-mplrcanng DNA and cause

frame-shifts in DNA and base substitution by analogs (Crueger and Crueger, 1989).

shon wavelength ulwviolet rays between ZW - 300 m, with an optimum

wavelength at 26Snm are effective in causing mutations The absorption maximum of

DNA is 265 nm The most important pmducfs of thls type of radiation are pyrimid~ne

dimers, formed behveen adjacent pyrimldrne bases on complernentvy strands of DNA.

Long wavelength ulmvialer rays betwem 300 - 400 m are less lethal mutagens.

However, rf eelis are exposed to Ulrs type of radiation in the presence of various dyes,

increased mutatlon frequency is induced (Crueger and Crueger, 1989) Ionizing radiation

includes x-rays, y-rays, p-rays. These m e s of radiations are seldom used for

mutagenesis ss the rays cause a much greater percentage of single and double strand

breaks in DNA than the other mutagens, whsch can result m malor smctural changes in

the chromosome. A vanefy of chemicals are lmom mutagens and are used in genetic

studies. These chemicals are classitied according to their mode of action. Frame-shifl

mutasens ~ntercalate intn the DNA molecule, causing errors in the reading h m e and

result in the f m a t i ~ of faulty proteins or no pmteins at all. fiamplcs of this type of

mutagen are aeridine dyes, such as acridine orange, proflavine and acnflavine. Although

useful in research, frame-shifl mutagens are not vny suitable for isolation of mutants ro

shain development, because they have little or no motagcnic &t in bactena and yeasts

(Crueger and Cmeger, 1989).

Base analogs, such as 5-bromouracll and 2-aminopurine, act as mutagens by

being incorporated into replicating DNA in place of the corresponding bases thymine

and adenine because of thelr suucrural similarity. These cause transitions to occur,

resulting in the wrong b s e paic being incorporated dnto the replicated DNA Condltians

for the development of thls type of mutants arc costly and as such, base analog muLlgens

are rarely used m practical apphcations.

Many carornogenie, or red yeasts have also been genetically altered usmg N-

methyl-N-nlm-N-nitrosoguanadine (NTG). An el nl., (1989) evaluated the effcctivpness

of UV light, ethylmcthanesulfonate (EMS) and NTG in generating greater p l p e n t

praductng mutants of Phafia rhodoiymo. NTG was repolfed to be the best mutagen.

However, most of the mutants were unstable. In another attempt to obtain hyper

pigment producing mutants, Lewis el a/. (1990) exposed Phqffio rhadorymo to NTG and

then screened thc astananthin-averproduecn using beta-ionone. Acheampong (2000)

s~wessfully heated Rhodolomlo mbnrTPl wlth NTG in order to produce mutants with

enhancedplgmcntation and a belter capacity lo utilize cheaper subsbates for growth.

1.7Astarsnthin:

Astaxanthin is the mat" carotenoid pigment found m some aquate animals. This

red-orange p~gment 1s closely related to other well-lmown caroteno~ds such as beta-

carotene or lutein, but has a stranger anttoridant activiry (10 times h~gher than bcta-

carotene). Studies suggest that astuanthm can be 1WO times more effective as

antioxidant Ulan vitamin E. In many of the aquatic animals where it can be found,

astaxanthin has a number of essential biological functions, mging from protection

against anidatran of essential polyunsaturated fatty acids to enhance immuntry and

growth. In species such as salmon or shrimp, astananthln a wen considered as essential

for normal gmwth and survival, and has been attributed to have vitamin-like properties.

Some of these unique pmpcrties have also been found to bs effective in mammals and

open very promising possibilities for nutrit~onal and pharmaceutical applications of

astaranthin in humans. It can be found in many of scafoods such as salmon, trout,

shrimp, lobster and fish eggs. It is also found in a number of bird species. Astaranthin

cannot be synUlesizcd by animals and must be pmvided in the dlet as is the case with

other camtenoids. While fish such as salmon an unable to convert other dletary

cmtenoids into astananthin, some species such as shrimp have a limited capacity to

convcli closely related dietary corotenolds into astananthm, although they will benefit

stmngly fmm being fed artuanthln directly. Mammals are also unable to synthesize

astaxanthin. Some mlcmrganisms can be quite rich in astaxanthin.

A ubiquitous micro-algae, Haemolococcus pluviolir is believed to be tbc

orgamsq which can a~cumulate the highest lcvels of astnxanthin in biological system.

The funeuon of astananthln appears to be to pmted the algae from adverse environment

changes, such as increased W-light photonidation and evaporation of the water pools in

whsch it lives. K o e m a r n e ~ e ~ s algae can accumulate as high as 10 to 30 g of astaxanthin

per kg of dry biomass. Thls level a IW to 3000 fold higher than in salmon fillets. Some

strains have even been obsmed to accumulate as much as 70 to 80 g of astaranthin per

kg of dry h~omass. Estentied astaxanthin from Hoemotoeoccus pluvialrs alagal meal s

the preferred form in several ord prophylactle and therapeutic formulatiom for muscular

dysfunction, such as exertional rhabdomyolysls (also known as exeltianal myopathy,

tying-up syndrome, mhuia, or Monday morning sickness) m horses (Lsgnell, 1999), as

well as for mastitis (mammary iflammation) m dairy cows (Llgnell, 1999)

Astaxanthln is one of a group of natural pigments known as camtenoids. The

astaranthin molecule is similar to that of the famtltar carotenoid, beta-camtene. The

small dcfferences in structure of these confer large differences in the chemical and

biological properties of these hvo molecules In palicular, aswanthin exhibits superior

antioxidant propmles to befa-carotene in a number of in vitra studies (Rrimky, 1992).

Higher survival rate in red sea-bream was found to be that astaxanthln enhanced liver

cell m c t u r e Glycogen storage in red tilapla tncnascs femhzation and survival rates of

eggs. Higher growth a s dunng the early-feeding period of young salmonids have all

been associated with dinary astaxanthtn supplementation (Sommer et 01. 1991: Tonissen

and Christiansen 1995; Kawalrami et ol. 1998). When astaranthln was included in

poultly feeds, dietary astaxanthin was reported to improve egg production, the general

health of hens and also increase in the hatching percentage, reslstlnce to Solmonello

infectla& and shelf life of eggs. (Ligndl et 01.19981.

1.8 Significance of Camtsnoids:

The food and phmamlogtcal industms are potential users of large amwnts of

natural antioxidants. One of the advantages for the fond industry 1s that these

antioxidants may be used as pnselvatives against both enzymatic and spontaneous

oxidation of foods, thereby extending their shelf ilfe. Astaranthin, which belongs to the

carolenoid gmup, is a very valuable nahiral red dye used as B feed additive for deepening

the ptgmentation of salmon and organic chicken eggs Inttial resulta also show that

astaxanthin 8s a promising eanca prevenung agent and hence has potential for use as an

additive for promoting gwd health (Tnnaka, 1995).

In nabre, llke other pigments, astaxanthtn a synthesized only by mlcroalgae and

then passed up the food chain. Salmon and other manne animals cannot m&e the

compound themselves and must get it in their food Tra&Uonaliy astaxanthin has been

added to commercial aquaculhln dmts to improve the pigmentatton of the flesh of fish.

This use w a r n s by far the largest market m terms of volume and market value.

However a number of sbdies (Kiaui H. and Bauemfemd, J.C., 1981) have shown that

astananthin was much more than a pigment and in fact had vaamm-like pmpertles. As a

result, astaxanthln is now also used to enhance the immune response of fish and shrimp

to secure maxrmum survival and gmwth. Recent studies (Ito 1 ol., 1986) with young

shrlmp and other fish species have shown a superior uptake of nabral astaxanthin from

mlcroalagae compared to the synthetic form Another rearon for aquaculturists to prefer

nabral astaxanthm is the growmg demand fmm consumers for fish being fed natural

pigments, identical to those fish &at acquire natural astaranthm from the envrmnment.

The plnL to red color of the flesh of salmontds 1s an imponant f~ctor in consumer

preference for coloured fish. Colour is not an tnmnslc component of the fish but results

hom the deposition of diemy camtenoids. Astaxanthin is an abundant carotenold 10 the

marine environment. Salmonids, llke most animals, are unable to synlhesire or

biologically vansfom eamtenoid precursors into the pigments found in thelr tasues.

Wsld salmon obtaln their carotenoids from marine zooplanlaon, nekton, and their natural

foods. Pen-taised salmonlds, m turn, must derive thrs pigmentatlon from sources in their

feed.

The dominant pigment source in aquaculture is synthetic astaxanthtn and

eanthuanthm, cornmial ly produced by Hoffmz La Roche (Basie, Swimland),

which are marketed under the trade names of 'Camphyll pink' and 'Carophyll red',

respectively (Tomssen d el., 1989). However, the use of synthetic feed colorants is

quickly declining due to strict regulations and the increasing reluctance of consumers to

accept chemicals as food additives.

In recent yean, yeasts have been used as a pigment source for fish. The species

Phafia rhodozpo possesses high levels of carotenoids, afwhich astaxatthin is the most

abundant. In feeding eials, the incorporation of thts yeast's pigment into the diets has

achieved htgh levels of pigment deposlnon in rainbow trout, lobsters and salmon

(Johnson and Lewis, 1977). Howcver, three major obstacles have prevented the

commercial use of Phofia rhodoiymo as a natural source af camtenoids in fish feeds: a

rlgid cell wall, which limits the pigment extractability, a slow p w t b rate and poor

digestihillty of the whole Phofia cells by the fish

A strain of theRhodotomln species. Rhodoromlo mbro TPI, was also found to be

a good soume of pigments for rainbow tout Unlike Phofia rhodoryma, whole cells of

Rhodoromla mbro TPI were able to induce pigmentation. In addition, this has been

found to have a faster growth rate and easin pigment extractability than Phufie

rkodozyma ISangha, 1994).

1.10 The Genus RhodotonJa.

The genus Rhodotomlo belongs to the class Deutemmycotina, family

C~yptocoecaceao (Kreger van-Rij, 1984) and sub-family Rhodotoluloideae (Lodder and

Kreger-van Rij, 1954).

Yeasts an classified in the family Cryptococcaceae by the oonstant presence of

budding cells- although a pseudomycelium, me mycelium and anhrospores may be

formed. Culture cells are hyaline, red, orange or yellow due to cara tmid pigments, and

an seldom b m m or black. Dissimilation is strictly oxidative or ondative and

fermentative (Kreger van-Rij, 1984).

Members of the genus Rkodoromln have ovoidal, spheroidal or elongate cells.

They reproduce vegetatively by multilatenil budding and variants of same species form

psuedohyphae or true hyphae. Neither ascospores nor ballistospores an formed. Red or

yellow oarolenoid pigments are synthesized in malt agsr cultures (Knger van-Rij, 1984)

Regarding culNre appearance, some swains appear mucoid due to capsule formanon,

while others seem pasty or dry and wri&led (Kreger-van Ril, 1984).

1.11 Commorei=l Importance of RhodoforuloSpecicp:

The metabolic capablhtter of some Rhodotomlo species have indicated possible

appljcat~ons of this genus m the commercial industry Two Rhodotomla mbro strains

were found to d e p d e Chydrony-benzoate and as such could be used m otl sludge

mament (Wright andRatledge, 1991)

Omdztak (1993) reported the production of extra cellular proteases by a strain of

Rhodotomla mbro. It wss proposed that these proteases could be used to degrade the

proteins responsible for protein hares that form m wines and beers during storage.

1.12 The red yeast, Rhodomrula nrbra:

The species Rhodmmlo mbra was first disvovered in 1889 by Demme under the

name Socchnromyces rube,. Like all Rhodotomlo species, ascospores or ballistospores

are not produced and reproduction is by multilateral buddmg. As described by Kreger

van Rij (1984). Rhodotomlo rubro assimilates glucose, sucrose, hehalose, raffmose, D-

xylose, ribitol, melezitosc and succinlc acid. Galactose, maltose, cellabiase, L-arabinose,

D-ribose, L-rhamnose, D-rnannitol and citric acid are assimilated by same swins while

lactose, soluble elarch, elylhritol, inasital, mellbiose and nitrate are not assimilated

(Kreger van Rij, 1984).

Cells grown in malt extract or on malt agar v q korn short ovoidal to elongate, 2-

5.5 nm in width, and occur singly, in pairs, short chains or in dustsrs. Colony color

ranges fmm deep coral to pink or salmon-colored. Colony surface is glistening and

usually smooth, but is sometimes reticulate, cormgated and the tenlure varies h m sot?

to mucous Kreger van R4, (1984).

The corbhydrate panems of Rhotomla mbra whole cell hydrolysates show lhe

presence of fueose and mannose as lhe dominant sugars in this yeast, while heritol and

pentitol also occur in high concentrations (Weijman and Miranda, 1988). The total Iipd

content of Rhodolaruln mbrn a about 6.0% of dry welghf with palmitic acid, aleic acid

and ltnolctc acid as lhe major lipids (Pemet el nL, 1995). Thss camtenogenic yeast also

contarns about 1W mg camtcnoiddg dry wesght, whtch includes beta-carotene, beta-

zeacarotene, torulene and tomlarhodin as the major components (Perrier er ol., 1995).

The GtC content a 60 - 63.5 mol% (Nakase and Komagata, 1971).

1.13 Deseriptlon of Rhodotorula rubra TPI :

Rhodotomla mbra shains have becn isolated from leaves, flowers, soil,

amtosphere and marlne sources (Cook, 1958; Ingram, 1955; Kreger van Rij, 1984).

Recently a new strain has been isolated from yogurt (Hari el ol., 1992). A new strain of

red yeast contaminating a home-fermented yogurt was tsolated and, uslng the Analytical

Profile lnde(AP1) clinical yeast system, was identified as Rhodmmlo rubro

(Hari d aL, 1992). The results confirmed by M i c m h g k Inc Norlhtield, VT using a

technique involving cell wall fany ac~d analysis, The isolate was named Rhodotomla

mbra TPI (Hari el 01.. 1992). Rhodotomlo mbro mutants were isolated by Achempong

(2000). He used thne different mutagens including UV irradiation, ethyl methane

sulfonats (EMS) and nitrosogumdine NTG. He found NTG to be a bcltcr mvtagcn and

he was able to isolate 8 mutants of R.mbro TPI

Like other Rhodoromnl mbro strains, Rhodotomlo mbro TP1 does not form

ascospores or ballistospores, and reproduces by mult~lateral budding (Sangha, 1994).

However, in one shldy, Sangha, (1994) observed the presence of ascospores m thts strain

of yea*. As such this experiment needs to be repeated. As described by Hari el a/.,

(1992). Rhodolomlo rubro TP1 asslmllatcs melezitose, melebiose, maltose, m m t o l ,

trehalose, D-ribitol, raff~noos elhie acsd, sucmse, arabinose, D-xylose, succinic aetd,

soluble starch, galactose, and ntmte. It is unable to assimilate glucose, elythritol,

inositol, rhamnose, cellobiiose, and lactose.

Cells grown in yeast erhacUmalt extract (YM) bmth are circular or ellipsoidal

and average 2 to 4 nm m drameter. Colony color is best described as salmon-colored and

the colony surface is glistening and smooth. The absorption specrmm of the pigment

from Rhodoforula mbro TPI shows that the pigment belongs to the family of

caratenoids. Rr values of the pigment on a thin-layer chmmatography plates were

similar to those obtained for standard astananthin, while a mass specmmeuy analysis

showed a molecular mass stmilar to that of aslaranthin (Han etal. 1992).

1.14 Potential Commercial Applications ofRhodom~ulo rub111 TPI:

Sangha (1994) found Rhodotomlo rubro TPl to be an eflicient so- of

pigments and nuhients for aquaculhlred rainbow mut. The yeast was found to be more

economically favorably over PhqDio rhodozymn, whtch has also been successful in

pigmenting pen-raised salmonids However, Rhodo~onrla mbm TPI has a faster growth

rate with greater levels of pigment praduc~on compared ta Pha& rkodoqma under

similar conditions of growth Moreover, whole cells of R. mbra TPI were able to

ptgment rainbow muts but cells of Ph. rhodoqmu showed no pigmentation (Ha" el al.

1993). They also found that Rhalolorulo rubro TPI could be successfully grown on

vanous indush-ial and agricultoral by-pmducts for biomass production. This has

important ~mplications, as the cost of growing suffictent amounts of yeast cultures for

commercial use has always bem a concern. These raw material by-products are readily

avadable, relatively low m cost while pure sugars like glucase and sucrose, whlch are

often used for microbial gmwth in laboratory situations, are too expenswe for use on an

tndustrial scale

1.15 Research Objeeths .

An increased production of carotenoids by micmorganisms swh as red yeasts will

make as industrial applications cost effective and competitive. With this in mind, mutant

smms o f R mbra TPl were examined wlth the following objectives.

(i) To determine optimal gmwth conditions for pigment production under the influence

ofpH, light, temperawes, and different sources of c a h n and nitrogen.

(ti) To investigate efficient methods for optimal cxmction of piwent8 fmm mutants and

parent cells using Freeze-d~%ng, E m h Press, SonicaUon and enzymatic cell breakage

methods in order to determine their efficiency on pigmmt recovery.

CHAPTER 2

METHODS AND MATERIALS

2.0 Materials

2.1 Chemicals:

Acetone, dlmefhyl sulphonide @MSO), sodium chloride, sulphuric acid, sadlum

hydroxide, petmleum ether, hexane, rodme c~ystab, citrate phosphate, ethyl methane

disulphate (EDTA), uima base (Tns HCI), dithiothreitol and beta mercapto ethanol

which were purchased h m Fisher Sctentitic Company Ltd , Falr Lawn, N.J , U.S.A. Ail

the chemicals were ofAnalar Grade and were used without further plmtication.

2.2 Sourres of Mieroarganisms:

The test soain used in the experiments was Rhodolorulo mbro TPI h m earher

collection from Dr. T. R Patels Laboratory, Department of Biology, Memorial

University of Newfoundland (MtJN), NL, Canada. M u m s strains, Mutant 1 (MI),

Mutant 2 (M2) and Mutant 3 (M3) were tsolated earlier by Acheampong (2000) working

m fhe same laboratory. These mutantt, maintained on Rose Bengal Agar plares

(pumhared from Difco Laboratories, Detroit, MI, U.S.A.) and stored at 40' C. These

were transferred once a month onto new plates. Rhodosporidium lonrloids (10657) and

Rhodotorula nznto (10658) were from Amnican Type of Culture Collection (ATCC).

2.3 Pent Extract and Cane Molasses:

Peat extract was a gifi from Dr. A.M. Manin's Laboratory, Department of

Biochemlsuy, MUN. Cane molasses was procured from Lalle Nand Inc., Monucal, PQ.

2.4 Lysing Enzymes:

Lysing enzymes from Tnchoderma hornranurn, A r p ~ ~ I I u s species, Cytophaga

spcctes and Rhizoctonio solnni were purchased from Slgma Chemical Company, St.

Louis, MO, U.S.A.

2.5 Media:

Yeast Malt Broth (YMB), Potato Deitmse Agar (PDA), Bacto Czapex Don Broth

(BCDB) and Rose Bengal Agar base (RBA) were purchased fmm Difca Laboratones.

1.6 Methods:

1.6.1 Preparstlon afMedia and Inocdum:

Yeast malt (YM) broth was prepared according to the instructions given by the

manufacturer. Laop-fulls of yeast fmm RB agar plates were aseptically added to 10 ml

of saline water and vortored This suspension was used for inoculating gmwth media.

2.6.2 Growth of Cultures snd Harvesting of Yeaat Cells:

Yeast cells were grown m liquid media of diffmnt typss Erlenmeyer flasks (2 L)

containing 500 ml liquid YM broth were inoculated with yeast suspsnslon and were

incubated at 28" C far 5 days in a Psychrothem Temperahm Control Shaker (New

Brunswick Seientiftc Co. Inc., Ediron, New Jersey, U.S.A). The c u l h w were agitated at

150 rpm. Liquid cultures were ccnVifiged at 10,OCil rpm fm 10 minutes to pellet the

cells. The pelleted cells were used for pigment extraction after washing three times m a

saline solution.

2.6.3 Optimimtion of Growth Conditions:

Different gmwth canditlons such ss subsfrate concentration, initial pH of the

culture medium, trmperaturc, llghf fermentation time, initial optical density of the

inoculum and agitation speed were tested to determine the optimum gmwth parameten

for the mutan& of R.mbro TPI.

2.6.4 Growth on Molasses and Peat Substrates:

(i) Ctude molasses diluted at a ratio of 1: 10 was used to detnmlne the effect of

carbon source on pigment production. YM broth medium (500 ml) contained 50 ml cane

molasses, as a supplement of earban source was incubaled in 2 L f laLs at 2S0 C on a

shaker at a speed of 150 rpm for 4 days. Aliquots (1 ml) were removed in 3 hour

intelvals and optical density was measured on a Pharmacia LKB Novaspec I1

spectmphotomctcr at a wavelength of 600 nm.

(ti) Peat extract diluted at a ratio of I: 10 was used to determine the effect of

nitrogen source on pigment production. YM broth (500 ml) contained 50 ml peat exfract

as a supplement of nitrogeneous source Broth culhlres were incubated at 28" C on a

shaker at a speed of 150 ~pm. Aliquots (1 ml) were removed m 3 hour intervals and the

ophcal density was moasured using a spectrophotometer.

2.6.5 Growth Measuremenl and GcnernUonTlmes:

Gmwth and biomass of the wild type R.rubra TPI and the mutants were measured

using methods such as (i) aptteal density measurement, (ii) dry weight and w d weight

determinations. Gmwth in liquid medla were examined as follows: YM broth (500 ml) m

2 L flasks were incubated at 28' Can a shaker (at 150 rpm) afler inoculation with 2 ml

suspension of the yeast. Aliquots (1 ml) were removed at 3 hour intervals and optlcal

density readings wore used to establish wwth curves. Generation times were calculated

uslng Ule logarithmic growth phase of each culture. Readmgs were taken in triplicates

for each yeast sample. Afler 5 days of tncubation the yeast cells were collected by

cenh~fugation in a pre-weighed centrifuge bottles, washed twtce with salme, weighed

and dried m a hot air oven at 80' C (Oven, Blue M el&e company, Blue Island,

Illinois, U.S.A.). The dry wetght was recorded after h e constant readmgr were

observed. Growth table gives the generation time values given by T = ( tA) i log (yln),

where x = eellsiml at time ti and y = cellslml at time ti.

1.6.6 ENert of Temperalurean Pigment Production aftheR. rubmTP 1 mutants:

To study the effect of different temperatures on growth and plgmnt production,

the culture flasks (70 ml liquid medium in 250 ml flasks) were incubated at 15, 20,25,

28,30 and 35- Can a shaker for 5 days. Growth was determined by wet weight and dry

weight ofthe cells.

2.6.7 Effect of Inld~l pH of the Growth Media on Pigment Praduetlon of Mutants:

To study the effect of different initial pH on growth and pigment production, the

pH of the growth medium was adjusted bctween pH 3 N 10. Thls was arhieved by

addlng NaOH (1 M) or by adding HCI (I M) to the broth. Yeast cell suspension (2 ml)

was added to 100 ml of YM broth conratned in 250 ml flasks. These flab were

mubated at 28' Can a shaker far 5 days. Growth was determined by the optical density

method as well as by the wet weight and dry weight methods.

2.6.8 Effect of Light an Growlh and Pigment Production of R.rubra TPI Mutants:

To study the effect of light an growth and pigment production, the culture flasks

were incubated in dark or in the presence of ltght on a shaker at 28" C for 5 days.

Biomass-yield was obtained by using wet weight and dry weight methods.

2.6.9 Pigment Extraction:

2.6.9.1 Ertrsetlan using French Press:

Wet cells (4 g) were placed m the French Press Cell (SLM Instruments, Chicago,

Illinois, U.S.A ) and chilled by placing the cylinder in a k c p e r (- 70' C) for I5 minutes.

Parually frozen cells were ruptured at 20000 ps . The broken eel1 mass was callected m a

125 ml flask and 20 ml of acetone was added to it. Afler shLlng the cells suspension

thoroughly the mixture was centrifuged at 5000 rpm m Sorvall RCJB Plus centrifuge

(Dupont-Sorvall Insmments, Newark, DE, U.S.A). The supernatant was decanted into a

clear flask and 20 ml fresh acetone was added to the pellet. It was then mined and

centrifuged as before. The extraction protocol is shown in Fig. 4. The acetone exaicts

were pooled (60 ml, appmr.) and fillernl through Whatman No I filter paper

Caratenoid containing acetone solution was added to 50 ml of n-hensne and mixed in a

soparatory h n e l . Sodium chloride was (0.5%, LOO ml) added to marimlze the

cxnactlon of the eamtonoids. Camtenoid conviinlng henane solutton was eoncentiated

using an evaporator (Rato vapour-f(, Bnnlunann, Buch~ Laborotonums, Onbno) to 3 ml

Yeast Cells - Bmken by French press d EEzs I

Add acetone, 20 ml I C

Cenmfuge, 5 min, 5000 rpm

Add acetone, 20 ml - Pellet .'

v o w

1 Cenmfuge, 5 min, SOW rpm

/\ Pellet Supernatant ($2)

I + Add amtone, 20 ml - Centrifuge, 5 mi", 5WO rpm

Vortex

/\ Pollet Supernatant (33)

\ Add s l and s2 to 83

Transfer into 250 rnl separatory fume1 - (60 ml appron,)

4 Add 50 ml n-herane + LOO ml NaCl solution (0 5%)

Aqueous phase / Organic \ phase (henane layer)

1 Concentrate, Rota- .-- Collect in a clean flask vap 3ml

Fig 4. French press procedure for pigment extractton.

The ahmqtron specrmm was recorded tn Ulo region 4W to 600 nm using a

spectrophotomcfer ( S h i m a h photo specmmeter W-260, Kyoto, Japan).

2.6.9.2 Extraction by Freeze Drying:

In hemdrying methods, LO g frozen cells were drted using a lyophiltzer

LABCONCO, Freeze Dry System, Indiana, U.S.A.). The dried powder (I g) was treated

with 6 ml of warmed dlrnethyl sulphoxtde in a 40 ml test tube. The Nbe was kept in the

dark at mom temperamre for 20 minutes by covering tt with aluminum foil. The mixture

was centrifuged at 5000 Ipm for 5 mi" and the supernatant was collected. The pellet was

extracted with 5 ml of additional acetone and cenmfuged as before The supernatant was

collected and the pellet was mated once again with 5 ml acetone and eenulfuged. The

supernatants obtained were pooled together (IS ml) were filtered through a No.]

Wharman filter paper. Penoleurn ether (30 ml) and IS ml water were added to this

tiltered supernatant in a separatory funnel Afler thorough mixing the organic phase was

allowed to separate. The bottom aqueous phase was removed and discarded The organic

phase (30 ml) containing carotenoids was dncd with anhydmus sodium sulphate

Na, SO4) and then concentrated usmg an evoporator to 3 ml as showed in Rg. 5.

2.6.9.3 Extraction by the Method of Sonleallon:

In sonication method fresh cells ( I gm) were s u s ~ n d e d in 2 ml acetone and

sonicated for a period of 3 minutes at intervals of 30 seconds using Braun-Sonic, B

Braun, Model 2000 son~cator. The suspension was cenmfugpd for 5 minutes (5004 vm).

Separate the supernatant (31) from the pellet and add 2 ml of acetone to it and vortened.

Fmeze dried . Add warm DMSO, 6 ml

Cells I g

1 Centrifuge, 5 mi", 5000 rpm - Incubated, dark, 20 mins.

Add acetone)5 m- Centrifuge, 5 min, 5000 rpm

#'\ Add acetone, 5 ml Pellet 2 Supematant ($2)

1 Centrifuge, 5 min, 5W0 rpm

#'\ Pellet3 Supmatant ( d k a ~ ~ & , + ~ ~ , , e l

1 Add 30 ml petmleum elher + 25 ml dlstillcd water

Aqueous phase Organic phase

1 AddNaaSOd (anhydmus)

I

5. ~ n e z e dlylng pmcedvre for pigment extraction.

Thls suspension was again sonicated and centrifuged as before and added the

supematant ( ~ 2 ) to %I. Fmm this acetone emaction mixhlre 1 ml was taken to run the

spectrum for the analysis of the pigment

2.6.9.4 Ertrsction using Enzymes:

In enzymatic cell breahgage method I g of wet cell mass was suspended in 2 ml of

Tns HCI (pH 7) buffer or Cimte Phosphate buffer (pH 7) in a centrifuge tube (15 ml).

Lystng enzyme (3.5 mg) was added to the tube and was mubated for 24 hours i n s water

bath (Precision scientific Company, U.S.A.) at 25" C. Rcaccion mixtures were

centrifuged for 10 mmutes, at a speed of 5000 rpm. Tbe supematant was then decanted

off and 2 ml acetone was added to the pelleted cells. It was Ulen vottencd and sonicated

for 3 mtnutes and centrifuged agar" as before. The acetone layer (supmatant 1, sl) was

collected in a fresh bottle, and the pellet was rsusponded in 2 ml acetone. Afler

thorough mixmg, it was once again cenmfuged and the supernatant $2 was obtamcd,

then mixed wlth sl m a round bottom flask and concentrated to 3 ml using an evaporator

Ftg . 6).

1.6.9.4.1 Frpeze and Thaw method:

In this method yeast cells were fmzen at -70" C far 3 hours and were then thawed

Thawed cells (I g) were separately suspended in 2 ml of Tns HCI buffers with pH

ranging between 7 and 9 or citrate phosphate buffer (pH, 5 to 7). These suspensions were

treted with a 3.5 mg lytic enzyme (Rhyzoctoniosolani) and w m incubated for 24 hours

at 25" C inn water bath. The incvbatcd oell suspcnslon was sonicated for 3 minutes and

2 ml of acetone was added to ~ t . This suspension was centrifuged st 5WO rpm, for 10

Wet cells, l g + 2 ml buffer + 3.5 mg enzyme --, Mix, mcubate 24 houn, 25 deg C

I Centrifuge, 5000 rpm, 10 min

Sonicatlon, 3 minuter -- Add 2 ml acetone, Vonex

Supematant S l Pellet

Sonication, 3 rmnutes - Add 2 ml acetone, Vortex

Cenmfuge, 5000 rpm, LO mln

Supematant S2 Pelld

1. Add SI and S2,4ml . Run the spectrum

Fig 6. Enzymatic cell-brpakagc procedure for pigment extraction

30

mmutes. The supmatant was decanted offand the pellet was suspended in 2 ml aceme.

This was vortered and was sonicated for 3 mmutes. The sonieated cell suspensian was

centrifuged at 5000 rpm for LO minutes. The supernatant ($1) was separated from the

pellet and 2 ml of acetone was again added to the pellet. This cell suspension war

sonicated for 3 minutes and was centrifuged as mentioned above The supernatant (s2)

was mired with $1 (4 mi). S p m m of this mixhre was obtained behvecn 200 - 600

nm. All the experiments were done m mplicates.

2.6.9.4.2 Effect Of Thiol Group on Pigment Recovery:

Reducing agents such as dithiothrcitol (Dm and bets mercapto ethanol @ME) were

used at different concentrations m evaluate their effects on pigment extraction Wet cells

(1 g) were separately added to 2 ml cttrate phosphate buffer (pH, 7.0) m four different

tubes. The concentrations ofBME in Ulc buffer were 50 mM, 150 mM and 200 mM One

g of cells were separately added to 2 ml citrate buffer (pH, 7.0) in four different tohes.

The concentrations of DTT m the buffer were IS, 20, 25 and 30 mM respectively. To

this cell suspension 3.5 mg lysing enzyme was added and incubated for 24 hours at 25"

C. The cell suspension was sonicated far 3 minutes and then 2 mi acetone was addcd to

it. This was then centrifuged at a speed of 5000 rpm, for 10 minutes and decanted. Two

ml of acemnc was then addcd to the pelleiied cells. The cells were vortened and

sonicavd for 3 minules and centrifuged at 5000 rpm for 10 minutes. The supematant was

separated fmm the pl le t and 2 ml of acetone was added to the pellet It wa. then

voncxcd, sonicated for 3 minutes and ~ ~ ~ h i f u g e d again. The two supernatants were

m~xed and the spectnun for the sample-mlxhue was recarded for the analysts of the

pigments.

2.6.10 Measurement ofpigment:

The tots! camtenoid caneentration in yesst cells was calculated using the formula,

Cororenoid(/lg)idv yeosl(g) = EW

where A s the absorbance maxima at 474 nm, V is thc total volume of the sample (ml),

E is the extinction coefficient and W is the dry welght of the cells. Siee the crude

enhacts usually oonfained a vancty of camtenaids an average coefflcient of 2100 was

used m the caiculations and the concatrations of the individual pigments were

calculated using the method according to An el of. 1989. The absomancc values of the

pigment extracts in acetone were measured by specmphofometer me maximum

absorbance determined by scanning fmm 600 to 300 nm in a S h i m a h Ultra Violet 260

Recording specmphotometer. ident~ficatian of the individual pigments was done by

comparison of their absorption maxima with those of standard earotenoids reported by

other researchen (Dsvies, 1976; Bauerfeind and Klaui, 1981).

2.6.11 Thin Layer Chromatography:

The pigments were seperated by means of Thtn Layer C h r m t o p p h . Pre-ted silica

gsl (Whatman lnfsmationai Lfd., Maidslone, England) plates were used to

chromatograph the samples. The solvent used was a 10% toluene mired m 90%

peholeum ether ("1"). The spotted TLC plate was developed in this solvent until the

sulvent fmnt was about 1 cm below the top of the plate. The spots were visualized under

ulm-violet rays and also by iodine vapours. Rr (Retardation factor) values were

calculated by using the ratio of the distance traveled by the substance to the dtstance

m l e d by the solvent.

CHAPTER 3

RESULTS AND DISCUSSION

3.1 Gmwth and pigment production by R.mbro mutants:

The gmwth rate was detemlned m YM broth. R. mbro mutants (MI, MZ and

M3). showed less growth than the parent P I . The generation tunes for mutants MI and

MZ were 12.0 and 11.52 hours, respectively. Flgure 7 lllustratcs the generation times for

the wild type and mutant yeasts. The time required for the population to double m the

case of TPI was less, mdicatlng faster growth rate compared O the mutants. R. mhro

TPI had a shorter generation time and greater biomass yield than that of P. r k o d o ~ o ,

in an earher investlgatton (Sangha, 1994)

The growth cums for the mutants and the wrld type organisms at 15' C are

shown in Fig.8. All the isolates showed a lag period of about fittern hours as s h o w in

the figure Figure 9 examines the growth curves at 25' C. The cell yields were gnaw

for cultures grown at 25" (Table I). The tlme to reach stationary phase for the mutants

MI, M2 and M3 were 42.8, 41.2 8nd 43.2 hours, respedvely (Fig. 10). M3 showed

mare tlme as parent TPl to reach stationary phase than the other two mutants, MI and

MZ. This figure shows the differences m tlmes to reach stationary phase by the mutants

and the wlld type yeasts.

3.1.1 ~ r o ~ t h on Cane Molasses and Peat Extracts:

Greater biomass yleld was obtained upon addltton ofpeat extract to the YM bmth.

Figure I I shows the effect of 1 %peat extract, on MI, MZ, M3 and TPI. The yields

TP1 Rm Rt M I M2 M3 Yeast sample I

L 1 Fig. 7. Generation times of different yeasts.

Yeast cells were growing in liquid media as desmbed under Materials and Method.. Each of the d m points represents the mean value of three determinations. The standard ermrrinthemeanforTPI.RmRt,ML.MZ andM3 arei0 .12 ,+023, i0 .20 ,*0 .31 ,* 0 25 and i 0.1 1 hour, respectively.

Erlenmayerflasks (500 ml) containing300 ml dthe llquld medium were tnoeulated with freshly gmm yeasts on RB agar and incubated on a Psyehmtherm, agitated a1150 rpm. Tho solid ourve is shown for MI only. The data points are averages of Uuee detemrnat~ons (standard dev~at~on, t 0.5 O.D. untts).

0 20 40 60

Time (Hours)

Fig. 9 Growth of different yeasts on YM-broth at 25' C

Growth conditions were simllsr to those gwen m the figure eaphon for Fig. 8 except tmpcraturc. The figure represents growth measured at dlffcrent time mntervals. The solid curve is shown for TPI, yeast only. The data polnts are averages of 3 detemlnations (stmdard deviation, -t 0.5 O.D. units).

I TP1 Rm Rt M I M2 M3 Yeast sample

L __ ___- I Flg. LO Time needed for achieving sfationary phase by different yeasts in liquid culhires.

Each of the data points represents the mean value of thrpe determmations. The standard amn m the mean for TPL, Rm, Rt, MI, M2 and M 3 are i 0.2, a 0.3, + 0.3, i 0 5, + 0.1 andiO.1, respectively.

Yeasts we= gown in a liquid media m flasks (500 ml) contaming 300 rnl YM-broth inoculated flasks were incubated at various temperatures (IS to 35' C) separately, m a Psychrothem shaker at I50 rpm. Cells were collected by centrifugation after 5 days. Wet weigh& and dry weights were detemtned in pro-weighed glass centrifuge tubes. Each experimental point represenb average of three deteminatlons. Emrs given are standard deviations.

Table I. Biomass ylcld (gn ) in wild type and mvtant yeasts at various temperatures.

Yeast

Tsmp. I Biomass wild

TPI Rm Rt MI MZ M3

I TPI Rm Rt M I M2 M3 Yeast sample

L- - _1 Pig. I1 Biomass yield of dlffennt yeasts gmwn in YM-bmth wlth 1 %peat sxbact.

Yeast cells were grown in liquid mllurcs in Erlmmeyer flasks (500 ml) containing 3W ml liquid media ~noeulnted wlth different yeasts. Culhlre flasb wore incubated at 25" C and shaken in a Psychrothem at I50 rpm. Yeas1 cells werp harvested by centrlfvgation as described under Materials and Methods Each exwirnpntd point vrpsenb an average of three readingf. The standard cmr in the mean forTP1, Rm, Rt, MI, M2 and M3 are t 0.25,0.32,0.42, 0 5 1 , 0 2 3 and 0.39 g/L, dry weight, rcspcctively.

were 7.87, 8.29, 11.42 and 9.54 &IL (dry wetght), respectively. The effect of 2 % peat

extract m YM broth was observed to be better for larger blamass yields. At thss

concentration M2 and M3 resulted in biomasr yield of 9 71 and 9.84 &IL, respectwely.

In contrast under sirnllar conditions the p m t TPI gave 10.12 giL, as shown tn

Fig. 12. Further increases in peat extract cancenhations for the growth medium did not

give mmsponding increases m the biomass ytelds except m the ease ofM1 (Fig 13)

The yeasts were able to utilize a wlde varieh/ of inorganic nitrogen sources with

an optimum growth in the presence of ammonium sulphate and ammonlum hydroxide

(Sangha, 1994) However, an organlc nitmgen source like peptone was assimilated

much better than an inorgane-nitrogen source (Sangha, 1994) Abour-Zeid and Yousef

(1972) also repolled similar behawor with Slrepfom~es eaespitosus. The yeast

p r e f d molasses may be because of the presence of lower amount of reducing sugars

m the peat hydrolysate. Anderson (1979) Condido ulilir on a commercral scale

ustng sulfile waste liquor. Nitrogen supplrmeotatton of sulphtte waste liquor m the form

of urea or ammonim sulfate and phospholus as phosphoric actd was found to enhance

the biomass ylcld and substrate consumption (Simard and Camemn, 1974).

figure 14 shows the effect of different concentrations of cane malasscs in YM

broth a biomass yield. With 1 and 2 % cane molasses the mutants and the parent

organism showed much greater cell gmwth than with the higher concenbalion (3 %).

The biomass yreld by mutanls showed canslderable variation as shown in Iigure 14.

Earlier workers found that cane molasses were better than beet molasses m

supporting the growth ofthe yeast (Pepplcr, 1979). It is poshllated that the bigher

TPI Rm Rt M i M2 M3 Yeast sample

I 2

Fig. I2 Biomass yield of yeast cells grown m YM-bmth with 2 %peat extrad.

Growth conditions were similar to Ulose described for caption for Fig. I1 except the concenlntlon of peat extract m the growth rnedlum.

Each exppPrimental data represents an average of three readings. Tie standard ermr of mean for TPI, Rm, Rt, MI, M2 and M3 are * 0.39, 0.23, 0.51, 0.42, 0 23 and 0.32 giL, dry weight, nspectively.

TP1 Rm Rt MI M2 M3 Yeast sample I

Fig. 13 Effect of 3 %peat extract on the biomass yield of yeast cells.

GroWh conditions were slmilai to those described for eaptron for Rg. 11 except the concenrration ofpeat edract in the gmwth medium.

Each experimental data represents an average of three readmgs. The standard error of mean for TP1, Rm, Rl, MI, M2 and M3 are i 0.23, 0.25, 0 32, 0.39,0.42 and 0 32 giL, dry we~ght, respectively

I 2 , , ; 5 6 1 , 2 , , 5 6

7% L 2 3 ? o ; 5 6 1 Cane molasses (%)

Rg. 14 Effects of cane molasses concentnitions on the biomass yleld in yeasts.

(1) TPI (2) Rm (3) Rt (4) MI (5) M2 (6) M3

Yeasu were grown in YM-broth plus different concenlratians of cane molasses as mdtcated. Liquid cultures were grown as described under Materials and Methods. Each experimental point represents an average of thmc rsadmgs. The standard errors of mean forTF'I, Rm, Rt, MI, M2 and M3 are 0.31, 0.42, 0.32, 0.25, 0.42 and 0.14 g& dry weight, respectrvely.

contcnt af sugars (55 - 62 %compared to 48 %for beet molasses) in cane molasses may

have a role m growth of yeast. Cane molasses are also richer in biotin, pantothenic acld,

thiamin, magnesium and calcium (Pcppler, 1979). These may also have a stirnulalory

effect on growth parameters of a yeast. Cane molasses have been used as a substrate for

the production of bio mass. Roh (1984) reported a fed batch system with molasses to

aptlmize cell yields and substrate utilization using S cerevrceue. Estevez and Almazan

(1973) used a continuous culture system with high test-molasses and crvde sugarcane

jutcc as the substrate and reported excellent biomass ytelds. Moreira a al. (1976)

supplemented molasses with urea and inorganic phosphotus to grow R, graeilir and

Condido ufilis.

In the present shldy also the highest biomass yield was obtained with the YM

bmth med~um (D~fco) than with YM broth supplemented wlth cane molasses. All three

mutants responded similarly. Ths presence of powth-Irm~nng impurities in the molasses

plus the deficiency of some nutrients may account for the differences in gmwth response

of the yeast. Molasses and wart also have been used to grow P. rhadolymly (Okagbue

and Lewis., 1985) while won has been used to boos( astaranthln production by the same

yeast (Johnson and Lewa, 1979).

3.1.1 Effects of the Media on Blornrss yield:

B a ~ t o Czapen Dor Broth (BCDB) was used to grow the yeast mltores. It was

f w d that there was very less growth and practically no pipent . Rose Bengal agar was

found to be better medium and pmmated greater gmwth when ampared to BCDB.

R nrbw TPI was found to gmw readily on the common laboratory medla while

P rhodorymo showed reduced growth on many of the medla tested, lncludlng PDA,

SDA and malt agar (Sangha, 1994). The mutants, MI, MZ and M3 did not grow e~ther

in Bacm Czapen broih or an Sabmud don agar plates. Under simrlsr and the same

conditions and the same submtes, the parent TPI showed reduced growth with no

pigment production.

3.13 Effects of Temperature:

The optimum temperature was found to be 28' C for the parent organisms and the

mutants. At this temperature, both, cell biomass as well a3 pigment pmductlon was

snhanced with all test organisms (Table 1). The extracted pigment from these cells was

analysed by using thin layer chromatography m b l e 2). From the table it is evident that

mutants produced four of the camtenoids present m the wild type, TPL but lacked

phympne The Rf values determined for the individual carotenoids also eotnctded with

those repond in literature (Achcampong, 2000). The differences in Rr values obsened

for pigments in mutants and wild-type yessts were very minute.

In a shldy using mutants of R. nueilaginoso, Villauheix (1960) reported that

toruleme, torularhodin, y-carotene and p-camtenc were the principal plgmmts of the

parental strain, whereas ~ h ~ t o s n e and phyfofluene were absent Nakaysma el ol., (1954)

also examined the pigments from several C~ptococcm and Rhodotomlo species and

concluded that quantities of the red and ye1lo.r p i p ~ n t s varied depending on rulnlrnl

condi~on specially, by tempsmhlre. According to these authors the concenhations of the

red pigments decreased at 5' C but increased as tho temperature was increased.

Table 2. Rrvalues of camtenoids fmm various yeast!

Yeast I TP1 l M l 1 M2 1 M3 Experimental Rf values

Pre-coated sillca gel plates were used to spat the yeast samples. The solvent is the mixture of 10 % toluene m oetroleum ether. Pigments were visualtzed by Iodine vapour

Inspection of the camtenoid composition of R wbro TPI as well as the mutagenized

cells allowed the identificatton of most of the earotenold previously described m other

Rhodotorula species (Bonner a ol., 1946: Hayman et al., 1974.. Simpson et o l , 1964).

In the present study, tomlene, tomlarhodin and p-camten were determined to be the

major pigments produced by the new yeast ,solate R mbro TPI, as reported earher

(Hari, 1994). In Rkodofomlo and Rkodasporrdrum species, carotenoids, tomlene and

torularhod#n are produced in hlgh amounts even though several othen caratenaids

including P, y and bcamtene, phytoene, phyiofuluene and p-zeacamtene may balso be

present (Ciegler, 1965, Hayman et al. 1974).

In a study to re-examine the pigments produced by R glulinis straln 48-231

whleh had been sNdied earlier by Nakayama el oL, (1954). Stmpson a al., (1964)

reported that the total carotmid eoncentratton, on a dry weight basis, was nearly equal

at both room temperamre and 5" C, however, the lwel of todene and torularhadln

coupled with a decrease in thc levels of P-camtcne when the yeast was cultured at a

hlgher temperaare, 25- C. The gain in the levels of tmlene and tomlarhodin were

nearly equal to the decrease in the level of P-carotene According to Slmpson and group

(L964), these results suggest that y-carotene lies at the branch potnt in the earotenoid

biosynthesis sequence, and that intermediates can be channelled through it either to P-

carotene or to the red pigments, torulene and tomlarhodin, depending on the growth

temperature. Similarly, in R, pvLdv 62-506, it was shown that there was sn lncrease in

the level of tarulcnc and tomlarhodin as the level ofy<aroten~ decreased.

3.1.4 Efferts of Inltinl pH:

Optimum btomass production was achieved when the initial pH range was

between 5 and 7 (Table 3). The d~fferences in biomass yields of all the mutants and the

parent were ststtstically significant In an earlier study (Sangha, 1994) it was found that

the amount of growth decreased for R. Nbro TPI as the pH increased from 7.2 to I0

This was also the case in the present study. Sangha (1994) also reported that yeasts had

difficulty in growth at pH between 3.0 and 4.9.

3.1.5 EIfrets of Light:

It is known that ltght influences pigment pmductlon in btological systems. Thc

effect of light enhanced blomass yields and pigment production in the parent and the

mutant yeasts. Mutants MI, M2 and TPI gave high camtenold concentration than

mutant M3. Figure I5 shows the effect of light on total carotenoid concentrations m

yeasts g m m at 28' C m YM broth. The light source was a hlngstcn lamp in a

Psychmthem. Effect of darkness on pigment praductlon was observed by growing the

yeast in dark brawn flask and incubated in a psychrothem with llghts turned off. The

ptgmrnt concentrations in yeast gmwn were very low in darkness compared to t h a of

samples grown under light. Figure 16 shows the tom1 carotenoid conccnaition (TCC) of

all the yeast samples tested. Parent TPl and M2 showed almost the same TCC, 148.4

and 152.5 llglg (dry wetght), respectively.

Girard et a1 (1994) obselved that yellow m u a m of P. rhodarymo accumulated

high cancentcattons of p-otcne while the white mutants produced no camtenoids. In

Rhodororulo species, y-carotene produced in then biosynthetlc pathway is usually

Yeasts were gmwn m pn-adjusted pH liquid media flasks (500 ml) containing 300 ml YM-broth. Inoculated flasks wpre incubated at 25' C in a Psychrotherm shaker (150 rpm). Cells were collected by centrifugation afler 5 days. Wet weights and dry weights were determined in pre weighed glass centrifuge tubes. Each experimental polnt represents an average of three readings. Ermrs given are smndard dcviatlons.

Table 3. Effect of initial pH on biomass yeilds- m yeasts.

M3

Initial pH / Biomass ycild (gIL)

Yeast MI TPI MZ

TPI Rm Rt M I M2 M3 Yeast sample

Rg. IS Effect of light an total carotcnoid concenhation in diffennt yeasts.

Ysn*l. ucrc graun ~n Ilquhl rncJ.8 IN flasks ,501 ml) iuntalnlnp 300 ml YLI-brrlh al Jc*:nhsJ unJcr Mntrr 111 and I ~ l h u d r Thc llsnJirJ cnvrl 118 the mean far 11'1. IRnl Kt. hl l . hl? 3nJ blZ arez 4 6 , 5 3. 5 u. J 5 . 0 2 an34 I.,( 6 . Jry uctmt. r:\);.llvcl)

TPI Rm Rt MI M2 M3 Yeast sample

, . - . -- - 1

Rg. 16 Effect of darkness on total carornoid production in different yeasts.

Yr.r\c, \\ere grrsnn ~n Ih,plJ ntcd8a ~n llr*k, (510 ml, c tnlalnly 300 ml YM-hrah 1, dcrcrlbrd under h4aarl.1, and \lelhlld~ I h c ,trndari mor- ~n Ihc menn for TPI, Rm. Rt, h l l . >4? an.l\l l l r c - 5 2. 5 7. J 7. 54. J 9 3nJ 5 3 . l.py dq a r ~ g h ~ . r c i p c r ~ ~ ~ c l )

transformed to yield elther p-camtene or tomlsne (Goodwm, 1965). The tomlene can

then be oxidized to form tomlarhodin (Lmpson el al. 1964). In a study (Kayser and

Vollouueir, 1961). ~t was found that the isolated p-carotene aver producing mutants

were similar to 6-carotene accumulating mutants of the yeast R, glulinir.

Although the mumts appeared to produce pigments similar to those encountered

m the parent strain the total quantity was reduced in them. Gimd el ol. (1994)

postulated that low pigment production in mutant is due to inhibition of the early steps of

carotenogenesls and the enzyme, phytoene synthetase, may be affected. In the present

study mutanta MZ and M3 in which 0-camtene in the total eamtenoid content was

detected may represent similar condition.

Spectral analysis (Table 4) and the wild-type reported that all the mutants contain

tomlens, tomlarhodin and p-carotene. Simpson sr el. (1964), in their investigation of

pigment production in P rhodoryma rrponed that y-carotene n convened to Loml~le

which is in tum convened to tomlarhodin. Naliayama er ol. (1954) determined the

content of individual carotenoids present m several species of Rhodotomla and reported

that tomlene, tomlarhodin, p-camtene and y-eamtene to be the pnncspal pigments in

these yeasts Although all investigators agree on the presence of these Uvee components,

tomlene, tomlarhodin, and p-camtene, the data with respect to concentrations reponed

was variable. Perhaps, all these researchers uscd different strains of yeast and cultural

conditions in fhelr studies

Since ~t has been reponed that pigment composition depends on the strain of yeast

and particular cultural conditions (Nakayama el oi., 1954; Kavantkov r! 01.. 1978:

Bonner er aL, 1946), the variations in the concentetian of the different plgments should

Table 4. Absorption maxima (hmax) of camtenolds h m different yeasts

a* Goodwlh 1955; b' Liggen-lensen, 1965. Absorptson spectra were obtained on a Shimadzu speclrophotometer using visible light between 400 - 600 nm. MI: Mutant I.,MZ: Mutant 2 .,and M3: Mutant 3.

not be surprising. In the current work the major pigments produced by these mutants

have been identifled to be tomlene, tomlarhodin and p-camtene. The tindings of present

investigations are supported by the earlier work (Slmpson, 1964; Nakayama el ol., 1954;

Kavanikov et oL, 1978 and Bonner, 1946).

3.2 Erect of Extraction Methods on Pigment Recovery:

3.2.1 French Press:

Mechanical dismption of yeasts has haditlonally been accomplished by using

either a French press or a Bead beater. In the present shldy, the recovery of carolenolds

from wild-type yeasts, T I , Rm, Rt and m u m u MI, M2 and M3, were 326.7, 277 5,

291.6, 245.4, 242.5 and 193.2 pgig, dry welght yeast, respectively, using French press

method (Fig. 17). Mutants MI and M2 showedcomparableptgment recovery. The least

yield of pigment recovery was obfained with M3 i.e. 59.1 %compared to that fmm TPI

The wild type yeasu gave superior yields compared to the mutant yeasts.

3.2.2 Freeze-drying:

Ftgure 18 shows the recovery of carotenoids from freeze-dried cells of TPI, Rm,

Rt, MI,MZandM3. The yields were 282.2, 196.3,243.5, 158.6,2374and214.4 pdg,

dry weight yeast, respectively. In the present shldy ~t was obsmed that M2 gave higher

recovery than other huo mutants MI and M3 Compared to yield from TPI, the recovery

of carotenoids from M3 war 84 %while those fmm MI and M3 were 56 2 and 75.8 %,

respectively. The yeasts, Rt and M2 gave comparable yields of carotenosds with values

of 86.1 %and 84.1 %,respectively, compared m that fmm P I . M3 gave shghtly belieer

Rg. 17 Effects of French press melhod on pigment recovery in different yeasts

Known amounts of yeast cells w e n broken in a Frsnch press and tho broken cell mass extracted with acetone as dcscnbed under Materials and Melhads.

The values represent avnages of three determinations. The standard errors in the meanforTPL,Rm,Rt,Ml,MZandM3arc*8.1,4.7,9.2,5.4,6.2and4.2pgig,dry weight. respectively.

TPI Rm Rt MI M2 M3 Yeast sample

I Fig. 18 Pigment extractability using Freeze drying mcthod in yeasts

Known amounu of frazon yeast cells were lyophalized for 24 hours and the dried p ~ ~ d e r sa, crcaccd \wth D\I(O m d psrolcum d l e r Thr plgment w=r iullr..ad Ir) ~ c n t r ~ i ~ g r c ~ v l l ~ n d the totdl ;~r.>lcno~d c~nimrratlon udi arh!e\cd bv u,>ng spzctmphelu !meter rr.ulcr i r c1cr:nhd undcr hlncr#nl\ and hlcfhod, Thc value; rcnreienl arer..re<

~ ~

olfhne determinations. The standard m r s m the mean for TPI. Rm, Rt, Mi , M2 and M3 arc i 8.2,6.3,8.1,5.2,3.4 and 7.3 pgig, dry weight, respectively.

recovery of pigments than wild type yeast, Rm. Compared to other yeasts MI gave least

amounts ofplgment recovery, with a value of only 56.2 %.

3.1.3 Sonieation:

Figure 19 shows pigment moveties from various yeasts broken by sottication.

hgment recovencs from Rt, Rm and MI w m 98.7, 86.5 and 92.3 %, respwtlvely, when

compared to the parent organism, TPI. Wlld type yeast, Rt showed 1.3 % reduction and

MI showed 7.7 % reductton in their pigment r e c o v ~ y with values compared to that from

TPI. Pigment recovery frmn Rm and M2 were similar with values of 86.5 and 84 0 %,

respetively, when compared a the recovety from TPI. Least quantity of motenoids

were released fmm M3 with a value of 77.6 Kcampared to that fmm TPI.

Flyre 20 shows comparative recovery of earatenoids using different cxaiction

methods. It 1s evident from the figure that French press method is suprior compared to

the other two methods. The recovery of camtenoids from TPI, using French press method

was greatest (326 ~ l g , dry weight). In contrast other methods decreased the amounrs of

camtenoids released Example, yields using ronication method and freeredtying method

were 37 % and 86.4 %, respectively, compared to that from French press method.

Compared a the other extractton mcthods, pigment recoveries from MI and M3 were

greater with French press method Yields with sonicaton method for MI, was only 46 %

while that with freeredrytng method was 64.6 % of the value obtained with the F m c h

press method (Fig. 21). Similarly percentile pigment recovery from M2, revealed

decreased values with sonscation a d f r e a e dryrng methods. Values observed were 57.5

%and 47 %,respectively (Plg. 22). In the case of M3, surprisingly, the highest yield of

I "I "" "' MI M' " j Yeast sample

Fig. 19 Pigment extractabihly ustng sonication method in different yeasts.

Known amounts of yeast cells were treated with enzyme and incubated for 24 hours. This cell mass was sonicated for 3 minutes wth 30 second i n m a l s for each. This cell debns was treated with acetone and the pigment was collected by ccnvifugation as described under Materials and MPthods The values rcprescnt averages of three detennations. The standard m r s in thc mrvl for TI, Rm, RL MI, MZ and M3 arc * 7 6,4 3,5.2,6.4,5.7 and 7.2 pglg, dry yeast, respectively

Sonlcatton French press Freeze dryer --- --

Flp 20 Effects of extracoon methods an plgment recovery horn TPl, yeast

R g 21 Effects of extractton methods on plgmed recovery fmm MI, yeast

-- - - - - ' 1 - - 5 200 n , - g 150

g 100

0

B 50

0 Sonlcaflon French press Freeze dryer

ppp--

Fig 22 Effects of extractton methods on pigment recovery from MZ, yeast

earolenoids was obtained with freeze-drying method (Fig. 23). The percentile recoveries

obtained with the French press and sanitation method were 44.4 and 90 %, respectwely,

compared to that with the freeze-drying methods. Results of crtraclion of carolenoids

From Rm usmg the rhrec methods as dcscnbed above arc illmsuated in Fig. 24. Fmm thrs

figure it is evident that French press method a superior compared to the other Mio

methods. Pigmenis recovered using soncation and fneze dving methods represent 38.3

% and 70.7 %, respectively, when compared to quantify of pigment released uslng the

French press method. Figure 25 compares quantlttes of pigment released from Rt with the

three methods. Once again French press method caused highest amounts of pigments to

be released from this yeast The amounts of pigments released with sonteation and freeze

drying methods represented 41.6 % and 83.5 % compared to the French press method.

Hari (1994) compared pigment release from P rhodorymo and R. mbro usmg French

pressure mcthodi and found that the pigment yield from R Rubra was more (2.07 m a

of medium) than from P rhodoiymu(l.21 m a modium).

Several methods have been attempted to enhance pigment extraction from

Prhodozymo including mechanical breakage and chemical (acid or alkaline) hydmlysss

(Simpson er al. 1971). These investigators found both methods to be very laborious. It

was also observed that avid or alkaline hydmlysis resulted in denahnation of cmtenoids

@&vies, 1976). Phaff (1977) proposed that the digestion of yeast cell walls for the

extraction ofplgment protom employing microbial lytic enzymes

1 Sonication French press Freeze dryer

fig 23 EffeM ofrxVactlon methods on pigment recoveq &om M3, ycast

Sonffiatbn French press Freeze drys,

Fig. 24 Effects ofexhaction methods an pigment recovory h m Rm, yeast

1 Sancat,on French press Freeze dryer

Fig. 25 Effects of extraction methods on ptgment recovery tiom Rt, yeast

3.2.4 Extraction using Enzymes:

(i) Effect of different lyiie enzymes on pigment release:

Three different Lyttc enzymes used were fmm Rhyoetonro soloni, Aspergillus

species, and E-zchodermn horariwn. Table 5 shows the effect of these enzymes on the

recovery of emotenoids from differmt yeasts Fmm this table it is evldent that the

enzyme from Rhyzoclonia soloni is more efficient in lyslng the cell walls of the mutants

and the parent yeast TPI compared to lyfic enzymes fmm other yeasts.

(ii) Effect of Buffers:

Breakage of vdous yeasts at different pHs using TrisHCl (pH: 7.0, 7.5, 8 0, 8.5

and 9.0) and eihate phosphate (pH: 5 0, 5.4, 6.0, 6.6 and 7.0) buffers was examined.

Table 6 and 7 show the total earatenold recovery from dtfferent yeasu. It a apparent

from these two tsblen that optimum cell breakage occurs at pH 7 0 wlth maximum

pigment release. With Tris-HCI buffer (pH 7.0) MI, MZ and M3 gave 76 %, 68 %and

53.5 $4, respectively, of pigment recovery compared to that fmm TPI It is dso observed

that the recoveries decreased wlth higher pH valves of the buffers Vable 6). In the case

of civate phosphate buffer (pH 7 0) the pigment ylelds fmm MI, M2 and M3 were

197 7, 162.1, 141.7 and 101.1 pgig, dry weight, respectively. When pigment recoveries

h m MI, M2 and M3 at this pH are compared to that fmm TPI, Ulere is a reductton by

18.1, 2 8 4 and 48.9 %,respectively. In the case of M l the plgment recovery at pH 5.4

and 6.0 were similar (LO5 and 106 pgig).

Table 5 Effects of lysing enzyme fmm different sources on pigment recovery of yeasts

Ill, ) c ~ cellr (1 g, acre 8s 2 ml of lr8. II( I mJ neat4 ullh 3 mg o i rn r )n r lronl dnllcrmt ro~rccv wprrrrrl\ Th~r cell ,u,pens!.ln ua, ,usmratcJ &r ?J hour, ot 8nc~hx~un Tile plgmcnt u a i then e r w r ~ c d as Jls;r#hod ~n hl.lrr~rli and Methods

Standard errors are for averages ofthree determinations.

Table 6. Recovq of camteno~ds by using lylic enzyme (R soloni) in Tris-HCI buffer

Camtenolds @gig)

7 0 7.5 8.0 8 5 90 pH

The yeast cells (I nl were susoended in 2 ml of trls HCI and treated wlth 3.5 me of enzyme. Thls cell suspension was sonicated afler 24 hours of mcubation. The pzgment was then extracted as described m matertal and methods The procedure 1s same as mentioned in the caption of Table 3 except the eoncenmtions for each sample were varied between 7.0 and 9 0. 'Standard m n are for averages ofthree determinations.

Table 7 Recovery of earatenoids by lyttc enzyme (R solano in citate phosphate buffer

Carotenolds (pdp,, dry weight) pH

5.0 5 4 6.0 6.6 7 0 l l 0 l l f ~ - .- TPI -. 1143;41 129-6r7b 1.1-82-34 1 7 O i i J 2 1 4 7 7 ~ 4 1 -

' 7 5 7 z J l ' l t f 3 1 d l t j J - 3 J 1 7 J j s 7 6 !b;.l-Al- , h l l hl! n J z . s z h19- IS ! ? L Z . . I P 1 1 4 . ! - b i l 1 ~ 1 7 ~ 6 0

S 6 2 r a 7 h 3 5 ~ 4 5 , 7 4 - 4 2 h l t # z 3 f t ~ l ~ l . l z S 3 - / h13---

lltc !ca\c rel., I urrc ruqpendcj ~n 2 rnl o f '#"arc pl8oqlhla b ~ f l r r unJ

CrearrJ w ~ h 3 1 n ~ p of ennmr Tnl, .'r. I ru,pcnc~ r a - ron~cxcJ r P ~ r 2J h lLr% ol IO;U~JIIJII 'Ihe o ~ o n i n t sa. lllrn crtra;fcd 2s de-o~bcJ ~n rn~tcr8ill a d nlcthojs Ihc . " procedure a same as mentioned in the captlon of Table 3 except the concenwtions for each sample were varied between 5.0 and 7 0. 'Standard errors are for averages of three determinations

(iii) Effect of reducing reagents:

Different Molarities of redueing reagents were obtained using dithiolhreiotol

( D m , 5.0, 10, 15.0, 20.0, 25.0 and 35.0 mM) and betbmercapto ethanol (BME, 50,

100, 150, and 200 mM). Table 8 shows the effect of DTT on pigment recovery in TPI,

MI, M2 and M3 yeasts. The table shows that the best concentration of DTT for htgher

enzyme activity is 25 mM and the yields observed forML, M2 and M3 were 90.5, 85.9

and 79 %, respoctiveiy, compared to that from TPI. It a observed that the pigment

recovery increased wlth the increasing concenbations of tbe reducing agent (5 to 25

mM)

Table 9 shows effect af BME on pigment recovery in Ule isolates The pigment

recovery decreased in MI, M2 and M3 by 8.9, 14.6 and 26.2 %, respectively, when

compared to that from P I . From the table it is evident that the best recovery was

observed wtth BME concentration of 100 mM. When 150 to 200 mM BME was used

w ~ t h the lytlc enymes the pigment release was reduced. Of the two redueing agents,

Dm was found to be supenor m stimulating enzyme activity. The tom1 carotenold

recovered wlth DTT wing TPI, MI, M2 and M3 were 96.5, 87.4, 82.9 and 76.3 pdg,

respectively.

(iv) Effect of freezing nod thawing:

Table 10 shows that freeze and thaw mnhod enhances pigment release from cells

treated with lytlc enzymes and then sonicated. P I showed more recovery than the

mutants. Pigment recovery m frozen and thawed cells doubled or tripled when compared

to unfrozen yeast cells (Table 10). The recoveries in MI, M2 and M3 were about 85 %,

78 %and 73 $4, respectively, compared to that fmm TPI.

Table 8 Recovery of camtenoids by using lyttc enzyme (R. solon9 in Dithio lhreiotol (DTT) buffer

The yeast cells ( I g) w m suspended in 2 ml t"s HCI contained DTT wlth various concentrations (5 to 35 mM) and treated wlth 3.5 mg of enzyme. This cell suspension

was sonicated aIter 24 hours of incubation. The pigment was Ulen eimcted as dercrlbed

D1T Concentration (in mM)

io material and methods. * Standard emrs are for averages of t h e detemmattons.

Carotenoids (&gig, dry weight)

The yeast cells (1 g) were suspended in 2 ml trls HCI contained BME wlth various conceneatlons (50 to 200 mM) and treated with 3.5 mg of enzyme. This cell suspension was sonicated after 24 hours of incubation. The plgmcnt was then extracted as described in materm1 and methods.

* Standard errors are averages of three determinations.

Table 9. Recovery of cmtenoids by using lylic enzyme ( R solanil in Beta Mercapto Ethanol ( B m ) buffer

BME Concentratlon Carotenoids (pgig, dry weight)

Table LO. Recovery of carolenoids by ustng lyiic enzyme (R solon0 by freeze and thaw method

Caratenoids @g/g, dry weight) Isolate 1 Frcsh cells Method 1 Freeze and thaw Method TP I (82.5i26' ( 194.3 dl 7 M I 1 85.9 & 2.4 11655i2.3

Known amounts ofypas: cells were allowed to thaw aRer being h z e n and treated with enzyme. The cell suspension was then incubated for 24 hours and the pigment was then extracted by centrifugation &er sonication as described in material and methods. ' Slandard erron an avnages ofthreo determinations

Figvrc 26 illushatcs effects of increasing substrate (one gram of yeast cells) on

the activity of lytic enzymes. Fmm the figure it is obscwcd that with a constant amount

of lytlc enzyme, the pigment recovely increased as substrate amounts were raised from I

g to 4 g of yeast cells. Optimum prgment recovery resulted with 4 g of yeasts and 3.5 mg

of lytic enzyme.

Subshate concentration greater than 4 g per assay dld not increase pigment

recovery. Flgvrc 27 illustrates effects of increasing enzyme concenhatlon on the

recovery of pigments from frozen and thawed yeast cells. Optimum pigment release

occuned with enzyme concentrations of 2.5 - 3.0 mg per assay. In the case of TPI

manlmum pigment rclease was observed with 2.5 mg enzyme, while in the ease of other

yeants, this concentmtian was found to be 3.0 mg per assay. Higher enzyme

concentration did not Bow corresponding increaser in p i p e n t release. French presi

method showed belter p ~ p e n t movery with all the eolates. P l p e n t recovery in cells

broken by French press doubled when compared to enzymatic breakage (Table 11)

Sangha (1994) also f m d in a shtdy that R mbro cell wall was reststant to tho enzyme

treatment and prsctleally no pigment was extractable from the enzyme treated. Hence, P.

rodhorymo cells were much more amenable to tupture by the funcelase than to

mechanical ruphtre in a Freeh press. It was also noted Ulat, the enzyme had a pH

optmum of 4 to 5 and a temperahre optimum of 30' C. In the present study enzyme

activity was tested at 25O C using a water bath. Sangha, (1994) also found that the

enzyme treatment requires a more scrupulous control of experimental conditions in order

to be effective than the French press rupture method. Also, the economic feasibility of

enzyme treatment is questionable a3 reported by

1 + T P ~ *- ~. .--

Fig. 26 Effects of cell biomass on the activity of the lytic enlyme.

Diffmnt amounts of v a s t cells (wet weleht) were treated with the same amount , - , of (3.5 mg) enyme and were incubated for 24 hours and the plgment was extracted as described m Material and Methods. Amounts of carolenoids released were qualltatlvely measured by determining absorption by the extract preparations at 400 nm

Each data point represents an average of 3 readlags The smdard e m of the m a n for Tpl, M1,MZ and M3 areLO.l,O.Z,O.l, and 0.1, respectively

0.5 i 1.5 2 2 5 3 3.5 4 4.5 5 5 5 6

Enzyme concentrations (mg)

+TP? )* M I --M2 c31 .- - - -

Rg 27 Effects of enzyme concmbatlon on release of carotenolds h m yeast

Fined amount of cell mass was eeated with the d~fferenl volumes of cnzymc and incubated for 24 hour.. The plgmcnt was entmcted as described m the Marerials and Methods.

Each data point represents an average of 3 readmgs. The standard mrs of the meanforTP1, MI, MZ andM3 are+ 0.2,0.2,0.1 and0.3,respectivcly.

GenUtles and Haard (1991). Thee factors tell that the m y m a t e technique is less

commercially practical than the French press. Fmch press method showed better

piwent recovery with all the isolates. Plgmcnt recovery in cells broken by French press

doubled when compared to euymatlc breakage (Table I I). Sangba (1994) also found m

a stody that R mbrn coll wall was resistant to the enzyme heahnent and practically no

pigment was enhactable fmm thc enzyme heated cells. Gentles and Haard (1991)

heated P. rhodorymo with the enzyme funcelase and reported that the yeast capsule, not

the cell wall, was removed by enzyme treahnnt. Thts would explain the ~ ~ ~ ~ e p t ~ b ~ l t t y of

the P. rhodoqma cell wall, by the snlyme heatment psrhaps, that the apparent

differences m the srmctore of capsule in these two red yeasts (Gentles and Haard,

(1991). In the present study, cells ware incubatd for 24 hours before extracting the

pigment. Frsnch press luptored method showed more extractability than ulth the

enzymatic cell breakage,

Table 11. Recovery of carotenolds by enzymatic breakage and Prenchpress

Carokno~ds (~dg , dry weight) Isolate

'Standard emrs are for averages of h e determiermiatlons

CONCLUSIONS

The yeast cells were gmwn under different growth eandltlans and observed that

the opttmum temperature for all the mutants was 28'C Parent TPI yielded 12.5 giL

biomass and other the mutants MI, M2 and M3 13.1, 11.0 and 9.5 glL, respecttvely

Yeast malt bmth wlth 2 % peat extract as a nthogenous source was observed to be a

good substrate concentration. The cane molasses concentration 1 to 2 % as a carbon

source also gave good biomass yield, whereas bacto czapen dox broth was found c be

lnhsbitor ofthe grow& and pigment pmduction in all the yeast samples The initial pH of

the medla was found to be 5.0 to 7.0 for the opttmum gmwth. In all the growth

condiuons light enhanced the ptgment production.

The French pnss method was found to be an eilieient extraction method for the

mutants as well as the wtldfype yeasts. The total camtenold concenmitians for MI, M2

and M3 a n 250.6, 245.4 and 193.2 pgig (dry yeast), respectively Mutant 2 gave high

amount of pigment when a was extracted with F n e v dryer method. Sonication method

alone dtd not give much extraction of the pigment in all the mutants The cells that were

hpated with lysing enzyme fmm Aspergillus species gave higher yields of pigments.

Lysing enzymes tested wlth Tris- HCI and Citratepbosphate buffers at pH 7.0 resulted

in relatively greater pigment release The freeze and thaw method also enhanced the total

catenoid coneentratlon compared to Gesh, unfrozen/thawed yeast cells Yeast cells

when treated with dsffcrent eoncentrattons of thiol reagents llke beta mercapto ethanol

(100 mM) and D~thio thiitol(25 mM) showed less pigment rccoucry.

In the presence of 1 to 2 %cane molasses or 2 %peat extract supplements m YM

broth, better growth yields combined with higher pigment production were noted. All

yeast cultures grew well between pH 5.0 and 7.0, at 28' C.

F m the above it a concluded that French press method a superior erbdction

technique as to other methods. Enzymatic methods dld not Improve the

extractsb~l~ty of pigments when compared to other methods For bcner understanding of

the biology of the mutant sbdlns the following directions may be adopted:

(i) Proximal analysa.

(~i) Regulauon of the biosynthetic pathway for camtenaidr.

(iii) Character~zatlon ofthe key e"Zymes in the biosynthetic pathway fm camtenoids.

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