Chemical composition of farmed and micropropagatedKappaphycus alvarezii (Rhodophyta, Gigartinales),a commercially important seaweed in Malaysia

Yoong Soon Yong & Wilson Thau Lym Yong & Su En Ng &

Ann Anton & Suhaimi Yassir

Received: 4 June 2014 /Revised and accepted: 19 August 2014# Springer Science+Business Media Dordrecht 2014

Abstract Micropropagation technologies play an importantrole in enhancing the nutritional composition of seaweeds.Kappaphycus alvarezii explants, obtained from two types ofseedling production system viz. micropropagation and farmpropagation, were analyzed. Results obtained from the post-farm cultivation seaweeds showed significantly higher totallipids in micropropagated compared to farm-propagatedK. alvarezii. In the mineral and trace element analyses,micropropagated K. alvarezii yielded significantly higher cal-cium, magnesium, beryllium, cobalt, copper, lithium, manga-nese, and zinc compared to farm-propagated K. alvarezii. Alower concentration of metal contaminants was detected inmicropropagated K. alvarezii compared to farm-propagatedK. alvarezii. Both sources of K. alvarezii showed high SFAcompared to MUFA and PUFA, where C16:0 and C18:0were found to be in abundance. The study suggestsmicropropagated K. alvarezii is a better food source forconsumption compared to farm-propagated K. alvareziiand justifies the rationale of using micropropagation tech-nique for seedling production in the seaweed industry.

Keywords Kappaphycus alvarezii . Rhodophyta . Seaweed .

Proximate composition . Fatty acids .Minerals and traceelements

Introduction

The cultivation of the seaweed, Kappaphycus alvarezii, con-tinues to expand in Malaysia due to the high demand ofcarrageenan. Increasing demand for K. alvarezii has led to ashortage of seedling supply, and micropropagation is one ofthe potential solutions. Micropropagation technology is newto the Malaysian seaweed industry and the results of applyingthis technology to seedling production are still unknown.Artificial nutrients and growth regulators which are added tothe growth medium during micropropagation may change thenutritional compositions of the K. alvarezii.

Studies on the nutrient content of Malaysian K. alvarezii,previously known as Eucheuma cottonii, were reported byMatanjun et al. (2009) and Mok et al. (2012), where its prox-imate, fatty acid, and amino acid composition were analyzed.However, there was a discrepancy in the results, and analyseswere limited to farm-propagated seaweeds only. According toKumar et al. (2007) and Lee et al. (2011), K. alvarezii is wellknown for its ability to absorb and accumulate trace elements inits tissue. This implies that there is a likelihood of variation inthe mineral and trace element content of micropropagatedK. alvarezii as compared to field grown specimens.

In this study, nutrients were added in the culture media toimprove the growth of seaweed micropropagules duringmicropropagation. This was done in order to enhance thenutritional composition of the seaweeds. Therefore, the nutri-tional information for micropropagated and farm-propagatedK. alvareziiwere determined and compared, in order to inves-tigate the effect of micropropagation technology on the nutri-tional composition of the seaweed.

Y. S. Yong :W. T. L. Yong (*)Biotechnology Research Institute, Universiti Malaysia Sabah, JalanUMS, 88400 Kota Kinabalu, Sabah, Malaysiae-mail: wilsonyg@ums.edu.my

W. T. L. Yonge-mail: wilsonyong80@yahoo.com

S. E. Ng : S. YassirFaculty of Science and Natural Resources, Universiti MalaysiaSabah, Jalan UMS, 88400 Kota Kinabalu, Sabah, Malaysia

A. AntonBorneo Marine Research Institute, Universiti Malaysia Sabah, JalanUMS, 88400 Kota Kinabalu, Sabah, Malaysia

J Appl PhycolDOI 10.1007/s10811-014-0398-z

Materials and methods

Seedlings of K. alvarezii, which weighed 45±5 g each, wereobtained from the sources of micropropagation and farmpropagation and cultivated in a seaweed farm located inSemporna, Sabah, Malaysia, using the long-line method.Micropropagation of K. alvarezii was carried out accordingto the optimized protocols developed by Yong et al. (2014a),followed by acclimatization (Yong et al. 2014b) prior seafarming. After 4 weeks of cultivation in the seaweed farm,10 crops of K. alvarezii from each source of propagation wereharvested and cleaned to remove epiphytes and unwantedparticles. The samples were oven dried at 60 °C until constantweight prior to being analyzed for their nutritional informa-tion. All data were tested, except fatty acid composition, fornormality prior to being analyzed with one-way analysis ofvariance (ANOVA) using SPSS version 16 software in orderto establish the differences considering p<0.05.

Proximate analysis Ash content of each seaweed sample wasdetermined by heating the dried seaweed samples at 700 °Cfor 3 h (AOAC International 2000). For total lipid determina-tion, seaweed lipid was extracted according to Ortiz et al.(2006, 2009), while total protein was determined using theKjeldhal method with a nitrogen conversion factor of 6.25(AOAC International 2000).

Mineral and trace elements In order to digest and extractmineral and trace elements from seaweed samples, methodsfromLarrea-Marin et al. (2010) and Rodenas de la Rocha et al.(2009) were modified and applied. Approximately 500 mg ofdried sample was weighed and placed into a 50-mL glass tube.About 10 mL of 69 % nitric acid (HNO3) was then pipettedinto the glass tube, and the vessel was gently shaken untilstable with no additional bubbles formed at room temperature.The samples were then boiled with agitation for 5 min. Afterthe samples were cooled to room temperature, they were keptin a water bath shaker for 24 h at 80 °C with continuousshaking at 100 rpm. The samples were then left to cool toroom temperature before adding 1 mL of 30 % hydrogenperoxide (H2O2) to each vessel. The vessels were agitatedfor predigestion. The samples were further digested by boilingwith agitation for another 5 min. After digestion, the sampleswere left to cool to room temperature before topping up to50 mL with distilled water. The solutions were then filteredwith a 0.2-μm syringe filter and analyzed with inductivelycoupled plasma optical emission spectrometry (Optima 5300DV ICP-OES, PerkinElmer).

Fatty acid composition Extracted crude lipid was added to1 mL of hexane before methylation. In order to analyze thefatty acid composition using gas chromatography-mass spec-trometry (GCMS), methylation was carried out by adding

2 mL of 5 % hydrogen chloride methanol into the hexane-diluted lipid. Themixture was agitated for 3 h and followed byadding a drop of water to stop the reaction. The hexanecontent was then recovered and dried using a rotary evapora-tor. The transformed fatty acids were then dissolved in 3 mLof hexane and filtered through a 0.2-μm syringe filter. Thefatty acid methyl ester (FAME) samples were injected into aGCMS system consisting of an Agilent 7890A gas chromato-graph system coupled with an Agilent 5975C mass spectrom-etry detector. A capillary column HP-5MS (30 m×0.25 mm)with a 0.25-μm film thickness of coated material was used.Injector temperature was set at 250 °C, and the temperatureprogram was as follows: start at 90 °C and hold for 3 min,from 90 to 180 °C at 3 °C min−1 and hold for 5 min, from 180to 290 °C at 3 °C min−1 and then hold for 15 min. A post-runof 10 min at 290 °C was sufficient for the next injection. Gaschromatography was performed in the split mode with ratio100:1. Helium gas was used as carrier gas and maintained at1.0 mL min−1 constant flow rate. Identification of compoundswas carried out by referring to NIST library, whereas therelative compositions were computed with reference to theabundance of the compounds in chromatogram, and no statis-tical analysis was carried out. Each analysis was carried out intriplicate, together with a blank solvent.

Results and discussion

The proximate analysis for both micropropagated and farm-propagated K. alvarezii are shown in Table 1. The ash contentof samples in this study ranged between 38 and 40 %, whichwas similar to a previous study byMatanjun et al. (2009). Theash content in both farm-propagated and micropropagatedK. alvarezii were similar to each other, and there was nosignificant difference between them. High ash content inseaweed is related to its ability to absorb minerals and traceelements from its surrounding environment (Pena-Rodriguezet al. 2011). For this reason, seaweeds are often used as ameans to recover heavy metals from water sources (Kumaret al. 2007).

The average lipid content for both control andmicropropagated K. alvarezii were less than 4 %, where the

Table 1 Proximate composition of both farm-propagated andmicropropagated K. alvarezii

Farm propagated, % Micropropagated, %

Ash 38.86±0.32 38.05±1.10

Lipid 2.06±0.15* 3.00±0.29*

Protein 9.81±0.30 9.95±0.21

Values are expressed as mean±standard deviation, n=3

*p<0.05; significantly different

J Appl Phycol

highest total lipid content was extracted from themicropropagated K. alvarezii (3.00 %±0.29) and was signif-icantly higher (F(1,4) =23.852) than farm-propagatedK. alvarezii (2.06 %±0.15). Lower lipid content extractedfrom the farm-propagated K. alvarezii may be due to thepresence of epiphytic algae on the seaweed during harvesting.The micropropagated K. alvareziiwere found healthy and freefrom epiphytes whereas the farm propagated were heavilycovered with epiphytes and showing signs of “ice-ice” dis-ease. Tissues of epiphyte-infected seaweed are usually ob-served to be degraded by bacteria (Vairappan et al. 2008).As secondary infectors, the bacteria obtain their energy sourcefrom the host tissue—either carbohydrates, lipids, or evenboth. This may explain the lower lipid content found infarm-propagated K. alvarezii compared to micropropagatedK. alvarezii.

The crude protein content of micropropagated K. alvareziiwas 9.95 %±0.21, and farm-propagated K. alvarezii was9.81 %±0.30. The contents of crude protein of both farm-propagated and micropropagated K. alvarezii were similar toeach other, and no significant difference was found. Theseresults are similar to an earlier study carried out by Matanjunet al. (2009) with 9.76%±1.33 of crude protein obtained fromK. alvarezii.

A total of 19 elements were measured (Table 2). Both farm-propagated and micropropagated seaweeds were found tohave high concentrations of sodium (Na), potassium (K),calcium (Ca), and magnesium (Mg). MicropropagatedK. alvarezii showed significantly higher content of Ca(F(1,28)=27.723) and Mg (F(1,28)=408.709) than the farm-propagated K. alvarezii (Table 2).

The micropropagated K. alvarezii had significantly higherconcentration of Be (F(1,28)=8.686), Co (F(1,28)=1,048.000),Cu (F(1,28)=89.559), Li (F(1,28)=40.893), Mn (F(1,28)=10,720.000), and Zn (F(1,28)=1,797.000) compared to thefarm-propagated K. alvarezii. According to the MalaysianFood Act (1983, amended in 2010), Cd and Pb are categorizedas metal contaminants. Among the screened seaweeds, Cd(F(1,28)=217.057) was significantly higher in farm-propagatedK. alvarezii compared to micropropagated K. alvarezii; and noPb was detected.

Higher concentrations of Ca, Mg, Be, Co, Cu, Li, Mn, andZn in the micropropagated K. alvarezii as compared to thefarm-propagatedK. alvareziimay be due to the absorption andaccumulation of these elements from the supplements provid-ed in the culture media. During micropropagation,K. alvareziimicropropagules were supplemented with Na, Fe, Mn, Zn,Co, K, Mg, Ca, Cu, and Boron (B). Thus, these elementscould have accumulated in the seaweed tissues.

According to Mok et al. (2012), mariculture products yieldhigher Cd than aquaculture products, which reflect the exis-tence of Cd in seawater. Cd exists naturally in trace level inzinc and lead ores. Due to the development of various

industries, Cd has spread widely and raised our concern.According to Satarug et al. (2003), Cd is found widely spreadthrough phosphate fertilizer, sewage sludge, and industrywastewaters. Exposure levels of about 30 μg day−1 or moreby an adult will have significantly higher risk of bone fracture,cancer, kidney dysfunction, and hypertension (Satarug et al.2003). Although the concentration of Cd in both farm-prop-agated and micropropagated K. alvarezii was found tobe in the accepted range of the Malaysian Food Act(1983, amended in 2010), the consumption ofmicropropagated K. alvarezii can be considered lessharmful than farm-propagated K. alvarezii, due to lowerCd contents in micropropagated K. alvarezii. Althoughthe contents of heavy metals in this study were withinthe limit set by the Malaysian Food Act in bothmicropropagated and farm-propagated seaweeds, contin-uous monitoring is necessary in order to ensure theseaweeds are safe for consumption.

A total of 14 fatty acids (Table 3) were detected where4 of them are saturated fatty acids (SFA), 5 of them aremonounsaturated fatty acids (MUFA), and the rest arepolyunsaturated fatty acids (PUFA). In addition, two typesof cyclopentyl fatty acids were detected in trace amountsin both farm-propagated and micropropagated K. alvarezii.

Table 2 The mineral and trace element content of both farm-propagatedand micropropagated K. alvarezii obtained from post-farm cultivation

Farm propagated, ppm Micropropagated, ppm

Sodium (Na) Saturated Saturated

Potassium (K) Saturated Saturated

Calcium (Ca) 762.50±18.88* 793.48±12.75*

Magnesium (Mg) 2664.53±50.51* 3053.43±54.77*

Beryllium (Be) n.d.* 0.01±0.00*

Cobalt (Co) 0.44±0.07* 2.26±0.21*

Cadmium (Cd) 0.48±0.02* 0.36±0.02*

Chromium (Cr) n.d. n.d.

Copper (Cu) 0.98±0.15* 1.57±0.19*

Iron (Fe) n.d. n.d.

Lithium (Li) 0.82±0.03* 0.95±0.07*

Manganese (Mn) 1.05±0.03* 2.59±0.05*

Nickel (Ni) n.d. n.d.

Lead (Pb) n.d. n.d.

Selenium (Se) n.d. n.d.

Zinc (Zn) 5.20±0.41* 16.70±0.97*

Vanadium (V) 0.64±0.06* 0.39±0.11*

Aluminum (Al) n.d. n.d.

Silver (Ag) n.d. n.d.

Values are expressed as mean±standard deviation, n=5

n.d. not detected

*p<0.05; significantly different

J Appl Phycol

These cyclopentyl acids, 11-cyclopentylundecanoic acidand 13-cyclopentyltridecanoic acid, were reported earlieras family-specific fatty acids found in the seaweeds fromthe Solieriaceae (Miralles et al. 1990). Among the detect-ed fatty acids, palmitic (C16:0) and stearic acids (C18:0)were found abundantly in both farm-propagated andmicropropagated K. alvarezii, which account for about60 % of the detected fatty acids.

Higher concentrations of SFA as compared to their respec-tive MUFA and PUFAwere detected in both farm-propagatedand micropropagated K. alvarezii (Fig. 1). Muralidhar et al.(2010) found similar results, where out of a total of 14 detect-ed fatty acids, SFA accounted for more than 75% of total fattyacid content, with the majority contributed by palmitic acid.As compared to the results of Muralidhar et al. (2010), bothmicropropagated and farm-propagated K. alvarezii in thisstudy showed higher PUFA content.Kappaphycus sp. collect-ed from India was reported to have high PUFA (85.0 %),followed by MUFA (12.4 %) (Rajasulochana et al. 2010).The PUFA content (51.55 %) was about two times higherthan MUFA (23.28 %) and SFA (25.17 %) reported byMatanjun et al. (2009). Although the composition of fattyacids was different from earlier studies, fatty acids such asC14:0, C15:0, C16:0, C18:0, C18:1n9c, C18:2ω6, C20:4ω6,and C20:5ω3 are consistently present in both the earlier andthe present study. The variation in fatty acid compositionscompared to earlier studies may be due to different locations,

varieties, or even species (Muralidhar et al. 2010;Rajasulochana et al. 2010). In this study, although the samespecies and varieties were analyzed, a different fatty acidprofile was obtained as well. This suggests that fatty acidcomposition is driven by environmental factors.

According to Simopoulos (2002), it is recommended thatthe ratio of omega-6/omega-3 essential fatty acids should be 1.Excessive amounts of omega-6 PUFA are known to promotethe pathogenesis, including cardiovascular disease, cancer,and inflammatory and autoimmune diseases, whereas alow ratio of omega-6/omega-3 exerts suppressive effects.

Table 3 Details of the fatty acidcompositions of both farm-propagated and micropropagatedK. alvarezii

Values are expressed asmean±standard deviation, n=9

Control, % Micropropagated, %

Saturated fatty acid (SFA)

14:0 Myristic acid 1.28±0.14 1.06±0.10

15:0 Pentadecanoic acid 0.10±0.01 0.10±0.01

16:0 Palmitic acid 36.36±1.18 36.65±1.79

18:0 Stearic acid 21.58±2.79 25.94±3.63

Total 59.32±4.12 63.75±5.53

Monounsaturated fatty acid (MUFA)

16:1n7c Palmitoleic acid 0.68±0.08 0.37±0.09

16:1n7t Palmitelaidic acid 8.55±0.58 6.82±0.95

18:1n9c Oleic acid 2.08±0.18 1.67±0.75

18:1n13c cis-13-Octadecenoic acid 1.08±0.14 1.56±0.90

18:1n9t Elaidic acid 1.41±0.44 4.26±1.20

Total 13.80±1.42 14.68±3.89

Polyunsaturated fatty acid (PUFA)

18:3ω6 γ-Linolenic acid 0.19±0.02 0.13±0.02

18:2ω6 Linoleic acid 0.37±0.09 0.50±0.43

20:4ω6 Arachidonic acid 5.95±0.58 4.50±0.79

20:5ω3 Eicosapentaenoic acid (EPA) 10.29±0.99 7.05±0.94

20:3ω6 Dihomo-γ-linolenic acid (DGLA) 0.75±0.17 1.29±0.50

Total 17.55±1.85 13.47±2.68

ω6/ω3 0.71 0.91

Fig. 1 Fatty acid compositions of both farm-propagated andmicropropagated K. alvarezii, where all samples consist of high SFA,compared to MUFA and PUFA. The error bars indicate the standarddeviation, n=9

J Appl Phycol

In the current study, the ratio of omega-6/omega-3 ofmicropropagated K. alvarezii was 0.91, which is nearerto the recommended ratio compared to farm-propagatedK. alvarezii (0.71). Thus, the micropropagated K. alvareziiis suggested to have an optimal ratio of omega-3 and -6essential fatty acids compared to farm-propagatedK. alvarezii.

Conclusion

Micropropagated K. alvarezii yielded more mineral and traceelements compared to farm-propagated K. alvarezii, whichcould be attributed by the enrichment of culture media in theearly stage of propagation. Micropropagated K. alvarezii wasfound to have lower metal contaminants, especially cadmium.Further screening of other metal contaminants, such as arsenicand mercury, is recommended to ensure theK. alvarezii is safefor consumption. ThemicropropagatedK. alvareziiwas foundto consist of an optimal ratio of omega-3 and -6 fatty acidscompared to farm-propagated K. alvarezii from the fatty acidcomposition analysis. This suggests that micropropagatedK. alvarezii is a better food source compared to farm-propagated K. alvarezii and justifies the rationale of usingmicropropagation for seedling production in the seaweedindustry.

Acknowledgments The authors wish to thank the PEMANDU,Malaysia, for funding the research under the Seaweed ResearchGrant of Project EPP3 (GPRL/SPS/2). The authors wish to acknowl-edge Gofar Agro Specialties for their sponsorship in the form ofenrichment fertilizers.

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