Improvement of yield and quality of agar from Gracilaria edulis (Gmelin) Silva.

tMPROVEMENT OF YIELD AND QUALITY OF AGAR FROM GRACILARIA EDULIS

(GMELIN) SILVA

DISSERTATION SUBMITTED

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF FISHERIES SCIENCE (MARICULTURE)

OF THE

CENTRAL INSTITUTE OF FISHERIES EDUCATION (DEEMED UNIVERSITY)

MUMBAI-400 061

by

CHANDRASEKHARA RAO A (Mari. 55)

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I C A R

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CENTRAL MARINE FISHERIES RESEARCH INSTITUTE

INDIAN COUNCIL OF AGRICULTURAL RESEARCH COCHIN-682 014

INDIA.

JUNE 2001

DEDICATED

TO MY

BELOVED PARENTS

P hon e: (Off)

Telegram Telex

Fax

E -m ail

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( w if m ar^TtypT '^ n ^ )

q r ^ H 1603, c m ^ - 6 8 2 014

CENTRAL MARINE FISHERIES RESEARCH INSTITUTE

(Indian Council of A gricutlural Research) P O S T BO X No. 1603, E R N A K U L A M , C O C H IN -682 014

3 9 4 8 6 7 / ...E xt 3 9 1 4 0 7

C A D A L W iN E K M 0 8 8 5 -6 4 3 5 M F R I IN 9 1 -4 8 4 -3 9 4 9 0 9

m d c n i f f i ® rn d 2 .vs n l.n e t. in

D ated 30 June, 2001

CERTIFICATE

C e rtifie d th a t th e th e s is entitle d “IWIPROVEIVIENT OF YIELD AND QUALITY OF AGAR FROM G r a c ila r ia edt///s(Gmerm)Silva” is a record of in d e p e n d e n t b o n a fid e research w o rk carried o u t by Mr CHANDRASEKHARA RAO

•A:* d u rin g th e period o f s tu d y from S e p te m b e r 1999 to A u g u st, 2001 under o u r su p e rv is io n an d guid a n ce fo r th e d e g re e o f Master of Fishery Science (Mariculture) an d th a t the thesis has n o t p revio usly fo rm e d th e basis fo r th e aw ard o f a n y deg ree , d ip lo m a , a ssociateship , fe llo w sh ip o r a n y o th e r sim ila r title.

M ajor Advisor/Chairm an

Advisory Com m ittee

f(Xr>cvn K P v (T ^et^ ja y a s a t f l^ ) S c ie n tis t (S e n io r S ca le) F ish e ry E n v iro n m e n t and M a n a g e m e n t D ivisio n

(P. Kaladharan) S cientist (S e n io r Scale) F ishery E n v iro n m e n t and M a n a g e m e n t Division

’TGopinathan) S e n io r S cientist Fishery E n v iro n m e n t and

M a n a g e m e n t Division

ACKNOWLEDGEMENT

i e xp re ss m y d e e p s e n s e of g ratitude to m y supervising guide, Dr.P.Kaiadharan, S cie n tist (S enior scale), F.E.M .D,, C .M .F .R .l, Cochin for his valuable guidance, u n stin te d s u p p o rt an d e n c o u ra g e m e n t thro ugh out the period of m y study as well as in the p re peration o f m anuscript, 1 am greatly indebted to m em b ers o f advisory c o m m itte e , Dr. C .P .G o p in a th a n , S e nior scie ntist and D r.R eeta Jayasankar, S cientist (S enior scale), C .M .F .R .l fo r th e ir guidance, supervision an d e n c o u ra g e m e n t through o u t m y work.

I am g ratefu l to Dr. M ohan Jo se p h Modayil, Director, C .M .F .R .l for providing me with the fa cilitie s to carry out m y w o rk at the institute during th e course o f this study.

I e xp re ss m y d e e p fe lt gratitude to Dr. R. Paul Raj, O.I.C, P .G .P .M . for his tim ely help, e n c o u ra g e m e n t and valuable s u g g e stio n s to carryo ut m y dissertattion work.

1 e xp re ss m y g ratitude to Dr. K a lia perum a l, O.I.C., M a n d a p a m Regional Centre for perm itting m e to ca rryo u t m y w o rk there. I wish to e xp re ss m y gratitude to Dr.

P .K a lim uthu and Shri J .R .R a m a lin g a m fo r their tim e ly help and valuable s u g g estions.

I ta ke this o p p o rtu n ity to express m y gratitude to D r.T .N a re n d e r for his help during m y d isse rta tio n w ork.

I am d e e p ly in d e b te d to Y .Freile-P elegrin, D .R obledo, B aricuatro and Hurtado C a lu m p o n g fo r se n d in g me the reprints o f th e ir work an d also fo r their valuable sugg estions, f e xp re ss m y heart-feft thanks to Shri S .N a n d a k u m a r Rao fo r his co o p e ra tio n a n d help. M y th a n ks are due to M r.K .S u m a n th kum ar for his w h o le h e a rte d c o -o p e ra tio n and help. M y thanks are d ue to Mr.Ram alinga, M rs.JuIiet, Mr. R a jann a, Mr. V e nu gopal, M r.Subodh ku m a r Patro, Mr. Laxm ikant

Patnaik, Mr. Kiran babu, Mr. P.S aravanane, Mr. V.Venkiteshan, Mrs.Smitha, Miss Liya and Miss. S a ndhya fo r th e ir help during the course of my work,

1 take this opp ortunity to thank all m y seniors and juniors for their cooperation, suggestions and support,

I express m y th a n ks to Ms. Rosalie schaffer, Technical inform ation specialist, U .S.A and Jean Collins, FAO Librarian for th e ir prom ptness in providing me with the literature needed fo r m y study,

I ackno w le dge the Indian council of Agricultural research, N e w Delhi for awarding me with the fellow ship thro u g h o u t the tenure of my M.F.Sc. course.

cUM ila ic n

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ABSTRACT

Use of optim um levels of alkali/acid and other m odifications in extraction m ethods w e re determ ined to increase the yield and quality of agar from red seaw eed G racilaria edulis (G m elin) Silva. Presoaking of dry w eeds in w a ter for 2 hrs in c re a s e the yield but did not im prove the gel strength of colfoid.

P resoa king in w a te r fo r 12 hrs in crea sed considerably gel strength, m elting te m p e ra tu re and reduced sulfate co n te n t but not the yield. Addition of 3.0 N solution o f.N a O H to the 0.5 N and 1.0 N N a O H presoaked G.edulis on boiling resulted in yield o f 44.91% and 49.26% respectively and also reduction of sulfate levels in agar (1.5% w/v). How ever addition of NaOH during boiling did not im prove gel stren gth of agar from alkali presoaked G .edulis . A ddition of different concentrations of N aO H during boiling o f HCI presoaked G .edulis show ed im p ro ve m e n t neither in yield nor in th e quality of agar and also resulted in hydrolysis o f agar. Pretreatm ent o f 2.0-3.0 N NaOH at 80°C for 1 hour to G .edulis presoake d for 11 hrs in w a te r proved to be m o st ideal and optim ized extraction to obtain higher yield (14,16% ), m a xim u m gel strength (291 g/cm^), lowest sulfate (0.732% ) and h ig h e s t m elting point (99°C) of agar.

CONTENTS

1. INTRODUCTION 1

2. REVIEW OF UTERATURE 4

2 .1 S E A W E E D S AN D TH E IR U T ILIZA TIO N 4

2.2 S E A W E E D D ISTR IBU TIO N A N D R E S O U R C E S IN INDIA 5

2.3 S E A W E E D S AN D P H Y C O C O LL O ID S 6

2 ,4 .S E A W E E D IND U STR Y IN IN D IA A N D W O R L D 7 2.5 C O M M E R C IA L FE A S IB ILITY OF A G A R IN D U S TR Y 9 2.6 YIELD, G E LS T R E N G T H A N D T H E IR R E LA T IO N S H IP

TO Q U A L IT Y OF A G A R 10

3. MATERIALS AND METHODS 13

4. RESULTS 21

5. DISCUSSION 39

SUMMARY 42

REFERENCES 44

Table i - Effect of presoaking in potable water on agar yield and quality from Gracilaria edulis (Mean ± S.D.)

26 Table ii ~ Effect of presoaking in different concentration of HCI on

yield and quality of agar from_G/'ac/7ar/a edulis (Mean ± S.D.) 27 Table iii - Effect of presoaking in different concentrations of solutions

on the agar yield and quality from Gracilaria edulis (Mean ± S.D.)

28 Table iv - Effect of addition of NaOH during the extraction of agar from

alkal pre-soaked (0.5 N NaOH) Gracilaria edulis (Mean ± S.D.)

29 Table v - Effect of addition of NaOH during the extraction of agar from

aikaii pre-soaked (1.0 N NaOH) Gracilaria edulis (Mean ± S.D.)

30 Table vi - Effect of addition of NaOH during the extraction of agar from

acid pre-soaked (0.5 N HCI) samples of Gracilaria edulis (Mean ± S.D.)

31 Table vii - Effect of addition of NaOH during extraction of agar from acid

presoaked (1.0 N HCI) samples of Gracilaria edulis (Mean ± S.D.)

32 Table viii - Effect of alkali pre-treatment with raising temperature 80°c

for one hour before extraction on the yield and quality of agar (Mean ± S.D.)

33 Table ix - Effect of acid pre-treatment with rising temperature to 80° c

for one hour on yield and quality of agar (Mean ± S.D.)

34

Table-x: Agar yield significance at alkali pre treatment 35

at 80° C for 1 hr (2.0 N NaOH)

Table- xi: Gel strength significance at alkali pre treatment 36 at 80° C for 1 hr (2.0 N NaOH)

Table- xii: Sulfate content significance at alkali pre treatment 37 at 80° C for 1 hr (2.0 n NaOH)

Table-xii: Melting temperature significance at various 38

NaOH pre treatment at 80°c for 1 hr (2.0 N NaOH)

1. Seaweed collection area 14

Plate 1. W ashed seaweeds kept for Sun drying 15 Plate 2. Repeatedly washed and dried seaweeds 15 Plate 3. Completely bleached and dried seaweeds 17

Plate 4. Expeller (press) 17

Plate 5. Dried Agar sheets of various chemical manipulations 19

Plate 6. Gelometer 19

INTRODUCTION

A g a r is th e m a jo r c o n s titu e n t of the cell wall p o lysaccharid e of certa in red a lg a e (R h o d o p h y c e a e ), e sp e cia lly the m e m b e rs of fa m ilie s G e lid ia ce a e , G e lid ie lla c e a e and G ra cila ria ce a e . “ A g a r - A g a r "is the M alay w o rd fo r a gelling s u b s ta n c e e xtra cte d fro m E u c h e u m a sp, now known to be c a rra g e e n a n (K a la d h a ra n & K a lia p e ru m a l, 1999). T h e term aga r is now g e n e ra lly a p p lie d to th e a lg a l g a la c ta n , w h ich h a ve aga ro se , th e d isaccharide a g a ro b io s e as th e ir repeatin g units.

A g a r is th e firs t ph yco co tlo id id e n tifie d an d isolated fro m red s e a w e e d s as a p u rifie d e xtra ct. In 1658 a J a p a n e s e in n k e e p e r T a ro z a e m o n a c c id e n ta lly d is c o v e re d th e m e th o d of p ro d u c in g dry a g a r (FAO, 1990). T h e w o rld d e m a n d fo r a g a r h a s in cre a se d ra p id ly in re ce n t y e a rs (Buendia, 1998) and p re s e n tly th e s u p p ly o f a g a r co n ta in in g s e a w e e d s are being greatly reduced. It is e s tim a te d th a t th e c u rre n t p ro d u ctio n o f a g a r in th e w o rld is a b o u t 7500 - 1 0,000 to n n e s /y e a r a n d In d ia 's s h a re is h a rd ly 130 to n n e s /y e a r (D evaraj e f a/., 1999).

S tu d ie s o v e r th e p a s t 30 y e a rs h a v e re ve a le d th a t th e b a s ic structure o f a g a r is re g u la rly a lte rn a tin g s e q u e n c e o f 3 linked p -D -g a la c to p y ra n o s e and 4 linked 3, 6- a n h y d ro - a - L -g a la c to p y ra n o s e (Araki, 1966). T h is re p e a tin g pattern m a y be m a s k e d by th e p re s e n c e of L -g a la cto se , 4 ,6 -0 -(1 -ca rb o xye th ylid in e ) - D g a la c to s e , S u lfa te h e m ie s te rs an d 0 - m e th yl s u b s titu e n ts in variou s p o sitio n s on g a la c to s e u nits (D u c k w o rth and Y a p h e , 1971).

G e fid iu m sp is th e m o st p re fe rre d c o m m e rc ia l aga rop hyte du e to th e high q u a lity o f its agar. It has b e c o m e u n a va ila b le to industries b e ca u se of d e p le tin g wild s to c k d u e to o ve r e x p lo ita tio n . E asy a va ila b ility of both wild and c u ltu re d GracH aria sp s, h a s led to GracHaria b e co m in g th e principal s o u rc e of a g a r w o rld w id e (G ritchley, 1993).

A p p ro x im a te ly 60% o f th e w o rld 's p re s e n t p ro d u ctio n o f a g a r is d e riv e d fro m GracH aria s p (D u ra ira tn a m , 1987). C hile is th e largest p ro d u c e r of a g a r (FAO, '3 996 ). T h e c h e m ic a l an d p h ysica l p ro p e rtie s o f com m e rcial a g a r p re p a re d in J a p a n , N e w z e la n d , U .S.A., U S .S .R ., and S p a in V a ry greatly w ith th e

a g a ro p h yte s used. B a se d on th e quality, aga rs have been classified into two types; nam ely,

(i) Food Grade Agar; and (ii) B a c te rio lo g ic a l agar.

F o o d g ra d e a g a r c o n ta in s a b o u t 10 - 15% essentially neutral aga rose , 4 0 % c h a rg e d a g a ro s e an d 40 - 50% non -ge lling sulfated agar m o le cu le s. B a c te rio lo g ic a l a g a r w ith a b o u t 3% su lfa te an d it should contain high c o n c e n tra tio n s o f a g a ro s e . A g a ro s e is re sp o n sib le fo r gelling pow er (A rm isen and G atatas. 198 7). B a c te rio lo g ic a l a g a r is e xp e c te d to contain gel strength a b o v e 2 50 g m /cm ^. M ostly p ro d u c e d a g a r in India is o f fo o d grade agar. Indian d e m a n d fo r fo o d g ra d e a g a r is m e t p rim a rily b y d o m e s tic production (C oppen, 1989) b u t 1 4 - 1 6 to n n e s o f h ig h e r-g ra d e a g a r is im p o rte d annually. J a p a n is th e m a jo r e x p o rte r to India. T h e tota l a g a r p ro d u c tio n in India ranges betw een 110 and 132 t, u tilizin g a b o u t 8 80 - 1100 t of dry w e e d p e r ye a r (K aladharan and K a lia p e ru m a l, 1999).

G e n e ra lly G ra cila ria sp. yie lds low q u a lity a g a r due to high sulfate c o n c e n tra tio n and th e re fo re th e y are called 'a g a ro id s' or ‘ g ra cila ria g u m ’ (Craigie,

1990). It is w e ll kn o w n th a t th e q u a n tity and q u a lity o f phycocolloid varies not on ly a m o n g s p e c ie s (C ote and H a n isa k, 1986), b u t th e y are also in flu e n ce d by e n v iro n m e n ta l fa c to rs (C raigie an d W e n , 1984), s e a s o n a l variations (Oza, 1978;

L a h a ye a n d Y a p h e 1988; R o b le d o e ta /., 1997; Freile - P e le g rin e ta l., 1999), and e x tra c tio n m e th o d s (C ra ig ie an d Leigh, 1978; A rm is e n a nd Galatas, 1987). In G ra cifa h a sp. a lk a lin e p re -tre a tm e n t is n e ce ssa ry to c h a n g e the L-galacto^e 6- s u lfa te in to 3 ,6 - a n h y d ro - L - g a la cto se to in c re a s e th e extract gel strength (A rm ise n a n d G a la ta s, 1987). V a rio u s w o rke rs had reporte d the extra ctio n of a g a r in India fro m s e a w e e d s by th e tre a tm e n t with d ilu te m ineral acids (B ose et al., 1943; T h ivy, 1958; K a p p a n n a an d Rao, 1963).

A t p re s e n t fo o d g ra d e a g a r produced in In d ia fe tc h e s a b o u t R s.300 - 350 / Kg, w h e re a s th e b a c te rio lo g ic a l aga r is s o ld at Rs.800 - 1000 / Kg.

m a kin g q u a lity o f a g a r th e s o le c rite ria fo r its p ric e value. Q uality o f a g a r is

de cid e d by th e gel stre n g th a nd s u lfa te content. G el stren gth is the c a p a c ity of gel to hold th e s u b s ta n c e on s u rfa c e w ith o u t breaking su rfa ce of gel. H e nce any v a lu e a d d itio n to th e in d ig e n o u s ly produce d a g a r such as su lfa te content reduction and gel stre n g th in cre a se , not at e xp e n se o f yield wil! d e fin ite ly m ake on e in d u s try e c o n o m ic a ily v ia b le an d can save fo re ig n excha nge by lim iting the im p o rt o f b a c te rio lo g ic a l agar. H e re an atte m p t has been m ade to increase the gel stre n g th an d to im p ro v e th e q u a lity of agar w ith ch e m ic a l m anip ulatio n from G ra cila ria e d u lis .the m o s t c o m m o n a g a ro p h y te in Indian aga r industry.

REVIEW OF LITERATURE

2.1 SEAW EEDS AND THEIR UTILIZATION

S e a w e e d s are o n e o f th e co m m e rcia lly im p o rta n t ren e w a b le m arine re so u rce s th a t b e lo n g to th e p rim itiv e g roup of non -flo w e rin g plants w hich grow s u b m e rg e d in in te rtid a l and s h a llo w w a te rs in th e sea, and also in b ra kish w a te r e s tu a rie s (U m a m a h e s w a ra R ao, 1969). B ased on m orp holo gy, cell wall and p ig m e n t c o m p o s itio n , s e a w e e d s are cla ssified into green, red and brown s e a w e e d s ( C h a p m a n ,J .V ., 1970).

T h e red algae s u ch as Gelidietla, G elidium , P te ro cla d ia and G ractla ria yie ld a g a r. S o m e o th e r red algae viz. H y p n e a sp, E u c h e u m a sp, C h o n d ru s sp, an d G ig a rtin a s p are th e m a jo r s o u rc e fo r th e p roductio n of c a rra g e e n a n . A lg in is o b ta in e d fro m brow n a lg a e like S a rg a s s u m sp., Turbinaria sp., H o rm o p h y s a sp., C ysto se ira sp., L a m in a ria sp., U n d a ria sp. and M acro cystis sp. (A non, 1 9 8 7 ). T h e s e p h y c o c o llo id s are used as gelling, stabilizing and th ic k e n in g a g e n ts in food, c o n fe c tio n e ry , p h a rm a c e u tic a ls , dairy, textile, paper, p a in t an d v a rn is h in d u s trie s etc. A p a rt from th e se , p ro d u cts such as m annitol, Iodine, L a m in a rin and F u co id in are also o b ta in e d from m arine algae (K a iia p e ru m a l. 1993; K a lia p e ru m a l e t al., 2000). M a n y protein rich se a w e e d s such as Ulva, E n te ro m o rp h a , C a ulerpa, C odium , M o n o s tro m a (Green Algae);

S a rg a s s u m , H y d ro c la th iru s , L a m in a ria , U n d a ria an d M a cro cystis (Brown algae);

P o rp h yra , G ra cila ria , E u c h e c u m a , L a u re n d a and A c a n th o p h o ra (Red algae) are u sed as h u m a n fo o d (Anon, 1987). In c o u n trie s like Japan, C hina, Korea, M alaysia, P h ilip p in e s and o th e r S o u th e a s t A sian c o u n trie s in th e fo rm o f soup, salad, c u rry etc. T h e food v a lu e o f se a w e e d s d e p e n d s on th e ir protein, m inerals, tra c e e le m e n ts a n d V ita m in s p re s e n t in th e m (S E A F D E C . 1998).

S e a w e e d s are u tilize d in d iffe re n t p a rts o f th e w orld in diversified fie ld s su ch a s a n im a l feed, fe rtiliz e r fo r land c ro p s etc. (D eve et al,, 1977), A c c o rd in g to S re e n iv a s a Rao e t al. (1980), S e a w e e d s a re used as a s o u rc e of ene rgy. In p re s e n c e o f m e th a n e bacteria, b io g a s p roductio n from Ulva was a c c e le ra te d .In In d ia fre s h ly c o lle c te d c a s t a sh o re s e a w e e d s are used as m anure fo r c o c o n u t p la n ta tio n e ith e r d ire ctly, or in th e fro m o f a c o m p o st e sp e cia lly in c o a sta l a re a s o f T a m ilN a d u an d K e ra la (U m a m a h e s w a ra Rao, 1972).

A c c o rd in g to C h e n n u b h o tia (1996), se a w e e d s m a n u re w a s supe rior to th e c o n v e n tio n a l o rg a n ic (farm yard) m anure. T h e high a m o u n t o f w a ter s o lu b le p o ta sh , o th e r m in e ra ls a n d tra c e elem e nts p re s e n t in se a w e e d s are readily a b s o rb e d by p la n ts and th e y c o n tro l d eficiency diseases. A cco rd in g to liu, sijan and th e c o -w o rk e rs (1997), G racilaria and p raw n polyculture practice im p ro ve w a te r q u a lity to s o m e e x te n t and prevent b a c te ria l disease o f prawn.

Use o f G ra c ila ria a s b io filte r in p o ly c u ltu re practices is in d e e d two w ay beneficial, as it w ill n o t o n ly h e lp fu l as b io filte r b u t a lso useful to lift e c o n o m ic s ta n d a rd s of p raw n fa rm e rs th ro u g h h a rve stin g . C u ltiva tio n o f G ra cila ria p a rvisp o ra in shrim p fa rm e fflu e n ts e n h a n c e d g ro w th o f a lg a e (Nelson e t al., 2001). S e a w e e d s rich in io d in e s u c h as A s p a rg o p s is tax\form is and S a rc o n e m a sp can also be used for co n tro llin g g o itre d is e a s e (C h e u n u b h o tia , 1996). T h e tra c e ele m e n ts and growth h o rm o n e s p re s e n t (K a la d h a ra n an d S rid har, 1999) in th e liquid seaw eed fertilizer act as g ro w th p ro m o te rs and in c re a s e th e yield of cro p p la n ts by 20-30% .

2.2 SEAW EED DISTRIBUTION AND RESOURCES IN INDIA:

M o re than 10,000 s p e c ie s o f m a rin e a lg a e have been reporte d from all o v e r th e w o rld . T o ta l w orld p ro d u c tio n of s e a w e e d s is e stim ated to 6-7 million to n n e s of w h ic h n e a rly 90% c o m e s fro m A sia p a cific regio n (H urta do-P once , A.,

1997). A c c o rd in g to FAQ d a ta b a s e (1998) out of 7 m illio n to n n e s of seaw eeds, 4 m illion to n n e s are brow n s e a w e e d s and 1.9 m illion to n n e s are red seaw eeds and rest o f th e m are green alg a e . In India luxuriant g ro w th of several sp e cie s of green, b ro w n , an d red algae o c c u r along th e south e a s t c o a s t of T a m ilN a d u , from R a m e s w a ra m to K a n ya ku m a ri, G u ja ra t, L a ksh a d w e e p and A n d a m a n N icobar islands. F a irly rich s e a w e e d s b e d s are p re se n t in th e v icin ity of Bom bay, Karwar, R a tna giri, G o a , V a rka la , V izh in ja m , V is h a k a p a ttin a m a n d in coastal lakes tike P u lik a t a n d G h ilka (U m a m a h e s w a ra Rao, 1969; C h en n u b h o tia , 1996;

K a lia p e ru m a l, 1993). A b o u t 7 0 0 s p e c ie s o f m a rin e a lg a e h a v e been recorded fro m d iffe re n t p a rts o f Indian c o a s t including L a ks h a d w e e p and A n d a m a n - N ic o b a r is la n d s , o f th e s e n e a rly 6 0 s p e c ie s are c o m m e rc ia lly im portant se a w e e d s (U m a m a h e s w a ra Rao, 1969; K a lia p e ru m a l et al., 2 0 0 0 ). From th e seaw eed re s o u rc e s s u rv e y ca rrie d o u t in th e in tertidal and s h a llo w w a te r areas of e a st and

w e s t c o a s t a n d a ls o L a k s h a d w e e p a rc h ip e la g o , so fa r by the C .M .F.R .I, an d other re se a rch o rg a n is a tio n s s u c h as C .S .M .C .R .I and N.I.O. it is estim ated th a t total s ta n d in g cro p o f all th e s e a w e e d s in Indian w a te rs is m o re than on e lakh to n n e s {wet w e ight) c o n s is tin g of 6 0 0 0 to n n e s {wet w eight) of aga r yielding seaw eeds.

16000 to n n e s (w e t w e ig h t of a lg in yie lding s e a w e e d s (alginophytes), and the re m a in in g q u a n tity o f e d ib le and o th e r s e a w e e d s (D evaraj et al.,1997).

2.3 SEAW EEDS AND PHYCOCOLLOIDS;

P h y c o c o llo id s e x tra c te d fro m s e a w e e d s have greater sig nifican ce;

p h y c o c o llo id s re fe r to th o s e p o ly s a c c h a rid e s e x tra c te d from both fre sh w a te r and m a rin e a lg a e (A rm is e n an d G a la ta s , 1 9 8 7 / P o ly s a c c h a rid e s e xtra cte d from m a rin e red a n d b ro w n algae, s u c h as agar, ca rrg e e n a n and algin are e c o n o m ic a lly im p o rta n t an d h a v e co m m e rcia l sig n ifica n ce , S ince these p o ly s a c c h a rid e s e x h ib it high m o le c u la r w eight, high visco sity and e xcellen t gelling, s ta b iliz in g an d e m u ls ify in g p ro p e rtie s (Ji M in g h o u , 1990).

In In d ia s e a w e e d s are n o w used m o s tly as raw m aterial fo r the p ro d u ctio n o f a g a r a nd s o d iu m a lg in a te (C oppen, 1991). E stim ated world p ro d u c tio n o f a g a r w a s 7 0 0 0 - 1 0 ,0 0 0 to n n e s va lu e d at U S $ 200 m illion in 1989 (H a rta d o -P o n c e , 199 7) J a p a n is th e m a jo r c o n s u m e r (a b o u t 2000 to n n e s per year) m o s tly c o m in g fro m d o m e s tic pro d u ctio n . U .S .A c o n s u m e s 1000 tonnes pe r year, w h ic h is m o s tly (80% ) s u p p lie d by Chile, M oro cco, and Spain. The E u ro p e a n u n io n n e e d s 1300 to n n e s p e r ye a r (C opp en, 1989).

T h e b ro w n s e a w e e d s are s o u rc e of algin. C hina, K o rea and Japan c u ltiv a te th e b ro w n a lg a e fo r fo o d , and not fo r algin. T h e U.S gets its a lg in a te ' fro m kelp h a rv e s te d fro m th e w ild in C alifornia, M e xico p ro d u ce s som e, but the E ln in o p h e n o m e n o n a d v e rs e ly a ffe c ts p ro d u ctio n (C o p p e n , 1991), A g a r-a g a r is often u s e d w h e re firm gel is n e e d e d and algin fo r so ft and vis c o u s product (C h e n n u b h o tia , 1 9 9 6 ). T h e w o rd ' a lg in a te ' is a g e n e ric term , m eaning the va rio u s d e riv a tiv e s o f a lg in ic a o id (C oppen, 1991). A lg in ica cid p re s e n t in s e a w e e d m a in ly a s c a lc iu m salt, w ith le sse r a m o u n t a s m ag n e siu m , so d iu m and p o ta ssiu m s a lts (A n o n , 1987). T h e m o s t im p o rta n t co m m e rica l derivative of

alginicacid is sodium alginate. O ther derivatives produced include the potassium, ammonium and calcium salts, propyleneglycolalginate and alginicacid itself (Kaliaperumal e t a/., 2000). T he ability of alginates to increase the viscosity of solution to which th e y are added in low concentration enable their use in a wide range of applications in the food and pharmaceutical industries (Coppen, 1991).

In India algin is prim arily used as a thickening agent in textile and printing industries (Kaliaperumal, 1993). The food industry alginates are used in the m anufacure o f a m ultitude o f dairy, bakery, m eat and other products, which take advantag es of their thickening, gelling and stabilizing properties (Coppen, 1991; Kaliaperum al, 1993). Carrageenan is extracted from Kappaphycus and Eucheuma. which are cultured in the Philippines, Indonesia, Fiji, Micronesia and C hina (Hurtado - Ponce, 1997). Other sources of carraneenan are Chondrus and Gigartina, Laurencia (Kaliaperumal, et. al., 2000). Laurencia papillosa can be used profitably as raw material (Doshi,ef a/., 1987) for production of carrageenan as this contains carrageenan, C hondrus and Gigartina are abundant in Canada and Atlantic coasts of Europe (Pillay, 1990). Three types of carrageenan used com m ercially are:

. Kappa carrageenan: - a hard gelling colloid extracted from Kappaphycus.

i. lota carrageenan: - a soft gelling colloid from Eucheuma.

ii. Lam bda carrageenan; - a non-gelling colloid extracted form Kappaphycus (FAO, 1990.)

2.4 S E A W E E D IN D U S T R Y IN IND IA AND WORLD:

Seaweed industry in India is totally based on the natural stock of agar yielding red seaweeds such as Gefidieffa acerosa and Gracilaria edulis and algin yielding brown seaw eed species such as Sargassum and Trubinaria (Coppen. 1991). A gar and sodium alginate are the phycocolloids produced in India. There are 40 seaw eed-processing units, of which 22 produce agar. India produces 110 - 132 tonnes of dry agar annually utilizing about 880 - 1100 tonnes of dry agarophytes, and 360-540 tonnes of algin from 3600 - 5400 tonnes of dry alginophytes (Kaladharan and Kaliaperumal, 1999).

In India although, utilization of seaweeds for the extraction of soda ash, alginicacid and iodine started during the second world war period, production of a g a r started in 1966 {Kaliaperumal, 1997); seaweeds exported until 1975 when the G o ve rn m e n t of India banned the export of seaweeds (Silas, e t al., 1987; Devaraj e t a!., 1997) in order to meet the requirement of local agar industry.

Im port of agar in 1987 into Thailand was valued at Bht. 112.9 million m ainly c o m e from Japan, but there has been increase from 94 tonnes to 134 tonnes in th e a g a r import from Chile in recent years. Malaysia imports of agar have been fa irly consistent at around 250 tonnes per annum. Indonesia import agar fro m a variety of sources, with Asian and South American countries.

India's d e m a n d fo r a g a r is m e t prim arily by dom estic production, but small am ount of ba cte rio log ical agar is im ported (Coppen, 1989).

A g a r producers in India follow a simple method of agar extraction (Armisen and G alatas, 1987). In this they boil the dry weed, the hot extract is filtered, cooled, fre e ze thawed, bleached and dried in the sun. The agar is m arketed e ith er in strips or as pow der (Coppen, 1991). Gelidiella acerosa yields industrial g rad e agar, w here as GracHaria yields food grade agar (Ji Minghou, 1990), All o v e r th e world agar is produced by two main processes:

i. F re e ze thaw ii. P ress dehydration

The m ain s te p s in processing o f agar from agarophytes m ay be summarised as:

i. E xtraction o f agar with hot w ater ii. Filtration to elim inate the w eed residue

iii. C o n c e n tra tio n and purification (Freeze thaw or gel press) iv. D rying

The main a d van ta g e s of gel press / press dehydration process is that it omits the freeze-thaw cycle, and the final product is always in powder form.

In fre e z e -th a w m ethod, square agar or strip agar is obtained (Ji Minghou, 1990). Algin is m a nu facture d as sodium alginate at the cottage industry level th ro u g h alkali deposition, precipitation by acidification, centrifuging, drying etc, (FAO, 1990). S a rg assu m and Turbinaha are the two main raw materials, used in algin industry. S argassum is preferred over Turbinaria as the quality and q u a n tity o f algin yield are better from the form er (Coppen, 1991).

Most of the units in India are ca p ab le of producing 20-30 t/yr and total algin production in India is 360-540 t/yr. (Coppen, 1991; Kaladharan and Katiaperumal, 1999), O ther w ellkno w n phycocolloid extracted from seaweeds is carrageenan, which is having co m m ercia l im portance. W orld carrageenan production has showed a 4% y e a rly growth from 1978 to 1993. The five carrageenan markets are Europe (36%), North A m erica (26%). Latin Am erica (17%), Australia (13%) and Japan (8%) (H artado-Ponce, 1997).

2.5 C O M M E R C IA L FEASIBILITY O F AGAR INDUSTRY:

A p p ro xim a te ly 60% of world present production of agar is derived from G racilaria sp (Durairatnam, 1987). Total agar production in the world touches 7000 to n n es, out of this 4200 tonnes derived from Gracilaria sp (Coppen, 1991). Six sp e c ie s of Gracilaria occuring in India have been reported to be potential s o u rc e of a g a r (Desikachary, 1967). Capacity of agar production units in India varies betw ee n 500 and 800 kg dry agar / month using 4 - 5 tonnes of dryweed / m o nth (Kaladharan and Kaliaperumal, 1999). They have also found that the m a te ria l is purchased from th e dealers at the price of Rs, 3000 - 4200 / ton for G racilaria and Rs. 12,000 - 1 6 , 0 0 0 / ton for dry Gelidiella. According to a recent su rvey on a g ar industry (Kaladharan and Kaliaperumal, 1999), the product of agar is sold w e e k ly to the B om bay m arket for Rs. 200 - 250 / kg of food grade agar and Rs. 40 0 - 500 / Kg. o f industrial grade agar. Approximately 10 lakh rupees m a y be required for the esta b lish m en t of small agar plant capacity of 2 - 3 tonnes (K a la d h ara n and Kaliaperum al, 1999).

2.6 YIELD, G E L S T R E N G T H A N D THEIR RELATIONSHIP TO QUALITY OF AGAR:

It is w ell known th a t the quantity and quality of the colloid varies not only among s p e cie s (Cote and Hanisak, 1986), but they are also influenced by environm ental fa c to r (Craigie and W en, 1984), seasonal variations (Oza, 1978), and extraction m e th o d s (Craigie and Leigh, 1978; Arm isen and Galatas, 1987).

Yanagawa (1936) offered the m ethod of treating the agar solution or algae with alkali to im prove th e quality o f agar of Gracilaria sp (FAQ, 1990). 'A ra ki'in 1956 showed the evidence, proving th e heterogeneity of the agar by separating the agar into two d iffe re n t polysaccharides named agarose and agaropectin using the acetylation m e th o d (Duckworth and Yaphe, 1971).

T h e seaw eed tre a tm e n ts prior to extraction are very important as they will co n ditio n to a high degree characteristics o f the agar obtained (Armisen and Galatas. 1987). Cultured Gracialaria edulis used fo r extraction of agar under pressure fo r 2 -4 hrs with addition of 0.5% KCI resulted slight increase in gel strength, gelling te m p e rature and melting point (Thom as and Krishnamurthy,

1976). A gar stu d ies in G racilaria bursapestohs {Grr\eWr\) SWva and Gracilaria coronopifolia indicate d that agar yield in inversely related to the total nitrogen content of thalli and gelstength of agar (Hoyle, 1978). The fluctuations of gelstrength of th e phycocolloid obtained from Gracilaria corticate showed a narrow range fro m 17-25 gm /sq.cm with a slight raise in the month of June, October, N o v e m b e r and D ece m b e r w hen algae attain peak growth (Oza, 1978).

The se asonal c h a n g e s in the properties of phycocolloids in their study suggest that strong gels from N eogardhie/la baiieyi in the w inter and in early summer months (Asare, 1980).

T h e agars extracted from Gracilaria are soft, non-rigid, plastic and interm ediate and th e strength of gelationcharacters could be improved considerably by alkali tre a tm e n t (W hyte and Englar, 1980). Depending on season and life sta g e s of aigae, variations in gelstrength, yield and gelation of agars o ccu r in G racilaria verrucosa (whyte e t a ! . , 1981.). Gracilaria cylindrica contain low su lfa te c o n te nt fa vo u rab le for gelstrength and the species appear to thrive under co n ditio n th a t been conducive to the m ass production (Doty and

Santos, 1983). Patw ary and V e n d e r Meer (1983) developed clones from wild Gracilaria tikvahiae, which are su perior in both growth characterstlcs and agar quality to wild ones. It is reported that agar of the Micronesian species Gracilaria edulis contain th e highest sulfate content and also a low gel strength (Nelson ef a i. 1983). F riedlander and zelikovitch (1983) obtained the positive correlation between the specific growth rate o f Gracilaria sp., Pterocladia sp., Hypnea musciformis and H ypnea cornuta and the phycocolloid content during the main growing season. Craigie and other (1984) established that alkali treatment modified agar from Gracilaria sjo e s te d tii to be the best agar produced from any Gracilaria sp. T h e y also found th a t Gracilaria plants richest in nitrogen gave the highest gelstrength. H ow ever C hristeller and Laing in 1989 noticed that significant increase in agar yield at low nitrogen concentration. Total agar content will be higher in algae grown in natural seaw ater than in enriched seawater (R otem , et. al., 1986). Cote and Hanisak (1986) stated that melting tem perature will have positive correlation with gelling tem perature and agar yield but it will negatively correlate with the am ount of sulfate present in agar.

According to Miller and Furneaux (1987) desulfation of agar occurs relatively slowly in rapidly growing m aterials o f Gracillaria secundata.

S e a son a l changes have been observed in alginicacid content of Cystoseira trinoidis and H orm o p h ysa triquetra (Kaliaperumal, et. al., 1988).

Durairatnam (1987) pointed out clearly that agar yield and gelstrength varies seasonally. Luhan (1992) observed distinct changed in agar yield & strength coinciding w ith ch a ng e s in environm ental conditions. There is a clear seasonality in the yield o f a g a r from the C ham pia nova, zea/and/er with lower values in winter and higher in spring (Miller et a i , 1996). Calumpong e t al., (1999) observed a strong correlation between agar yield and nutrient levels in ambient water. Freile- Pelgrin et al. (1999) found correlation between agar yield and seasonal changes.

T h e quality of phycocolloids such as agar-agar and carrageenan is usually d eterm ined by their gelstrength. Vijayanthi and Rengasamy (1989) devised a new appartus fo r the determ inaton of gel strength.

A ccording to Ji Minghou (1990), Gracilaria contains a considerable amount of highly sulfated galactans varying with species, growing season and location. He proved that alkali pre treatm ent process for Gracilaria sp is an extremely im p o rta n t m easure to im prove the quality of agar product, Coppen(1990) observed that chem ical treatm ent of agarophytes prior to extraction often produces a better agar in term s of quality of yield than one produced w ith o u t such treatm ent. A lkali pre-treatm ent has been found to be most useful; particularly fo r Gracilaria sp. Miza mouradi-Givernand (1992) stated that sulfate c o n te n t and 3,6 anhydrogalactose content in agar of Gelidium sesquipedale ne ga tive ly correlated with gelling properties of agar. Mathew e t al.

(1993) perform ed alkali pre-treatm ent to Gelidiella acerosa and obtained significant in cre a se in yield and quality com pared to acid pre-treatment.

According to A n o n g Chirapart and his team (1995) gelstrength of the crude agar extract incre a se d with the increase in concentration of sodium hydroxide treatment. Freile-Pelegrin and R obledo (1997) found that alkali treatment dram atically im pro ve d the quality o f agar in Gracilaria cornea.

A c c o rd in g to Mirza M ouradi-G ivernaud and co-workers (1999), agar rheological ch a ra cters are influenced by reproductive stages of algae. It is established th a t the phycocolloid gel strength is more related to the mean polysaccharide chain length but not its chemical substitutes (Thiery-Givernaud et a/,, 1999). Freile-Pelegrin (2000) studied extensively on the effect of storage time on gelstrength and melting tem perature of agar and also reported that these are negatively correlated. However, Lian (2000) opined that the yield and properties of agar varied with extraction m ethods. It is learnt that lot of factors are responsible fo r determ ining the yield, quality of agar extracted from Gracilaria spp.

In th e light of the above review the present investigation can fill the gap in info rm a tio n on

(i) R elationship betw een yield, gelstrength and sulfate content in agar extracted from the s e a w e e d Gracilaria edulis.

(ii) The o p tim u m requirem ent o f alkali or acid as well as other post harvest m anipulations to obtain m axim um yield and improved quality of agar from G.

edulis.

MATERIALS AND METHODS

3.1. Collection of seaweed:

Fresh seaw eed GracHaria edulis (Gmelin) Silva were collected from pamban area (78° 8 ’ E, 9° 17' N) M andapam (Fig. I). This marine red algae belongs to class: R hodophyceae, Order: G igartinales and Family: Gracilariaceae.

Entire plants o f G. eduUs^ grow ing attached to coral stones or sea grasses were hand picked during low tide period from the lagoon, They were washed with seawater and w e re transported to the laboratory.

3.2. Cleaning and Drying:

In th e laboratory plants were washed thoroughly in running freshw ater until th e algae were free from sand and other dirt particles. Care was taken to rem ove epiphytes, other algae, shells and caicarious material from seaweed, w ho se presence m ay alter the yield and quality of agar. The washed and cleaned m aterial w as dried in sunlight for 5 - days, alternating with soaking over night until th e sam ple becom e bleached and dried (Plate 1, 2, & 3).

3.3. Effect of Soaking:

T o study the effect of soaking, weighed 20 gm of dry and clean Gracilaria edulis and 400 ml of potable w ater was added (1:20 w/v) and kept them soaked fo r 11 hr / 12 hrs as required prior to boiling. Control sam ples were boiled directly. S im ilarly w eighed sam ples were soaked in dilute solution o f HCI / NaOH at co n c e n tra tio n s of 0.5N / 1 .ON fo r 12 hrs prior to extraction.

In e xperim ents with acid / alkali pretreatment, samples soaked in potable w ater fo r 11 hrs w ere transferred to 0.5N / 1 .ON acid /alkali for 1 hr and were m aintained at 80°c ± 2. Exactly after I h r the acid / alkali solution was decanted from th e beakers, w ashed well with fresh water and finally with distilled water to rem ove th e tra ce s of acid / alkali before boiling with distilled water.

fYo -L-

0 50 m co oo

' oo

■o

?05 C/)O)

. Cio

3-o

•Ao

Plate 1. W ashed seaweeds kept for Sun drying

3.4. Extraction of Agar:

For th e extraction of agar, method described by Matsuhashi (1971) was followed. A c c u ra te ly weighed 20 gm of dry G. edulis, chopped into small bits.

They were boiled separately in 1000 ml beakers after adding 400 ml distilled water and a d justing the p ^ to 6.3 - 6.5 in an autoclave at 1 kg /cm^ pressure for 2 hrs. The hot e x tra c t was recovered after filtration through muslin cloth and pressed in an e xp e lle r (Plate 4). T h e residue was re-extracted with 100 ml of hot distill water. T h e filtra te s w ere poured into plastic trays and left to becom e gel at room tem perature. T h e gel was streaked with iron scalpel. These gels were then frozen at -10°c fo r one day and later thawed. The thaw ed materials were placed on net screens ke p t slanting for sun drying for 3 days.

In th e experim ents o f agar extraction from alkali / acid presoaked samples in a cid ic / alkaline m edium , instead of distilled water. The solutions of 0.5N, 1 .ON, 2.ON or 3 .ON concentration of HCI / NaOH (400 ml) was added. After the extraction th e p ^ o f filtrate was adjusted to 6.3 - 6.5 with addrtion of acetic acid / NaOH.

3.5. Determination of yield:

A g a r sam ples after com plete drying w ere weighed accurately to calculate p e rce n ta g e yield.

Yield % = _______ wt. of agar_______X 100 W t. o f dry seaweed used

T h e dry m aterials w ere stored in polythene bags (Plate 5) for further physical and c h e m ic a l analysis.

3.6. Determination of Gel strength:

A m o n g physical properties of agar Gel strength and melting point were determ ined. T o determ ine th e gel strength and melting point 1.5% w/v agar solution w as used. T h e solution w as heated in a w ater bath. After complete

' ’3 ^ i s ' • ~A -

V ■ 5r^'-irv^*i*««-'..i/.c--- ■»s£?‘:? ^ ^ .it '

Plate 3. Completely bleached and dried seaweeds

dissolving of agar in w ater then th e solution was allowed to form gel at room temperature fo r 12 hrs. G e lo m e ter w as used to determ ine the gel strength (Funaki and Kojim a, 1951} o f agar. G elom eter (Plate 6) w as placed gently on the surface of the gel so th a t th e cylindrical plunger {1cm^ cross section) at bottom touched perfectly on gel. W e igh ts w ere added gradually on the pan of gelometer until it broke g e n tly th e gel in 20 seconds. The w eight was noted and taken as gel strength and e xp re sse d in gm/cm^.

3.7. Determination of merting temperature:

For th e d e term ination o f melting tem perature, method suggested by Whyte et al., f l9 8 1 ) w as follow ed. T h e agar solution 1.5% (w/v) was heated in water bath. T h e solution was stirred using glass rod while boiling. After dissolution re m ove d the solution from w ate r bath and allowed to cool at room temperature. A fte r th e gei form ation, the gel w as again kept in water bath, Therm om eter bu lb w a s placed in th e center of the gel and then the temperature was raised up to 50®C, Spherical glass beads {8 mm (j)) w ere added one by one for every 1"’C raise in tem perature. T h e tem perature w as noted when the glass beads started sinking. T h a t te m p e ra tu re was taken as melting tem perature of the agar.

3.8. Determination of Sulfate content:

S u lfa te c o n te nt in a g a r sam ple was determ ined according to the following m ethod (Ji M inghou, 1990),

Reagents:

A: S a tu ra te d Mg (N 03)2 in HNO3 B: G lycerol: Ethanol (1:2)

C: S olution o f NaCI - HGl [NaCI (67gm) + HCI {8ml) + H2O (200 ml)]

Plate 5. Dried Agar sheets of various chemical manipulation

W e ig h e d accurately 100 mg of dry agar sannple using an electronic balance (Sartorius). In a crucible th e sam ple was taken. To this 2 ml of reagent A was added to th e sa m ple and it was allowed to evaporate in the hood. After complete e va po ra tio n of fu m e s th e s e crucibles were placed in the muffle furnace at 400 C for 5 hrs. A fte r 5 hrs, sam ples removed from muffle furnace were cooled at 80 C, A fte r cooling of sam ples I.ON HCI was added to the sample. The solution was filte red through filter p a pe r fS/o.1 (W hatm an).

T h e filtra te w as collected in 50 ml volum etric flask. The filtrate was made up to 50 m l and transferred to 150 ml beaker. 5 ml of reagent ‘C and 10 ml of reagent ‘B' w ere added to the filtrate. This mixture was stirred for 1 minute using a m e cha n ical stirrer. Then to this solution 0.2 gm of BaCi2 was added and stirred for 1 m inute.

A fte r se ttle m e nt of solution the solution w as again stirred for 15 seconds. O ptical d e n sity was ta ke n at 425 nm, using a spectrophotom eter (GBC, UV/ VIS/911 A), A stan dard su lfa te solution was prepared by dissolving 0.1479 gm of Anh. N a 2S 04 in one litre distilled water. From this standard sulfate solution, aliq u ots in th e range o f 0,5 - 7 mg/lit w ere prepared and the sulfate contents w ere d e te rm in e d as above,

A b lank w as prepared with 10 ml of reagent B as above, A standard graph w a s plotted and a m ou n t of sulfate content in the agar samples were calculated fro m graph,

3.9. Statistical analysis:

All th e extractions, physical and chem ical analysis were performed at least 5 tim es. D ata w ere processed for statistical analysis by the one-way analysis of va ria n c e (A N O V A ) and significant difference calculated using the SPSS/PC and M S E X C E L software.

RESULTS

I. EFFECTS O F P R E -S O A K IN G ON Y IE L D AND Q UALITY OF AGAR:

A g a r yield from G racila ria edulis w as varying between 10.10 — II.6 1 % at d iffe re n t penods, of p re-soaking in potable w ater (Table-1). A gar yield increased slightly (1 1 .44% ) w hen p re-soaking period w as reduced to 2 hrs. and agar yield re du ce d ( 1 0 . 10%) w hen pre-soa king period w as prolonged to 12 hrs.

Although agar yield in control (non so a ked ) tre a tm e n t showed 11.61%, the gel strength obtained w a s low {54gm /sq.cm ).

For d iffe re n t presoaking periods gel strength varied between 54- 90gm/sq.cnn, G el strength of agar w as the highest (90 gm/sq.cm) when presoaked in p o ta b le w a te r for 1 2 hrs. (Table -I).

A g a r yield and gel strength w ere significant at different pre-soaking periods (P <0.05). A N O V A results sh o w e d that 2 hrs presoaking in potable water was preferable fo r a g a r yield, and 12 hrs tre a tm e n t preferable for better gel strength. A g a r yield and gel s tre n g th w ere negatively correlated (r= -0.8898), Sulfate content in a g a r ranged from 3.534 - 4,944% (Table-1). Sulfate was minimum (3,534°o) in 1 2 hrs. p reso a king period (Table-1),

M e ltin g te m p e ra tu re s of ag ar w ere in the range of 66-77°C for different p re -so a kin g periods. M elting point was m axim um (77°c) at 12hrs period of presoaking (Table-1). C orrelation betw een sulfate content and melting point of agar obtained fro m v a rio u s pre-so a king tre a tm e n t w ere negatively (r= -0,1828) correlated (T able-l). M elting point and sulfate content were significant (P<0.05) at different p re s o a k in g periods. A N O V A showing 12 hrs treatm ent was the best for sulfate reduction and a g a r with high melting point (P <0,05),

II. EFFECTS O F P R E S O A K IN G IN D IFF E R E N T CONCEN TR ATIO NS OF HCI ON YIELD A N D Q U A L IT Y O F AGAR:

W h e n G. eduhs w as presoaked in 0,5 N HCI the yield from G. edutis registered m a x im u m (15.844% ). W h e n concentration of HCI was increased for presoaking, yield of a g ar also increased. A t different concentrations of HCI pre­

soaking agar yield va rie d in th e range of 4.3761 -15.844 % and the gel strength of agar was in th e ra ng e of 21-60 gm /sq.cm (Table-ll). A t 0.5N HCI gel strength was 22 gm /sq.cm and sulfate c o n te n t ranged from 4.182-4.71% . Sulfate content was m inim um (4.1 8% ) in 1 .ON HCI presoaked sam ple (Table-il),

III. EFFECT O F P R E S O A K IN G IN D IF F E R E N T C O N C E N TR A T IO N S OF NaOH SOLUTION O N T H E A G A R Y IE L D A N D QUALfTY:

P re s o a k in g of dry G.eduHs in different concentrations of NaOH solution registered h ig h e r agar yield in 0.5N NaOH (12.4%) than in l.ON NaOH

( T a b l e 111)

G el s tre n g th of agar ranged from 61-135 gm/sq.cm. Gel strength was m axim um in l.O N N aO H (135 gm /sq.cm ). A gar yield and gel strength were negatively c o rrela te d (r= -0.971). A N O V A results show ed that yield and gel strength w ere s ig n ific a n t {p<0.05) in different concentrations of NaOH presoaking. S u lfa te c o n te n t in agar was in the range of 3.6442-4.796 %, rTiinimum being 3 .6 4 4 % in l.O N N aO H presoaked sam ples.

A lk a li p re s o a k in g o f G. edulis had im proved melting point (82 C) of agar (Table-ll!), S u ifa te c o n te n t w a s show ing sign ifica nt (p<0.05) reduction with increasing c o n c e n tra tio n s o f N aO H presoaking. A N V O A results indicated that 1 ON NaOH p re s o a k in g w as ideal fo r sulfate reduction. Sulfate content and melting point w e re n e g a tiv e ly co rrela te d (r= -0.09)

IV. EFFECTS O F A D D IT IO N O F NaO H DURING TH E EXTRACTION OF AGAR FROM ALKALI P R E -S O A K E D (0.5N ) NaOH ON AGAR YIELD & QUALITY

A d d itio n o f d iffe re nt concentrations of viz. 0.5N, 1.0N, 2N NaOH or 3.ON NaOH during boiJing to alkali presoaked (0.5N NaOH). G. edu/is yielded agar in the range o f 1 2.46 - 44.41 % (Table IV). A gar yield attained higher levels (44.41%) in 3 .ON N aO H addition during extraction. Agar yield was highly significant (p<0-05) with addition o f different concentration of NaOH as mentioned above. A N O V A results revealed th a t addition of 3 .ON NaOH during extraction of agar could incre a se the yield significantly.

Gel strength of a g a r increased when 0.5N NaOH was added during extraction (44 g m /sq .cm ). Gel s tre n g th ranged from 19 gm/sq.cm in 3.ON NaOH treatment to 126 g m /sq .cm w hen N aO H was not added (Control) indicating addition of N aO H beyond 0.5N during extraction as unfavorable (Table-IV).

Sulfate content in agar w as in ra n g e of 2.28-3.756 % (Table-IV). Sulfate content remained m in im u m (2,28% ) in 3 .ON N aO H addition during the extraction of agar.

M elting point ranged from 51>65°C (Table-IV) registering the maximum (59'’C) in 0.5N N aO H addition during extraction. A N O V A showed that addition of 3 .ON N aO H during extraction was the ideal fo r sulfate reduction and addition of 0 5N N aO H dunng extraction favorable fo r gel strength enhancement (Table IV). A g a r yield and gel s tre n g th w ere negatively correlated (r= -0.81439).

V. EFFECTS O F A D D IT IO N O F N aO H D U RING T H E EXTRACTION OF AGAR FROM ALKALI P R E -S O A K E D (1 .ON NaO H) ON AGAR YIELD AND QUALITY:

A g a r yield re gistered m axim um (49.26%) in 3.ON NaOH addition during extraction (TabJe-V). A g a r yield was in th e range from 18.4 4-4 9 .2 6 ^.

Agar yiefd w as sig n ific a n t (p<0.05) at different concentrations of NaOH addition during the extraction of agar. A N O V A also indicated th a t 3.0N NaOH treatment was most su ita ble fo r b e tte r a g ar yield. A s shown in T a b le V, gel strength of agar was varying in th e ra nge of 18-1 28 gm /sq.cm . Gel strength w as maximum in the

control (128 g m /sq .cm ) treatm ent, which did not receive additional NaOH (Table- V) Get strength and yield show ed negative correlation (r= -0.77128). Sulfate content was minimunn (1.578% ) in 3.0N NaOH addition during the extraction of agar. Sulfate c o n te n t varied in the range from 1,578 - 4.202 %, the lowest being in the 3.ON N aO H addition during extraction. Melting point was minimum ( S r c ) in agar extracted fro m 3 .ON N aO H added G. edulis during extraction and maximum (85°C) in th e control (Tab!e-V).

VI. E FF E C T O F A D D IT IO N O F N aO H DURING T H E EXTRACTION OF AGAR FROM TH E A C ID P R E -S O A K E D (0.5N HCI) ON AGAR YIELD AND QUALITY:

A d d itio n o f varied concentrations of N aO H during extraction o f agar from acid (0.5N HCI) preso a ke d G. eduJ/s show ed yield of agar could not be improved (yield ra ngin g from 6 .49-35.94% ) by adding NaOH during extraction (Table VI). A d d itio n o f 3.0 N N aO H extraction showed that all the agar samples were hydrolyzed. Yield could not be determ ined. Gel strength in 0.5N NaOH addition sh o w e d 22 gm sq.cm b u t lower than the control (Table-VI). Sulfate content w as m in im u m (4.24%) in 2 .ON NaOH addition during extraction. In 3.ON NaOH addition all th e sa m ple s w ere hydrolyzed. Similar trend was obtained when v a rio u s c o n cen tra tio n s of NaO H w as added to 1.0N HCI presoaked G.

eduirs during a g a r extraction. H ydrolysis of agar occurred during extraction (Tab(e-V(().

VIII. E F F E C T O F ALKALI P R E -T R E A T M E N T W IT H RAISING TEMPERATURE TO 8 0 “C F O R 1 hr O N TH E AGAR YIELD AND QUALITY:

A t d iffe re n t concentration of NaOH pre-treatm ent and increased tem perature (80°C ), th e yield of agar ranged from 10.152 - 14.156%. Agar yield was m a xim u m (14.1 56 % ) in 2.ON N aO H and m inim um yield (10.152%) in 3.ON NaOH (Table-V I II). A g a r yield show ed significance (p<0.05) at different concentrations o f N aO H p retreatm en t at 80*^0. A N O V A results showed that 2.0N NaOH p re -tre a tm e n t at 80°C w as ideal fo r agar yield. Gel strength varied from 69-291 g m /sq .cm . 3 .ON N aO H pre-treatm ent at 8 0 "C increased gel strength to

291 gm /sq,cm (Talbe VIII) and w as m inim um (168 gm/sq.cm) in 0.5N NaOH pretreatment, w hich is higher than th e gel strength achieved by control {69 gm/sq.cm). G el strength show ed significance at different (p<0.05) treatments.

Agar yield and gel strength w ere negatively correlated (r= -0.03435). Sulfate content varied fro m 0,732 % - 5.548% with the least values (0.732%) in 3.ON NaOH p re-trea tm en t at 80°c.

M elting point at d iffe re n t concentrations of NaOH pre-treatment at 80°C te m p e rature ranged from 6 8 -9 9 °C (Table-Vlil) with the maximum in 3.ON NaOH p retre atm en t (99°C).

A N O V A results s h o w e d th a t 2.ON NaOH pretreament at 8 0 °0 was highly preferable fo r quality (Gel strength) & yield im provem ent (p<0.05; Od=- 0,057 agar yield); cd ^ 3,305 (gel strength); cd 0.048 (sulfate %); cd = 2.075 (melting point '’C)

IX. E FFECT O F A C ID P R E -T R E A M E N T AT HIGH TE M P E R A TU R E 80°C FOR 1 hr. ON YIE LD A N D Q U A LIT Y O F AGAR:

A t d iffe re nt co n c e n tra tio n s of HCI pretreatm ent 0.5N, 1.0N, 2,ON and 3 ON at QO°C te m p e ra tu re resulted hydrolysis of agar except in control agar could not be extra cte d (Table IX).

TREATMENT

Control

YIELD (%)

GEL STRENGTH

gm/sq.cm 1.5% agar

(w/v)

SULFATE CONTENT

{%)

MELTING POINT

(°C)

1 1,612 ± 0.25 54,0 ± 3.36 4,944 ± 0.05 66 ± 4.14 2 hrs. 1 1.44 ± 0.521 61.0 ± 7.36 4.86 ± 0.06 70 ± 4.27 4 hrs. 10.492 ± 0.32 65,0 ± 3.80 4 .1 2 8 ± 0 .0 6 75 ± 8 .5 0 8 hrs. 10-274 ± 0.40 77.0 ± 5.26 3.846 ± 0.05 75 ± 6.80 j 12 hrs. 10,10 ± 0,26 90.0 ± 8.06 3.534 ± 0.08 7 7 ± 1.14

TREATMENT I

i

YIELD

' (%)

GEL S TR ENG TH

gm/sq.cm 1.5% agar

(w/v)

SULFATE C O N TENT

(%)

MELTING POINT

r c ) Soaked in

Potable W ater 12.364 ± 0 .1 3 60,0 ± 4.4 4.71 ± 0.06 70 0.5 N HCI 15.844 ± 0.37 22.0 ± 1.6 4.602 ± 0.12 NIL 1,0 N HCI 4,376 ± 0.22 21.0 ± 1.09 4.18 ± 0.04 NIL

TREATMENT 1

; ( °) i

GEL STRENGTH

gm/sq.cm 1.5% agar (w/v)

■ ■

SULFATE C ONTENT

{%)

MELTING POINT

r c ) Control

(Soaked in Potable Water)

12.5 ± 0.09 61 ± 2.58 4.796 ± 0,06 69 ± 1.92

Pre 0 5 N soaked !

12.4 ± 0.15 125 ± 2.07 3.759 ± 0.0 3 80 ± 2.4 0

in 1

NaOH 1,0 N 12.124 ± 0,04 135 ± 2.24 3.644 ± 0.02 82 ± 1.64

TREATMENT YIELD (%)

GEL STRENGTH

gm/sq.cm 1.5% agar

(w/v)

SULFATE CONTENT

{%)

MELTING POINT

r c )

Control 12.464 ± 0.02 126 ± 2.95 3.756 ± 0.02 65 ± 1.92 0.5 N 20.092 ± 0.14 44.0 ± 1.14 3.234 ± 0,03 59 ± 1.58 1,0 N 31.946 ± 0.19 40.0 ± 1.51 3.102 ± 0.03 57 ± 1.14 2.0 N 34.032 ±0.11 23.0 ± 1.64 2.808 ± 0.02 55 ± 1.30 3.0 N 44.410 ± 0.42 19.0 ± 1.58 2.28 ± 0.02 51 ± 1.48

TREATMENT YIL-r.D (%)

GCL STRHNGTH

gm/sq.cm 1.5% agar (w/v)

SULI'ATH CONTENT

(%)

MI-LTING POINT

(“ Q Control 18.446 ± 0,19 128 ± 1,78 4.202 ± 0.02 85 ± 1.30

0.5 N 28.324 ± 0.38 33.0 ± 1.14 3.90 ± 0.03 64 ± 1.58 1.0 N 34.052 ± 0,07 21.0 ± 0.89 3.85 ± 0.02 58 ± 1.14 2.0 N 36.272 ± 0.16 19.0 ± 1,14 1.72 ± 0 .2 2 53 ± 0.87 3.0 N 49.262 ± 0.13 18.0 ± 0.70 1.578 ± 0.02 51 ± 0.83

TREATMENT YIELD (%)

GEL STRENGTH

gm/sq.cm 1.5% agar

(w/v)

SULFATE CONTENT

(%)

MELTING POINT

r c )

Control 35.940 ± 0,38 32.0 ± 1.09 4.658 ± 0.02 58 ± 0.70 0.5 N 16.120 ± 0.02 22.0 ± 0.90 4.33 ± 0.03 53 ± 1.14

1.0N 9.798 ± 0 .0 3 18.0 ± 1.14 4.296 ± 0.01 NIL

2.0 N 6.492 ± 0.02 17.0 ± 1.14 4.24 ± 0,03 NIL

3.0 N NIL NIL NIL NIL

TREATMENT YIELD (%)

GEL STRENGTH

gm/sq.cm 1.5% agar

(w/v)

SULFATE CONTENT

{%)

MELTING POINT

(°C) Control

(0.0 N NaOH) 2 3 .6 ± 0 .1 1 4 29.0 ± 1.04 4.13 ± 0.12 57 ± 1.34

0.5 N 21.56 ± 0.15 22.0 ± 0.15 3.8 ± 0.07 NIL

1.0 N 7.654 ± 0.03 18.0 ± 0.03 3.42 ± 0.03 NIL

2.0 N NIL NIL NIL NIL

3.0 N NIL NIL NIL NIL

Table VIII - E FFECT OF ALKAU PRE-TREATMENT WITH RAISING TEM PER A TU RE 80°C FOR ONE HOUR BEFORE EXTRACTION ON TH E YIELD AND Q UAUTY OF AGAR (MEAN ± S.D.)

TREATMENT YIELD

(%)

GEL STRENGTH

gm/sq.cm 1.5% agar

(w/v)

SULFATE CONTENT

(%)

MELTING POINT

r c )

Control

(0.0 N NaOH) 11.772 ± 0 ,0 3 69.0 ± 1.80 5.548 ± 0.07 68 ± 2.7 0 0.5 N 13.143 ± 0.05 1 6 8 ± 1.51 4.208 ± 0.01 88 ± 2.72 1.0 N 13.226 ± 0,05 191 ± 1.92 3.556 ± 0.05 95 ± 0.83 2.0 N 14.156 ± 0,06 274 ± 2.73 0.844 ± 0.03 96 ± 1.3 0 3,0 N 10.156 ± 0.05 291 ± 5.45 0.732 ± 0.02 99 ± 1.00

TREATMENT YIELD (%)

GEL STRENGTH

gm/sq.cm 1.5% agar

(w/v)

SULFATE CONTENT

(%)

MELTING POINT

( X ) Control

(O.ON HCl) 12.166 ± 1.14 55.0 ± 1.38 4.9 ± 0 .0 2 7 2 ± 2 . 1 3

0.5N NIL NIL NIL NIL

1.0N NIL NIL NIL NIL

2.0N NIL NIL NIL NIL

3.0N NIL NIL NIL NIL

Table-X: AGAR YIELD SIGNIFICANCE AT ALKALI PRE TREATMENT AT 80 °C FOR 1 hr (2.0 N NaOH)

ANOVA Single -actor Source o;

Variation SS d f MS F P-value F crit

Between

Groups 50.90776 4 12.72694 4493.976 3.16E-29 2.866081 Within

Groups 0.05664 20 0.002832

Total 50.9644 24

Table- XI: G E L S T R E N G T H S IG N IF IC A N C E AT ALKALI PRE TREATM ENT AT 80 °C FO R 1 hr (2.0 N NaOH)

A N O V A Single -a cto r Source o1

Variation S S d f M S F P-value F crit

Between

Groups 161199 4 40299.76 4324.009 4.64E-29 2,866081

Within

Groups 186.4 20 9,32

Total 161385.4 24

Table- XII: SU LFA TE C O N T E N T SIG NIFICANCE A T ALKALI PRE TR E A T A T 8 0 FOR 1 hr (2.0 N NaOH)

ANOVA singiefactor Source o f

Variation SS d f MS F P-vafue F crit

Between

Groups 90.25178 4 22.56294 11258.95 3.27E-33 2.866081

Within

Groups 0.04008 20 0.002004

Total 90.29186 24

Table-Xlll: MELTING TEMPERATURE SIGNIFICANCE AT VARIOUS A NaOH PRETREATMENTAT 80"c FOR 1 hr (2.0 N NaOH) ANOVA Single Factor

Source of

Variation S S d f M S F P -v a lu e F crit

Between

Groups 2 9 6 7 .7 6 4 7 41 .94 201.611 7 .4 1 E -1 6 2,86601 Within

Groups 7 3 .6 20 3 ,6 8

Total 3 0 4 1 .3 6 24