Publications

[142]

1. Manoj Kumar, Puja Kumari, Vishal Gupta, P.A. Anisha, C.R.K. Reddy and

Bhavanath Jha (2010) Differential responses to cadmium induced oxidative stress

in marine macroalga Ulva lactuca (Ulvales, Chlorophyta). Biometals 23, 315-

325.

2. Manoj Kumar, Puja Kumari, Vishal Gupta, C.R.K. Reddy and Bhavanath Jha

(2010) Biochemical responses of red alga Gracilaria corticata (Gracilariales,

Rhodophyta) to salinity induced oxidative stress. Journal of Experimental Marine

Biology and Ecology 391, 27-34.

3. Manoj Kumar, Vishal Gupta, Nitin Trivedi, Puja Kumari, A.J. Bijo, C.R.K.

Reddy, and Bhavanath Jha (2011) Differential responses of intertidal red alga

Gracilaria corticata (Gracilariales, Rhodophyta) following desiccation exposure.

Environmental and Experimental Botany 72, 194-201.

4. Manoj Kumar, Vishal Gupta, Puja Kumari, C.R.K. Reddy and Bhavanath Jha

(2011) Assesment of nutrient composition and antioxidant potential of seaweeds

from Caulerpaceae. Journal of Food Composition and Analysis 24, 270-278.

5. Manoj Kumar, Puja Kumari, Nitin,Trivedi, Mahendra K. Shukla, Vishal Gupta,

C.R.K. Reddy and Bhavanath Jha (2011) Minerals, PUFAs and antioxidant

properties of some tropical seaweeds from Saurashtra coast of India. Journal of

Applied Phycology, DOI 10.1007/s10811-010-9578-7.

6. Manoj Kumar, A. J. Bijo, Ravi S. Baghel, C.R.K. Reddy and Bhavanath Jha

(2011) Selenium and Spermine as cadmium stress buster in the marine macroalga

Gracilaria dura (C. Agardh) J. Agardh. (Submitted).

7. Manoj Kumar, Nitin trivedi, C.R.K. Reddy and Bhavanath Jha (2011)

Imidazolium ionic liquid induced oxidative stress, DNA damage and its

abatement by Acadian marine plant extract in macrophytic marine green alga

Ulva lactuca. (Submitted).

8. Mahendra K. Shukla, Manoj Kumar, Kamlesh Prasad, C.R.K. Reddy and

Bhavanath Jha (2011) Partial characterization of sulfohydrolase from Gracilaria

dura and evaluation of its potential application in the improvement of the agar

quality. Carbohydrate Polymer 85, 157-163.

9. Vishal Gupta, Manoj Kumar, Puja Kumari, C.R.K. Reddy and Bhavanath Jha

(2010) Optimization of protoplast yields from the red algae Gracilaria dura (C.

Agardh) J. Agardh and G. verrucosa (Huds.) Papenfuss. Journal of Applied

Phycology, DOI 10.1007/s10811-010-9579-6.

10. Puja Kumari, Manoj Kumar, Vishal Gupta, C.R.K. Reddy and Bhavanath Jha

(2010) Tropical marine macroalgae as potential sources of nutritionally important

PUFAs. Food Chemistry 120, 749-757.

11. Reddy C.R.K., Manoj Kumar, Vaibhav A. Mantri and Bhavanath Jha (2008)

Seaweed protoplasts: status, biotechnological perspectives and needs. Journal of

Applied Phycology 20, 619-632.

Publications

[143]

12. Nitin Trivedi, Vishal Gupta, Manoj Kumar, Puja Kumari, C.R.K. Reddy and

Bhavanath Jha (2011) Solvent tolerant marine bacterium Bacillus aquimaris

secreting solvent stable alkaline cellulase. Chemosphere 83, 706-712.

13. Nitin Trivedi, Vishal Gupta, Manoj Kumar, PujaKumari, C.R.K. Reddy and

Bhavanath Jha (2011) An alkali-halotolerant cellulase from Bacillus flexus

isolated from green seaweed Ulva lactuca. Carbohydrate Polymers 83, 891-897.

14. Vishal Gupta, Ravi S. Baghel, Manoj Kumar, Puja Kumari, Vaibhav A. Mantri,

C.R.K. Reddy and Bhavanath Jha (2011) Growth and agarose characteristics of

isomorphic gametophyte (male and female) and sporophyte of Gracilaria dura

and their marker assisted selection. (Aquaculture- accepted)

15. Vaibhav A. Mantri, Mukund C. Thakur, Manoj Kumar, C.R.K. Reddy and

Bhavanath Jha (2009) The carpospore culture of industrially important red alga

Gracilaria dura (Gracilariales, Rhodophyta). Aquaculture 297, 85-90.

Participation in Conferences/Symposium/Training

[144]

CONFERENCES/SYMPOSIUM ATTENDED

1. Manoj Kumar, Puja Kumari, Anisha P. A., C.R.K. Reddy and Bhavanath Jha.

(2009) Cadmium induced oxidative stress biomarkers in Ulva lactuca (Ulvales,

Chlorophyta). Presented in 7th

Asia Pacific Conference on Algal biotechnology,

1st- 4

th December, Delhi University, New Delhi, India.

2. Manoj Kumar, Puja Kumari, Vishal Gupta, C.R.K. Reddy and Bhavanath Jha

(2008) Salinity induced changes in antioxidant enzymes and biochemical

components in the red alga Gracilaria corticata. Presented in Vth

Asian Pacific

Phycology Forum on "Algae in a changing world", held on 10th

-14th November,

Wellington, New Zealand.

3. Manoj Kumar, Vishal Gupta, C.R.K. Reddy, Vaibhav A. Mantri and Bhavanath

Jha (2008) Biochemical responses of Gracilaria corticata to salinity stress,

Gujarat science congress conference, held on 9th

March, Bhavnagar University,

Bhavnagar, Gujarat, India.

TRAINING PROGRAMME

1. Participated in a 10 days training programme for COMET assay for studying the

DNA damage in cell at IITR (Indian Institute of Toxicology Research), Luknow,

India. 2nd

-13th

August, 2010.

Journal of Experimental Marine Biology and Ecology 391 (2010) 27–34

Contents lists available at ScienceDirect

Journal of Experimental Marine Biology and Ecology

j ourna l homepage: www.e lsev ie r.com/ locate / jembe

Biochemical responses of red alga Gracilaria corticata (Gracilariales, Rhodophyta) tosalinity induced oxidative stress

Manoj Kumar, Puja Kumari, Vishal Gupta, C.R.K. Reddy ⁎, Bhavanath JhaDiscipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scientific and Industrial Research (CSIR), Bhavnagar 364021, India

⁎ Corresponding author. Tel.: +91 278 256 5801/2566970 / 256 7562.

E-mail address: crk@csmcri.org (C.R.K. Reddy).

0022-0981/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.jembe.2010.06.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 26 February 2010Received in revised form 31 May 2010Accepted 1 June 2010

Keywords:Antioxidant enzymesGracilaria corticataMineralsOxidative stressPhycobiliproteinsPUFAsSalinity stress

The biochemical responses of Gracilaria corticata (J. Agardh) J. Agardh to salinity induced oxidative stress werestudied following the exposure to different salinities ranging from 15, 25, 35 (control), 45 to 55 in laboratoryconditions. The growth was highest under 25 (3.14±0.69% DGR) and 35 (3.58±0.32% DGR) and decreasedsignificantly in both extreme lower (15) and hyper (55) salinities. Both phycoerythrin (PE) andallophycocyanin (APC) were significantly higher in hyper-salinity (45) with an increase of almost 70% and52% from their initial contents. Conversely, the level of increase of the same in hypo-salinitieswas considerablylower as compared with that of hyper-salinity. Both hypo- and hyper-salinity treatments induced almost twofold increase in the contents of polyphenols, proline and the activities of antioxidative enzymes such assuperoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR) especially for 6 dexposure. The Na+ ions readily displaced the K+ and Ca2+ from their uptake sites at extreme hyper-salinity(55) and accounted for substantial increase in the ratio of Na+/K+ and Na+/Ca2+ that impeded thegrowth under long term exposure (N6 d). The survivability at salinity 45 evenwith considerably higher ratio ofNa+/K+ and Na+/Ca2+ suggests the compartmentalization of Na+ into the vacuoles. Further, the micronutrients such as Zn, Fe and Mn were decreased at both high and low end salinities with highest at extremehyper-salinity. The C18:1(n−9) cis, C18:2(n−6), C18:3(n−3) and C20:4(n−6) were found in significantamounts in hyper-salinities. The C18:1(n−9) cis in particular increased by 60.25% and 70.51% for salinities 45and 55, respectively from their initial amounts. The ratio of total unsaturated to saturated fatty acids (UFA/SFA)also increased linearly with increasing salinity. These results collectively suggest the potential role ofantioxidative enzymes, phycobiliproteins, PUFAs and mineral nutrients to combat the salinity inducedoxidative stress in G. corticata.

3805x614; fax: +91 278 256

l rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

The red alga Gracilaria corticata (J. Agardh) J. Agardh is one of thecommon algae of the Indian coast and occurs predominantly in thelower littoral zone. It also inhabits occasionally in the intertidal rockpools as submerged population. The intertidal algae often get exposedto the atmosphere periodically during low tide regimes and experi-ence an oxidative stress on regular basis with the turning tides. Inmarine waters, salinity around 35 is the most common, but it couldalso vary from 10 to 70 as a result of evaporation or precipitation/freshwater influxes (Graham and Wilcox, 2000). Osmotic stress mostoften resulting from fluctuating salinities exerts considerable oxida-tive stress on seaweeds in the intertidal zone. The previous studieshave investigated the responses of estuarine macroalgae for eitherindividual or combined abiotic factors (light, pH, temperature,

nutrient load and salinity) in the context of growth andphotosyntheticperformance (Macler, 1988; Dawes et al., 1999; Israel et al., 1999; Choiet al., 2006; Phoopronget al., 2007). Subsequent studies have also dealtwith the possible effects of environmental stresses on floristicvariations of intertidal benthic macro algal communities (Helmuthet al., 2005).

It has been suggested that instant responses of marine plants toadverse environmental conditions involve excess production ofreactive oxygen species (ROS) such as hydrogen peroxide (H2O2),singlet oxygen (1O2), superoxide radical (O2

•−) and hydroxyl radical(OH−) (Dring, 2006). Increased physiological stress conditions lead tothe rapid formation of ROS that reacts with most cellular componentsand thus they need to be neutralized instantly once formed.Acclimation to altered osmotic conditions particularly to salinityinduced stress involves changes in physiological processes includingantioxidant enzymes [superoxide dismutase (SOD), catalase (CAT),ascorbate peroxidase (APX) and glutathione reductase (GR)] and non-enzymatic antioxidants (ascorbate, glutathione and carotenoids).All these processes function in coordinated manner in order toalleviate the cellular hypo/hyper osmolarity, ion disequilibrium and

Author's personal copy

Environmental and Experimental Botany 72 (2011) 194–201

Contents lists available at ScienceDirect

Environmental and Experimental Botany

journa l homepage: www.e lsev ier .com/ locate /envexpbot

Desiccation induced oxidative stress and its biochemical responses in intertidalred alga Gracilaria corticata (Gracilariales, Rhodophyta)

Manoj Kumar, Vishal Gupta, Nitin Trivedi, Puja Kumari, A.J. Bijo, C.R.K. Reddy ∗, Bhavanath JhaDiscipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scientific and Industrial Research (CSIR), Bhavnagar 364021, India

a r t i c l e i n f o

Article history:Received 9 July 2010Received in revised form 4 March 2011Accepted 11 March 2011

Keywords:Antioxidative enzymesDesiccationGracilaria corticataPolyaminesPUFAsReactive oxygen species

a b s t r a c t

Intertidal alga Gracilaria corticata growing in natural environment experiences various abiotic stressesduring the low tides. The aim of this study was to determine whether desiccation exposure would leadto oxidative stress and its effect varies with exposure periods. This study gives an account of variousbiochemical changes in G. corticata following the exposure to desiccation for a period of 0 (control), 1,2, 3 and 4 h under controlled conditions. During desiccation, G. corticata thalli showed dramatic lossof water by almost 47% when desiccated for 4 h. The enhanced production of reactive oxygen species(ROS) and increased lipid peroxidation observed during the exposure of 3–4 h were chiefly contributedby higher lipoxygenase (LOX) activity with the induction of two new LOX isoforms (LOX-2, ∼85 kDa;LOX-3, ∼65 kDa). The chlorophyll, carotenoids and phycobiliproteins (phycoerythrin and phycocyanin)were increased during initial 2 h exposure compared to control and thereafter declined in the succeedingexposure. The antioxidative enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX),glutathione reductase (GR), glutathione peroxidase (GPX) and the regeneration rate of reduced ascorbate(AsA) and glutathione (GSH) increased during desiccation up to 2–3 h. Further, the isoforms of antiox-idant enzymes Mn-SOD (∼150 kDa), APX-4 (∼110 kDa), APX-5 (∼45 kDa), GPX-1 (∼80 kDa) and GPX-2(∼65 kDa) responded specifically to the desiccation exposure. Compared to control, a relative higher con-tent of both free and bound insoluble putrescine and spermine together with enhanced n-6 PUFAs namelyC20:4(n-6) and C20:3(n-6) fatty acids found during 2 h exposure reveals their involvement in defencereactions against the desiccation induced oxidative stress.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

The red alga Gracilaria corticata (J. Agardh) J. Agardh occursextensively in intertidal zone of the Indian coast and regularlyexperiences the desiccation during low tide periods. The organ-isms living in the intertidal zone of tropical shores are subjectedto various types of abiotic stresses due to periodic exposure toa wide range of fluctuating environmental factors such as desic-cation, salinity, radiation, temperature and pollutants (Apel andHirt, 2004; Liu and Pang, 2010; Kumar et al., 2010a). The environ-mental exposure during low tide condition, demands the intertidalmacroalgae to prepare early for the desiccation followed by rehy-dration and associated cellular damage (Burritt et al., 2002). Thisconstant state of readiness requires a great deal of energy budgetand could be a contributing factor to the slow growth rates of algaedwelling at the upper littoral zone as compared to those at lowerlittoral zone (Stengal and Dring, 1997). The possible explanation

∗ Corresponding author. Tel.: +91 278 256 5801/3805x614;fax: +91 278 256 6970/7562.

E-mail address: crk@csmcri.org (C.R.K. Reddy).

for the success of an alga exposed to drought could be either beingphysiologically more tolerant or better at resisting the water loss(Ji and Tanaka, 2002).

During desiccation many of the intertidal seaweeds experienceextreme drying rates, reaching air dryness within hours (Schonbeckand Norton, 1979; Nelson et al., 2010), generally depends on the cli-matic conditions as well as the evaporating surface-to-volume ratioof the thallus (Lobban et al., 1985). Also, desiccation causes cellu-lar dehydration, which increases the concentration of electrolytewithin the cell, causing changes to membrane-bound structuresincluding the thylakoid (Kim and Garbary, 2007). It has been sug-gested that instant responses of marine plants to adverse milieuinvolve excess production of reactive oxygen species (ROS) suchas hydrogen peroxide (H2O2), singlet oxygen (1O2), superoxide(O2

•−) and hydroxyl radical (OH−) (Burritt et al., 2002). The abil-ity to withstand the oxidative assault imposed by ROS dependson the enzymatic and non enzymatic oxidants of the cell. Thisantioxidant system functions in a coordinated manner to alle-viate the cellular hypo/hyper osmolarity, ion disequilibrium anddetoxification of ROS which otherwise cause oxidative destruc-tion to cell (Wu and Lee, 2008; Liu and Pang, 2010; Kumar et al.,2010a,b).

0098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.envexpbot.2011.03.007

Differential responses to cadmium induced oxidative stressin marine macroalga Ulva lactuca (Ulvales, Chlorophyta)

Manoj Kumar • Puja Kumari • Vishal Gupta •

P. A. Anisha • C. R. K. Reddy • Bhavanath Jha

Received: 7 January 2010 / Accepted: 12 January 2010 / Published online: 30 January 2010

� Springer Science+Business Media, LLC. 2010

Abstract This study describes various biochemical

processes involved in the mitigation of cadmium

toxicity in green alga Ulva lactuca. The plants when

exposed to 0.4 mM CdCl2 for 4 days showed twofold

increase in lipoperoxides and H2O2 content that

collectively decreased the growth and photosynthetic

pigments by almost 30% over the control. The

activities of antioxidant enzymes such as superoxide

dismutase (SOD), ascorbate peroxidase (APX), glu-

tathione reductase (GR) and glutathione peroxidase

(GPX) enhanced by twofold to threefold and that of

catalase (CAT) diminished. Further, the isoforms of

these enzymes, namely, Mn-SOD (*85 kDa), GR

(*180 kDa) and GPX (*50 kDa) responded specif-

ically to Cd2? exposure. Moreover, the contents of

reduced glutathione (3.01 fold) and ascorbate

(1.85 fold) also increased substantially. Lipoxyge-

nase (LOX) activity increased by two fold coupled

with the induction of two new isoforms upon Cd2?

exposure. Among the polyunsaturated fatty acids,

although n - 3 PUFAs and n - 6 PUFAs (18:3n - 6

and C18:2n - 6) showed relatively higher contents

than control, the latter ones showed threefold increase

indicating their prominence in controlling the cad-

mium stress. Both free and bound soluble putrescine

increased noticeably without any change in spermi-

dine. In contrast, spermine content reduced to half over

control. Among the macronutrients analysed in

exposed thalli, the decreased K content was accom-

panied by higher Na and Mn with no appreciable

change in Ca, Mg, Fe and Zn. Induction of antioxidant

enzymes and LOX isoforms together with storage of

putrescine and n - 6 PUFAs in cadmium exposed

thallus in the present study reveal their potential role in

Cd2? induced oxidative stress in U. lactuca.

Keywords Antioxidant enzymes �Cadmium � LOX � Minerals � Oxidative stress �PUFAs � Ulva lactuca

Introduction

Of the toxic substances contaminating the aquatic

environment, heavy metals particularly cadmium,

lead and mercury are of great concern for humans as

well as for the environment because of their acute

toxicity and high mobility in food chain (Sokolova

et al. 2005). Cadmium (Cd2?), with no reported

biological function except one occasion as a cofactor

for carbonic anhydrase in marine diatom (Lane and

M. Kumar � P. Kumari � V. Gupta �C. R. K. Reddy (&) � B. Jha

Discipline of Marine Biotechnology and Ecology, Central

Salt and Marine Chemicals Research Institute, Council

of Scientific and Industrial Research (CSIR), Bhavnagar

364021, India

e-mail: crk@csmcri.org

P. A. Anisha

School of Environmental Studies, Cochin University

of Science and Technology, Cochin, India

123

Biometals (2010) 23:315–325

DOI 10.1007/s10534-010-9290-8

Author's personal copy

Original Article

Assessment of nutrient composition and antioxidant potential of Caulerpaceaeseaweeds

Manoj Kumar, Vishal Gupta, Puja Kumari, C.R.K. Reddy *, B. Jha

Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scientific and Industrial Research (CSIR), Bhavnagar 364021, India

1. Introduction

An increasing human population, global climate change and thediversification of terrestrial food resources for energy needs in recenttimes have raised serious global food security concerns (Rosegrantand Cline, 2003). Further, the globalization of markets has alsobrought about an increasing globalization of foods, diminishing theboundaries of human races and geographical regions of the countriesthroughout the world. There has also been a quest to explore andutilize foods from non-conventional sources, of both terrestrial andmarine origin, to enhance and supplement the nutritional quality ofhuman foods. Also, this in turn eases off the growing burden ontraditional foods. Marine macroalgae, commonly known as sea-weeds, are one of the living renewable resources of the oceans withpotential food applications. Consumption of seaweeds as seavegetables in human diets has been the common practice in severalAsian countries (Nisizawa, 2002). Presently, interest in supplement-ing the human foods with antioxidants particularly from naturalsources has been on the raise as synthetic antioxidants have beensuspected to be a possible cause for liver damage and carcinogenesis

(Farag et al., 2003; Tang et al., 2001). Therefore, there is a need forisolationandcharacterizationofantioxidantshavingleastsideeffectsfrom natural sources as an alternative to synthetic antioxidants.

The previous studies have demonstrated the potential ofenzymatic superoxide dismutase (SOD), catalase (CAT), ascorbateperoxidase (APX) and glutathione reductase (GR) and non-enzymatic (polyphenols, glutathione, ascorbic acid and carotenoids)antioxidants scavenging the reactive oxygen species (ROS) thusrelieving from the oxidative stresses and other associated healthrisks such as cancer, coronary heart diseases, neurodegenerativediseases and inflammation (Duan et al., 2006; Kuda and Ikemori,2009; Nagai and Yukimoto, 2003). The recent findings have alsorevealed availability of useful metabolites with medicinal propertiesfrom some marine biota (Blunt et al., 2005; Mayer et al., 2009).

Recently, the genus Caulerpa has attracted the attention ofresearchers due to its important secondary metabolite caulerpe-nyne (CYN) that is reported to exhibit the antineoplastic,antibacterial and antiproliferative activities (Barbier et al., 2001;Cavas et al., 2006). Further, it has also been shown to inhibit the celldivision of sea urchin eggs as well as cancer cell lines (Fischel et al.,1995; Lemee et al., 1993). Three species of Caulerpa namely C.racemosa, C. scalpelliformis and C. veravelensis have been foundgrowing luxuriantly in the intertidal region during October–February along the Veraval coast of Gujarat (north-western coast ofIndia). Among these three species, C. racemosa with a wide

Journal of Food Composition and Analysis 24 (2011) 270–278

A R T I C L E I N F O

Article history:

Received 17 July 2009

Received in revised form 5 July 2010

Accepted 31 July 2010

Available online 8 December 2010

Keywords:

Antioxidants

Biochemical constituents

Caulerpa

Minerals

Nutritional supplement

Pigments

Polyunsaturated fatty acids

Seafood

Food analysis

Food composition

A B S T R A C T

The proximate nutrient composition, mineral contents, enzymatic and non-enzymatic antioxidant

potential of three Caulerpa species were investigated. All three species were high in ash (24.20–33.70%)

and carbohydrate content (37.23–48.95%) on dry weight basis (DW). The lipid content ranged between

2.64 and 3.06% DW. The mineral contents varied marginally among the species but were in the order of

Na > K > Ca > Mg. The Na/K ratio among the species varied from 1.80 to 2.55 and was lowest in C.

scalpelliformis. A 10 g DW of Caulerpa powder contains 11–21% Fe, 52–60% Ca and 35–43% Mg, which is

higher than the recommended daily allowance (RDA), compared with non-seafood. The percentage sum

of PUFAs (C18:2, C18:3, C20:4 and C20:5) in total fatty acids was highest in both C. scalpelliformis

(39.25%) and C. veravelensis (36.73%) while it was the lowest in C. racemosa (24.50%). The n�6/n�3 ratio

among the species varied from 1.44 to 7.72 and remained within the prescribed WHO standards (<10).

Further, the higher enzymatic dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and

glutathione reductase (GR) and non-enzymatic antioxidant potential of Caulerpa species found in the

present study confirm their usefulness in terms of nutrients and antioxidants.

� 2010 Elsevier Inc. All rights reserved.

* Corresponding author. Tel.: +91 278 256 5801/256 3805x614;

fax: +91 278 256 6970/256 7562.

E-mail address: crk@csmcri.org (C.R.K. Reddy).

Contents lists available at ScienceDirect

Journal of Food Composition and Analysis

journa l homepage: www.e lsev ier .com/ locate / j fca

0889-1575/$ – see front matter � 2010 Elsevier Inc. All rights reserved.

doi:10.1016/j.jfca.2010.07.007

Minerals, PUFAs and antioxidant properties of sometropical seaweeds from Saurashtra coast of India

Manoj Kumar & Puja Kumari & Nitin Trivedi &Mahendra K. Shukla & Vishal Gupta & C. R. K. Reddy &

Bhavanath Jha

Received: 16 April 2010 /Revised and accepted: 17 August 2010# Springer Science+Business Media B.V. 2010

Abstract Twenty-two tropical seaweeds from the Rhodo-phyta, Phaeophyta and Chlorophyta were examined fortheir possible use as nutritional supplements. All seaweedscontained balanced Na/K and C/N ratio and high amountsof macroelements (Na, K, Ca, and Mg) as compared to theterrestrial vegetables. Among the microelements, Fe wasthe highest followed by Zn, Mn, Cu and other traceelements. Fatty acid distribution showed high level of n-6and n-3 polyunsaturated fatty acids (PUFAs), and theirratios were within the WHO prescribed limits. The higherratios of PUFA/SFA (>0.4) are in agreement with therecommendations of nutritional guidelines. Most of thespecies, especially the Chlorophyta and Phaeophyta, hadpermissible intake values of unsaturation, atherogenic andthrombogenic indexes comparable to milk-based products.Principal component analysis demonstrated a correlationbetween total phenolic content, total antioxidant activity,DPPH, and O2

•− radical scavenging activity, suggestingpolyphenols as the chief contributor to the antioxidantactivity in seaweeds. These results indicate that theseseaweeds could be a potential source of natural antiox-idants, minerals and high-quality PUFAs and may beefficiently used as ingredients in functional foods.

Keywords Antioxidant potential . Minerals . PUFAs .

Tropical seaweeds

Introduction

Increasing awareness among consumers about health-promoting foods has aroused interest in food supplementresearch worldwide. In addition to food supplements,consumption of exotic foods with proven nutritionalvalues has also been gaining prominence in severaldeveloped countries (Herrero et al. 2006). Many of thesefoods are presently promoted and marketed as function-al foods with premium price. The beneficial actions ofthese foods are reported to be mainly due to their function-al components such as minerals, antioxidants and n-3 fattyacids, which are either absent in the analogous traditionalfoods or present only in trace concentrations. Consequent-ly, there has been a quest to explore and utilize foods fromnonconventional sources of both terrestrial and marineorigin to enhance the nutritional quality of human foodswhich in turn also reduces the dependability on traditionalfoods.

Seaweeds with their diverse bioactive compounds (Leeet al. 2008; Zubia et al. 2009) have opened up potentialopportunities in pharmaceutical and agri-food processingindustries. The consumption of seaweeds as part of diet hasbeen shown to be one of the prime reasons for lowincidence of breast and prostate cancer in Japan and Chinacompared to North America and Europe (Pisani et al.2002). Seaweeds also contain sufficient amounts of protein,polysaccharides (e.g., alginates, fucans and laminarans),and amino acids of considerable nutritional importance.Algal lipids (1–3% dry matter) contain a high proportion ofessential fatty acids particularly n-3 polyunsaturated fattyacids (PUFAs). At present, marine fish and their oil are themajor commercial sources of PUFAs, but their suitabilityfor human consumption has been questioned from thebiosafety perspective.

M. Kumar : P. Kumari :N. Trivedi :M. K. Shukla :V. Gupta :C. R. K. Reddy (*) : B. JhaDiscipline of Marine Biotechnology and Ecology,Central Salt and Marine Chemicals Research Institute,Council of Scientific and Industrial Research (CSIR),G.B. Marg,Bhavnagar 364021, Indiae-mail: crk@csmcri.org

J Appl PhycolDOI 10.1007/s10811-010-9578-7

Optimization of protoplast yields from the red algaeGracilaria dura (C. Agardh) J. Agardh and G. verrucosa(Huds.) Papenfuss

Vishal Gupta & Manoj Kumar & Puja Kumari &C. R. K. Reddy & Bhavanath Jha

Received: 4 February 2010 /Revised and accepted: 17 August 2010 /Published online: 7 September 2010# Springer Science+Business Media B.V. 2010

Abstract This study reports on the optimization ofprotoplast yield from two important tropical agarophytesGracilaria dura and Gracilaria verrucosa using differentcell-wall-degrading enzymes obtained from commercialsources. The conditions for achieving the highest protoplastyield was investigated by optimizing key parameters suchas enzyme combinations and their concentrations, durationof enzyme treatment, enzyme pH, mannitol concentration,and temperature. The significance of each key parameterwas also further validated using the statistical centralcomposite design. The enzyme composition with 4%cellulase Onozuka R-10, 2% macerozyme R-10, 0.5%pectolyase, and 100 U agarase, 0.4 M mannitol in seawater(30‰) adjusted to pH 7.5 produced the highest protoplastyields of 3.7±0.7×106 cells g−1 fresh wt for G. dura and1.2±0.78×106 cells g−1 fresh wt for G. verrucosa whenincubated at 25°C for 4–6 h duration. The young growingtips maximally released the protoplasts having a size of 7–15 μm in G. dura and 15–25 μm in G. verrucosa, mostlyfrom epidermal and upper cortical regions. A few large-sizeprotoplasts of 25–35 μm, presumably from cortical region,were also observed in G. verrucosa.

Keywords Agarophytes . Commercial enzymes .

Gracilaria . Protoplast production . Rhodophyta

Introduction

Among the red algae, the genus Gracilaria has considerableindustrial importance as an agarophyte and is the principalsource of raw material to the agar industry worldwide(Zemke-White and Ohno 1999; Smit 2004). These agarindustries consume 72,300 dry tons of agarophytes annuallyand produce approximately 9,600 tons agar with a valueof US$173 million. Of this, Gracilaria alone accounts forabout 80% of the world’s agar market with a value ofUS$ 138 million (Bixler and Porse 2010). The increasingdemand for agar worldwide, coupled with short supplies ofagarophytes from wild stocks, has led to the developmentof viable field cultivation methods for their large-scalecultivation in the sea (Critchley 1993). Following the successin large-scale cultivation, cellular biotechnology techniquesare also being applied to improve the cultivated germplasmof this important resource (see reviews of Reddy et al.2008a, b, 2010). However, these techniques have largelyremained underdeveloped and are thus in their nascent stage.The high structural complexity and diversity in cell wallcomposition (Duckworth and Yaphe 1971; Rochas andLahaye 1989) have rendered the agarophytes in particularrecalcitrant to enzymatic digestion of the cell walls and havethus become an impediment for realizing the potentialsoffered by the application of biotechnology tools andtechniques for seaweeds in general. The skeletal and matrixpolysaccharides of cell walls of red seaweeds mainly consistof cellulose and agar. Therefore, the mixture of enzymeshaving cellulase and agarase are invariably required to digestthe cell wall components of agarophytic red algal species forpreparing protoplasts.

To date, protoplast isolation has been accomplished for48 red algal species, including agarophytes, belonging to 13genera (Reddy et al. 2010). In the genus Gracilaria, with

This paper was presented at the 7th Asia Pacific Congress on AlgalBiotechnology, New Delhi, 2009.

V. Gupta :M. Kumar : P. Kumari :C. R. K. Reddy (*) :B. JhaDiscipline of Marine Biotechnology and Ecology,Central Salt and Marine Chemicals Research Institute,Council of Scientific and Industrial Research (CSIR),Bhavnagar 364021, Indiae-mail: crk@csmcri.org

J Appl Phycol (2011) 23:209–218DOI 10.1007/s10811-010-9579-6

Pi

MD

a

ARRAA

KADGS

1

paocofiw(taotoatcs

ss

f

0d

Carbohydrate Polymers 85 (2011) 157–163

Contents lists available at ScienceDirect

Carbohydrate Polymers

journa l homepage: www.e lsev ier .com/ locate /carbpol

artial characterization of sulfohydrolase from Gracilaria dura and evaluation ofts potential application in improvement of the agar quality

ahendra K. Shukla, Manoj Kumar, Kamlesh Prasad, C.R.K. Reddy ∗, Bhavanath Jhaiscipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scientific and Industrial Research (CSIR), Bhavnagar 364021, India

r t i c l e i n f o

rticle history:eceived 26 September 2010eceived in revised form 31 January 2011ccepted 3 February 2011

a b s t r a c t

Sulfohydrolase extracted from Gracilaria dura was purified to homogeneity and investigated for improv-ing the quality of commercial agar. The purified enzyme (50 kDa) showed optimum activity at pH 8.0 andtemperature 35 ◦C. The agar treated with ∼50 U of purified enzyme exhibited 1.66-fold increase in 3,6-AG

vailable online 5 March 2011

eywords:garesulfationracilaria dura

content with 60% sulfate removal and also resulted an increase of ≥2-fold in viscosity and gel strengthwith a recovery of 90% agar. Further, the gelling and melting temperatures were markedly decreased to31 ◦C and 82 ◦C respectively over the control values of 39 ◦C and 90 ◦C. The scanning electron microscopyrevealed higher cross-linking and rigidity in the treated agar while FT-IR spectral analysis confirmed theincreased 3,6-AG content with decreased sulfate. Therefore, the possibility for cloning of sulfohydro-lase encoding gene(s) for its commercial production and exploitation in desulfation of agar could be an

ive m

ulfohydrolase eco-friendly and alternat

. Introduction

Agars, the sulfated galactans are the most abundant structuralolysaccharide of red algal cell walls and are generally considereds the mixture of agarose and agaropectin, and accounts for ca. 50%f algal dry weight (Yoon & Park, 1984). These sulfated galactansonstitute a large family of hydrocolloids composed of linear chainsf galactose (Gal) with alternating �-1,3 and �-1,4 linkages and dif-er in degree and position of sulfate esterification. The agar qualityn terms of gel strength is solely attributed to the 3–6, AG content

hile the sulfate moiety and the contaminating floridean starchstorage sugars of red algae) undermine the quality of agar. In addi-ion, presence of acidic groups such as sulfonic, pyruvic and uroniccid in agar deteriorate its gel strength by blocking the formationf double helices between the molecules. The proportion and dis-ribution of the aforementioned substituents in the skeletal chainf the galantans modify the physico-chemical properties of thegar (Lahaye & Rochas, 1991; Yaphe & Duckworth, 1972). Amonghe others, viscosity and gelling capacity are the key features thatonfer diverse industrial applications for these hydrocolloids as

tabilizers, thickeners and gelling agents (Usov, 1992).

The Gal-6-sulfurylases found in agarophytes and the analogousulfohydrolase activities extracted from carrageenophytes repre-ent a novel class of enzymes owing to the chemical reaction

∗ Corresponding author. Tel.: +91 278 256 5801/3805x614;ax: +91 278 256 6970/7562.

E-mail address: crk@csmcri.org (C.R.K. Reddy).

144-8617/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.carbpol.2011.02.009

ethod to alkali treatment.© 2011 Elsevier Ltd. All rights reserved.

they catalyze and have only been found in the red algal lineage.The conversion of porphyran to agarose and �-carrageenan to�-carrageenan has been demonstrated previously using partiallypurified enzyme from Porphyra (Rees, 1961a) and Chondrus crispus(Wong & Craigie, 1978) respectively. Recently, Sulfurylase I andII were purified from C. crispus and evaluated their potentials forconverting �- into �-carrageenan (Genicot-Joncour et al., 2010).

With increasing demand of agarose in biotech and pharmaceu-ticals industries, considerable emphasis has been made to removethe sulfate group from agar in order to enhance the gel strength.The widely used alkali pretreatment methods in the industries haveproven their potential to improve the gel strength (Freile-Pelegrin& Murano, 2005; Marinho-Soriano & Bourret, 2005; Meena, Prasad,Ganeshan, & Siddhanta, 2008; Mehta et al., 2010) though theyhave drawbacks of diminished polysaccharide yield and gener-ation of an effluent, which can have deleterious environmentaleffects if discharged untreated. Thus, there is a growing needto develop eco-friendly downstream processing technologies forrecovery of products from bioresources. Recently, a method forobtaining high quality agarose with high yield has been estab-lished in Gracilaria gracilis circumventing the alkali pretreatment(Rodriguez, Matulewicz, Noseda, Ducatti, & Leonardi, 2009). A sim-ple subjecting of temperate red seaweed C. crispus to dark for 10days enhanced the gel quality of carrageenan (Villanueva, Hilliou,

& Sousa-Pinto, 2009).

Nevertheless, there is no evidence for the sulfohydrolaseassisted desulfation of agar in the literature. In view of this, we havefor the first time attempted to purify and characterize the sulfohy-drolase from the agarophytic red alga Gracilaria dura (J. Agardh) C.

Seaweed protoplasts: status, biotechnological perspectivesand needs

C. R. K. Reddy & Manoj K. Gupta & Vaibhav A. Mantri &Bhavanath Jha

Received: 22 April 2007 /Revised and Accepted: 23 July 2007 / Published online: 19 September 2007# Springer Science + Business Media B.V. 2007

Abstract Protoplasts are living plant cells without cell wallswhich offer a unique uniform single cell system that facilitatesseveral aspects of modern biotechnology, including genetictransformation and metabolic engineering. Extraction of cellwall lytic enzymes from different phycophages and microbialsources has greatly improved protoplast isolation and theiryield from a number of anatomically more complex species ofbrown and red seaweeds which earlier remained recalcitrant.Recently, recombinant cell wall lytic enzymes were alsoproduced and evaluated with native ones for their potentialabilities in producing viable protoplasts from Laminaria.Reliable procedures are now available to isolate and cultureprotoplasts from diverse groups of seaweeds. To date, thereare 89 species belonging to 36 genera of green, red andbrown seaweeds from which successful protoplast isolationand regeneration has been reported. Of the total speciesstudied for protoplasts, most belonged to Rhodophyta with41 species (13 genera) followed by Chlorophyta andPhaeophyta with 24 species each belonging to 5 and 18genera, respectively. Regeneration of protoplast-to-plantsystem is available for a large number of species, withextensive literature relating to their culture methods andmorphogenesis. In the context of plant genetic manipulation,somatic hybridization by protoplast fusion has been accom-plished in a number of economically important species withvarious levels of success. Protoplasts have also been used forstudying foreign gene expression in Porphyra and Ulva.Isolated protoplasts are also exploited in numerous miscel-laneous studies involving membrane function, cell structure,

bio-chemical synthesis of cell walls etc. This article brieflyreviews the status of various developments in seaweedprotoplasts research and their potentials in genetic improve-ment of seaweeds, along with needs that must to be fulfilledfor effective realization of the objectives envisaged forprotoplast research.

Keywords Cell wall lytic enzymes .Marine macroalgae .

Protoplast isolation . Protoplast fusion .

Somatic hybridization . Selection of transformants

Introduction

Protoplasts are naked living plant cells devoid of cellwalls which provide a unique uniform cell systemunderpinning several aspects of modern biotechnology.Recently, due to public antagonism, especially in Europe,to recombinant DNA technologies (GM crops) there hasbeen renewed interest in exploiting protoplasts in somatichybridization, cybridization, protoclonal variation studies,proteomics and metabolomics in higher plants (Daveyet al. 2005). Interestingly, it was the observation of naturalenzymatic degradation of cell walls during fruit ripeningthat stimulated interest in investigations on preparation ofprotoplasts using cell wall lytic enzymes in higher plants.Treatment of either cells or tissues with specific cell walllytic enzymes results in total removal of their rigid andcomplex polysaccharide cell walls. Although protoplastsisolation from macrophytic benthic marine algae wasreported as early as 1970 using mechanical methods(Tatewaki and Nagata 1970; Enomoto and Hirose 1972;Kobayashi 1975), the success in producing a large numberof viable protoplasts became possible only after thedevelopment of an enzymatic method by Millner et al.

J Appl Phycol (2008) 20:619–632DOI 10.1007/s10811-007-9237-9

DO09237; No of Pages

C. R. K. Reddy (*) :M. K. Gupta :V. A. Mantri :B. JhaDiscipline of Marine Biotechnology and Ecology,Central Salt and Marine Chemicals Research Institute,Bhavnagar 364 002, Indiae-mail: crk@csmcri.org