Squalen Vol.7 No.3 Page Jakarta, December 2012 ISSN

ISSN. 2089-5690

BULETIN PASCAPANEN DAN BIOTEKNOLOGI KELAUTAN DAN PERIKANAN (BULLETIN OF MARINE AND FISHERIES POSTHARVEST AND BIOTECHNOLOGY) Volume 7

.

Number 3

.

December 2012

Ministry of Marine Affairs and Fisheries Agency for Marine and Fisheries Research and Development Research and Development Center for Marine and Fisheries Product Processing and Biotechnology

Squalen

Vol.7

No.3

Page 97-138

Jakarta, December 2012

Accredited No. : 428/AU/P2MI-LIPI/04/2012

ISSN. 2089-5690

FOREWORD

First of all, we would like to express our gratitude toward God Almighty for the Grace that the Squalen Buletin Pascapanen dan Bioteknologi Kelautan dan Perikanan (Squalen: Bulletin of Marine and Fisheries Postharvest and Biotechnology) Volume 7 Number 3 edition December 2012 has been succesfully published. Several changes have been made from this edition onwards including accreditation number, field scope, language and additional subheads. The accreditation number of this bulletin has been revised from 148/Akred-LIPI/P2MBI/03/2009 into 428/AU/P2MI-LIPI/04/2012 based on the Decree of the Head of Indonesian Institute of Sciences number 395/D/2012 that was issued on April 24th, 2012 regarding Re-accreditation of scientific magazine. This edition contains primary articles, i.e.Study on the Effect of Pollutants on the Production of Aaptamines and the Cytotoxicity of Crude Extract from Aaptos suberitoides; Screening of Thermostable Protease Producing Microorganisms Isolated from Indonesian Hotspring; Screening and Characterization of L-Glutaminase Produced by Bacteria Isolated from Sangihe Talaud Sea; Effect of Chopping Step and Drying Technique on the Quality of Alkali Treated Cottonii (ATC); Fucoidan from Brown Seaweed and Its Bioactivity The editorial staffs would like to acknowledge all of those who participated in the publication of Squalen Bulletin of Marine and Fisheries Postharvest and Biotechnology. We hope that readers find this bulletin beneficial to enhance discourse and knowledge especially in the area of marine and fisheries postharvest and biotechnology. Finally, comments and suggestions are welcome for further improvement.

Editor

i

ISSN: 2089-5690

SQUALEN BULETIN PASCAPANEN DAN BIOTEKNOLOGI KELAUTAN DAN PERIKANAN (SQUALEN: BULLETIN OF MARINE AND FISHERIES POSTHARVEST AND BIOTECHNOLOGY) Vol. 7 No. 3, December 2012

CONTENT

Page FOREWORD ..........................................................................................................................

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CONTENT .............................................................................................................................

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Study on the Effect of Pollutans on the Production of Aaptamines and the Cytotoxicity of Crude Extract from Aaptos suberitoides Ariyanti Suhita Dewi, Tri Aryono Hadi, Hedi Indra Januar, Asri Pratitis, and Ekowati Chasanah........ 97-104 Screening of Thermostable Protease Producing Microorganisms Isolated from Indonesian Hotspring Dewi Seswita Zilda, Eni Harmayani, Jaka Widada, Widya Asmara, Hari Eko Irianto, Gintung Patantis, and Yusro Nuri Fawzya ................................................................................. 105-114 Screening and Characterization of L-Glutaminase Produced by Bacteria Isolated from Sangihe Talaud Sea Tanti Yulianti, Ekowati Chasanah, and Usman Sumo Friend Tambunan.......................................

115-122

Effect of Chopping Step and Drying Technique on the Quality of Alkali Treated Cottonii (ATC) Singgih Wibowo, Muhamad Darmawan, Arif Rahman Hakim, and Seruni Marsella ....................... 123-130 Fucoidan from Brown Seaweed and Its Bioactivity Ellya Sinurat and Endar Marraskuranto.....................................................................................

131-138

CURRENT CONTENT KEYWORDS INDEX

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Study on the Effect of Pollutants ............(A. S. Dewi, T. A. Hadi, H. I. Januar, A. Pratitis, and E. Chasanah)

STUDY ON THE EFFECT OF POLLUTANTS ON THE PRODUCTION OF AAPTAMINES AND THE CYTOTOXICITY OF CRUDE EXTRACT FROM Aaptos suberitoides Studi Pengaruh Polutan terhadap Produksi Aaptamin dan Sitotoksisitas Ekstrak Pekat dari Aaptos suberitoides Ariyanti Suhita Dewi1)*, Tri Aryono Hadi2), Hedi Indra Januar1), Asri Pratitis1) and Ekowati Chasanah1) 1)

Research and Development Center for Marine and Fisheries Product Processing and Biotechnology, Ministry of Marine and Fisheries, Petamburan VI, Jakarta, Indonesia 10260 2) Research Center of Oceanography, Indonesian Institute of Sciences, Pasir Putih, Jakarta, Indonesia 14430 *) Corresponding author: [email protected]

ABSTRACT This experiment was conducted to study the effects of anthropogenic stressor on the spatial variability of secondary metabolites from marine sponge Aaptos suberitoides. Samplings were conducted at 7 sites in Marine National Park of Thousand Islands that are extended within 30 km off Jakarta bay on late February 2011. Sponges were collected and quantified by means of liquid chromatography coupled with photo-diode array detection, whereas, cytotoxicity of sponges extracts was determined against T47D (breast) cancer cell lines. Results showed that the spatial production of aaptamine and isoaaptamine did not correlate with the quality of their surrounding habitat, despite nitrite and nitrate levels significantly affected the bioactivity of crude extracts. Keywords: A. suberitoides, aaptamines, spatial production, cytotoxicity, pollutants ABSTRAK Penelitian ini dilakukan untuk mempelajari efek tekanan antropogenik terhadap variabilitas spasial metabolit sekunder dari spons laut A. suberitoides. Kegiatan sampling dilakukan di tujuh titik yang berada di area Taman Nasional Kepulauan Seribu yang tersebar sepanjang 30 km dari wilayah Teluk Jakarta pada bulan Februari 2011. Spons dikoleksi dan dikuantifikasi menggunakan kromatografi cair yang dilengkapi deteksi photo-diode array sedangkan sitotoksisitas ekstrak spons diukur terhadap sel kanker T47D (payudara). Hasil penelitian menunjukkan bahwa produksi spasial dari aaptamin dan isoaaptamin tidak berkorelasi dengan kualitas dari habitat disekitarnya, meskipun kadar nitrit dan nitrat mempengaruhi bioaktivitas ekstrak pekat secara signifikan. Keywords: A. suberitoides, aaptamine, produksi spasial, sitotoksisitas, polutan

INTRODUCTION : Located in the northwest of West Java, Indonesia, the reefs of Thousand Islands have been reported to undergo various levels of pressures from a number of human activities, such as fishing, coral mining, dredging, anchor damage, oil spills, resort construction and the discharge of industrial wastes. Jakarta, with its over 12 millions inhabitants, has contributed to considerable environmental disturbance, due to its domestic effluents (de Voogd & Cleary, 2006). For the purpose of assessment on the effects of nutrient enrichment on Thousand Islands’ coral reefs, bioindicators are needed to provide an early warning of threats. Sessile taxa are considered suitable as

bioindicators for environmental deterioration in marine and freshwaters due to the fact that they exhibit slow mobility and develop high adaptability toward environmental changes (Alcolado, 2007). Sponges as bioindicators for environmental deterioration have been used mainly to monitor heavy metals’ build-ups. Others reported their use to observe sedimentation, organic pollutants, inorganic pollutants, and temperature changes (Dewi, 2010). Sponges have attracted a considerable attention from diverse scientists mostly due to their capability of producing toxic secondary metabolites that are proposed to be utilized as chemical defensive agents such as antipredation, spatial competition, and control of epibiont overgrowth. Since the rate of predation is

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Squalen Vol 7 No 3, December 2012: 97-104

higher in temperate habitats, tropical sponges have evolved more effective secondary metabolites to deter predators (Becerro et al., 2003). Natural spatiotemporal variations of secondary metabolites from marine sponges have been reported in several papers (Page et al., 2005; Abdo et al., 2007), however, only fewer studies observed correlations between fluctuations of isolated bioactive compounds and environmental stressors in their habitats (Thompson et al., 1987; Wiens et al., 2003; Abdo et al., 2007). Among others, A. suberitoides is reported to be one of the most common marine sponges found in Thousand Islands (de Voogd & Cleary, 2006), which illustrates their constant exposure to pollutants throughout the year. The marine sponge A. suberitoides is classified as part of the order Hadromerida and family Suberitidae. They are known to have a unique class of alkaloids as major components namely the aaptamines, which bear a unique 1H-benzo [de] [1,6] naphtyridines structure. Aaptamine (1) and isoaaptamine (2), the major metabolites of A. suberitoides, were first reported by Nakamura et al. (1987) and Fedoreev et al. (1989),

respectively. Both compounds have been reported to possess anticancer activities against various cell lines i.e. HeLa (Ohizumi et al., 1984), P-388 (Shen et al., 1999), p21 (Aoki et al., 2006), K-562 (Gul et al., 2006); and murin colon (Bowling et al., 2008). Since both metabolites were also found in different genuses, it is suggested that sponge-associated microbes are the true producer of those bioactive compounds (Bowling et al., 2008). This study aimed to investigate correlations between the presence of anthropogenic stressors with the spatial patterns and cytotoxicity of crude extracts from A. suberitoides in Thousand Islands, Indonesia. MATERIAL AND METHODS Sampling Sites Samplings were conducted at 8 sites in Marine National Park of Thousand Islands that are extended within 30 km off Jakarta bay during east monsoon in 2011 (Fig. 1). The sampling locations (from north to south) are as follow: Rengit Island (R) [5o 29' 42.80"

Figure 1. Study sites in Thousand Islands are indicated with flags.

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S, 106o 34' 44.9" E], Sebaru Besar Island (SB) [5o 30' 13.20" S, 106o 33' 28.1" E], Pelangi Island [5o 35’ 16.9" S, 106o 35' 38.50" E], Kayuangin Bira Island (KB) [5o 36' 27.90" S, 106o 34' 5.6" E], Opak Besar Island (OB) [5o 40' 18.60" S, 106o 34’ 4.82" E], Karangbongkok Island (K) [5o 41' 20.58" S, 106o 33' 53.98" E], Semak Daun Island (SD) [5o 44' 4.16" S, 106o 34' 35.23" E] and Panggang Island (P) [5o 44' 49.5" S, 106o 34' 5.60" E]. Anthropogenic stressors mainly originate from domestic waste from the southern part of Thousand Islands (Jakarta bay) and oil rig discharge from the northern parts (Java Sea). Animal Material A. suberitoides was not found in Rengit Island. Therefore, sponges were only collected from 7 sites with three replicates for each site. Samples were harvested by hands in each site at a depth of 12-16 m. Collected samples were immediately kept frozen until extraction. Voucher samples are stored in the Research and Development Center for Marine and Fisheries Product Processing and Biotechnology, Ministry of Marine Affairs and Fisheries and the Research Center of Oceanography, Indonesian Institute of Sciences for taxonomic identification. Analysis of Water Quality Water samples were collected at a depth of 5 m. Chemical analysis of water quality (DO, nitrate, nitrite, ammonia, and phosphate) in each location were carried out in situ by using colorimeter Hach DR-890 and reagent kit (Hach, USA) with a series of concentration ranging from 0.001-15 ppm of O2; 0.001 – 0.5 ppm of NO3-; 0.0001 – 0.35 ppm of NO2-; 0.001 – 0.5 ppm of NH3 and 0.001 – 2.5 ppm of PO43-. Methods used were according to Kroger et al. (2009). Dissolved oxygen and acidity were measured by using Hach HQ40d portable meter (Hach, USA). Salinity was measured by using refractometer (Atago, Co. Ltd., Japan). Chemical Extraction and Quantification Approximately 5 g wet weight of each frozen sample was extracted exhaustively with 10 ml of pure methanol (JT Baker, USA, reagent grade). Approximately 1 ml of each extract was centrifuged to coagulate suspended solid. Each supernatant then concentrated with nitrogen and freeze-dried. After weighing, extracts were re-dissolved in methanol (JT Baker, USA, HPLC grade) to give concentration of 10 mg/ml. Five micro liters of extract was injected into HPLC system Shimadzu 10-AD with PDA detector and Shimpack VP ODS column (2.0 mm x 150 mm). Eluent used was 20% MeCN/H2O (1% TFA) at a flow rate of 0.2 ml/min. Concentrations of aaptamine and

isoaaptamine were determined by calculating the integral of the peak area and plot it to the standard curve. For the standards, identification was also performed by means of 1D NMR spectra (recorded by Jeol 500 MHz with cryoprobe system) in DMSO-d6 (Sigma Aldrich, USA) with comparison to that of literature. Cytotoxicity Assay In vitro cytotoxicity assay of aaptamines was performed against T47D breast cancer cell lines according to methylthiazol tetrazolium (MTT) method by Zachary (2003). Reagents used in this assay were purchased from Sigma Aldrich (USA). Breast cancer cell lines were obtained from the Laboratory of Industrial Technology Development and Biomedics, the Agency for the Assessment and Application of Technology, Indonesia (BPPT). Statistical Analysis Spearman’s correlation coefficient (rs) was used to examine correlations between aaptamines concentration and water parameters. Relationships between dependent values of aaptamines and independent variables of water parameters were examined by means of regression analysis. Biophysic patterns of both are presented in contour maps (software Surfer-Golden). RESULTS AND DISCUSSIONS Taxonomic Identification A. suberitoides has a massive and blunty lobate form with a smooth and sometimes elevated surface. The color is reddish black with vivid yellow for interior. The consistency is compact and spongy (Fig. 2A). Osculum is around 3-5 mm in diameter. The skeleton contains many spicules erect with points toward the outside (ectosome) (Fig. 2B). Whereas its spicules consist of strongyloxeas with two different size ranges, the larger ones are 700-1200 µm and the small ones are 110-300 µm (Fig. 2C). There is a difference with Laubenfels’s description (1954) about A. suberitoides particularly on the spicules. Our specimen has larger and longer spicules than Laubenfels’s. In microscopy observation, the length instead of the width was measured, due to the different habitat of our specimen than that of Laubenfels’s. Analysis of Water Quality The result of water quality analysis is shown in a contour map (Fig. 3). Analysis of water quality showed that dissolved oxygen was concentrated only in the

99


Figure 2. (A) Specimen of A. suberitoides; (B) Skeleton profile (100x magnification); (C) Spicule profile (200x magnification).

Figure 3.

Contour maps of water quality parameters in Thousand Islands waters during February 2011: A. Dissolved oxygen (DO); B. Phosphate; C. Nitrite; D. Ammonia; E. Nitrate; F. Total nitrogen; G. Acidity; H. Salinity.

northern part of the marine park. Phosphate contents demonstrated an increasing trend in the northern zone, whilst those of nitrogen were concentrated in the middle and southern zone. Organic build-ups were shown dispersed in the southern zone, whereas seawater acidity was spread out in middle and southern area. Salinity is relatively similar at all locations during sampling.

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Chemical extraction and quantification Aaptamine and isoaaptamine for standards were isolated and showed UV absorbance at 236; 255; 384 nm and 249; 265; 410 nm, respectively. The structure of aaptamine and its isomer is shown in Fig. 4. Identification by means of 1D NMR demonstrated similar data to that of literature (Nakamura et al.,


1982). Average yield of isolated of aaptamine per wet weight appeared at 0.08 mg/g and that of isoaaptamine appeared at 0.02 mg/g (Fig. 5). The highest differences were found betweeen concentrations of aaptamine within sites and replicates (p>0.05) (Table 1). On the other hand, concentrations of isoaaptamine exhibited significant differences within replicates and sites (p<0.05) (Table 1). Similarity test betweeen groups showed that aaptamine exhibited variability among replicates but not among sites (Tukey test, p>0.05) (Table 1). Whereas, isoaaptamine demonstrated variability both within replicates and sites (Tukey test, p>0.05) (Table 1). The concentrations of both aaptamine and isoaaptamine were found to have no significant correlation with water quality (p>0.05) (Table 2). Cytotoxicity of Crude Extracts The bioactivities of crude extracts were measured based on their cytotoxicity against human breast cancer cell lines (Fig. 6). The highest cytotoxicities were shown by crude extracts from Pelangi and Kayuangin Bira islands by killing approximately 75% of cancer cells. The lowest cytotoxicity was displayed by crude extract from Sebaru island (50% viable cells).

However, this fluctuation of cytotoxicity was not considered significant (p>0.05) (Table 1). On the other hand, nitrite (p<0.05, r2=-0.75) and nitrate levels (p<0.05, r2=0.90) strongly affected the bioactivities of crude extracts (Table 2). No correlation between cytotoxicities and the concentration of aaptamines was found based on the Spearman test (Table 3). There are two major causes of pollutants in Thousand Islands, i.e. oil rig discharge in northern zone and domestic waste from the Jakarta bay. Although we expectantly observed concentrated pollutants in both zones, however, brought by the east monsoon in October, the sea surface currents disperse pollutants westward, leading to the accumulation of pollutants in middle zone. Similarly, aaptamine and isoaaptamine showed trend of build ups in the same zone, although no significant difference was found among sites. Correlations between water quality and concentration of both compounds were not found. This is probably due to the fact that the pollution gradient along the sampling site was relatively similar so that no variation of aaptamines was detected. Interestingly, the presence of inorganic nitrogen in the ecosystem affected cytotoxicity of crude extracts,

R1O N

H 3 CO RN

(1) R=H, R 1 =CH 3 (2) R=CH 3 , R 1 =H

Figure 4. Chemical structure of aaptamine (1) and isoaaptamine (2).

Figure 5. Mean concentration of aaptamines from A. suberitoides collected along the sampling sites (p>0.05, N=21).

101


suggesting that other secondary metabolites possibly responsible for their activity. Furthermore, the cytotoxicity of crude extracts is probably related to the role of associated bacteria in A. suberitoides that is known as the true producer of secondary metabolites. Bacterial symbionts, especially

cyanobacteria, in sponges have been reported to play role in nitrogen cycle (Fiore et al., 2010). In this case, bacteria utilize available nitrate in the environment and contribute to nitrogen fixation. Hence, we suggest that inorganic nitrogen level in seawater is probably affecting the activity of sponge-associated

Table 1. Effect of locations and replicates on the concentration of aaptamines and their cytotoxicites against breast cancer cell lines. Bold values indicate significant correlations Source of Variation

df

Locations

6

634901.5

4.59

0.01

1565160

Replicates

2

705451.3

5.1

0.02

4487257

12

138439.8

Error

Isoaaptamine MS F P

MS

Aaptamine F

T47D F

P

MS

1.28

0.34

341.34

2.52

0.12

3.66

0.06

86.28

9.96

<0.01

1226461

P

34.26

Table 2. Spearman correlation for the effect of water quality on the concentration of aaptamines and the bioactivity against breast cancer cell lines. Bold values indicate significant correlations.

Isoaaptamine

r

Aaptamine

P r2

Isoaaptamine 1 --0.04

P r2 P

0.94 0.04 0.94

T47D

2

Aaptamine 0.04 0.94 1

T47D 0.04 0.94 -0.36

---0.36 0.43

0.43 1 ---

Figure 6. Mean viable cells of T47D after the addition of crude extracts from A. suberitoides (p<0.05, N=21) Table 3. Spearman correlations between aaptamines and bioactivities of crude extracts DO Isoaaptamine

r

Aaptamine T47D

2

Nitrite

Ammonia

Nitrate

Nitrogen

-0.6

-0.46

-0.39

-0.07

0.06

-0.07

p 2 r

0.12 0.18

0.29 0.11

0.38 -0.21

0.88 -0.5

0.9 -0.66

0.88 -0.5

p 2 r

0.7 0.11 0.82

0.82 -0.07 0.88

0.64 -0.75 0.05

0.25 0.21 0.64

0.11 0.9 0.01

0.25 0.21 0.64

p

102

Phosphate

pH 0.28 0.54 0 1 0.09 0.84

Salinity 0.02 0.97 0.04 0.93 -0.46 0.3


cyanobacteria in producing bioactive compounds. However, further research needs to be conducted to test this idea.

production of bioactive compounds in this particular marine sponge.

In the ocean, pollution of inorganic nitrogen mainly comes from domestic and industrial run off in addition to aquaculture practice that may lead to three major environmental hazards such as acidification and eutrophication, as well as impairment on aquatic animals (Camargo & Alonso, 2006). Several other sponges have been reported as potential bioindicator for environmental degradation due to municipal wastewater based on their relative abundance, for instance: Amphimedon viridis, Cliona celata, Clionaviridis, Mycale microsigmatosa, Scopalina ruetzleri (Alcolado, 2007) and Cliona delitrix (Carballo et al., 1994).

ACKNOWLEDGMENTS

Various authors have also reported spatial variation of secondary metabolites in sponges due to environmental factors. Thompson et al. (1987) firstly proposed that environmental conditions, instead of genetic differences, affected the composition of diterpene in Rhopaloeides odorabile. Also, variability in salicylihalamide A from Haliclona sp. in different locations was reported to correlate with water temperature (Abdo et al., 2007). On the other hand, other research reported that spatial variability of bioactive compounds in marine sponge Mycale hentscheli was not directly correlated to water temperature, depth, or salinity (Page et al., 2005). Furthermore, correlation of secondary metabolites production in Acanthella cavernosa (Jumaryatno et al., 2007) as well as Aplysina fulva (Nunez et al., 2008) and their ecological conditions is remained undetermined. According to Cooper (2009), there are five selection criterias for an appropriate bioindicator for water quality assessment on coral reef i.e. specificity, monotonicity, variability, practicality, and relevance. This research showed that although the monitoring of aaptamines production and bioactivity of methanolic extracts is considered practical and relevant, however, further research is needed to evaluate the rest of the above criteria in polluted areas to assure the effect of changes in nutrient loading. CONCLUSIONS No direct correlation between the production of aaptamines and marine pollution was detected in this research, presumably due to the similarity of environmental state along the sampling area. The correlation between inorganic nitrogen contaminant and the bioactivity of crude extract suggests that inorganic nitrogen pollution generates specific stress response in A. suberitoides that initiates the

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Screening of Thermostable Protease..... (D S. Zilda, et al...)

SCREENING OF THERMOSTABLE PROTEASE PRODUCING MICROORGANISMS ISOLATED FROM INDONESIAN HOTSPRING Penapisan Mikroorganisme Penghasil Protease Tahan Panas yang Diiisolasi dari Sumber Air Panas Indonesia Dewi Seswita Zilda1)*, Eni Harmayani2), Jaka Widada2), Widya Asmara2), Hari Eko Irianto1), Gintung Patantis1) and Yusro Nuri Fawzya1) 1)

Research and Development Center for Marine and Fishery Product Processing and Biotechnology 2) Biotechnology Study Program, Gadjah Mada University * Corresponding author: [email protected]

ABSTRACT Although many proteases had been studied and characterized, only a few of them are commercially available. Protease thermostability is one of the crucial properties for industrial application. This research aimed to isolate and to screen the potential isolate which produce thermostable protease. There were 6 isolates (BII-1, BII-2, BII-3, BII-4, BII-6 and LII), isolated using solid Minimal Synthetic Medium (MSM) supplemented with 1.5% skim milk, that have, protease activity. Based on the 16S-rRNA gene sequencing analysis, isolates BII-1, BII-2 and BII6 were identified as Bacillus licheniformis, isolates BII-3 and BII-4 were identified as Bacillus subtilis, while isolate LII was identified as Brevibacillus thermoruber. Three isolates (BII-6, BII-4 and LII) were then further investigated for the second screening step using liquid MSM supplemented with 1% skim milk. The isolates (BII-6, BII-4 and LII) optimally produced protease when they were cultivated at 35, 30 and 50 oC respectively after 22 h of incubation. Protease produced by BII-6, BII-4 and LII had optimum temperature of 65, 60 and 85 oC, optimum pH at 78, 8 and 9 and stable up to 100 min at 55, 60 and 75 oC respectively. Keywords: thermostable protease, Bacillus subtillis, Bacillus licheniformis, Brevibacillus thermoruber ABSTRAK Meskipun banyak protease yang sudah dipelajari dan dikarakterisasi, hanya beberapa yang tersedia secara komersial. Ketahanan protease terhadap panas merupakan salah satu sifat penting untuk diaplikasikan di industri. Penelitian ini bertujuan untuk mengisolasi dan menapis isolat potensial yang menghasilkan protease yang tahan terhadap panas. Ada 6 isolat bakteri (BII-1, BII-2, BII-3, BII-4, BII-6 dan LII) yang diisolasi dengan menggunakan media padat Minimal Synthetic Medium (MSM) dengan penambahan 1,5% skim milk mampu menghasilkan protease. Analisis susunan gen 16S rRNA terhadap keenam isolat menunjukkan bahwa hanya ada 3 isolat berbeda yang teridentifikasi sebagai Bacillus licheniformis (BII-1, BII-2 dan BII-6), Bacillus subtilis (BII-3 dan BII-4) dan Brevibacillus thermoruber(LII). Tiga isolat (BII-6, BII-4, dan LII) selanjutnya diteliti lebih lanjut untuk penapisan kedua menggunakan medium cair MSM dengan penambahan 1% skim milk. Isolat-isolat ini menghasilkan protease secara optimal ketika dikultivasi pada suhu 35, 30, dan 50 oC untuk masing-masing isolat setelah inkubasi selama 22 jam. Protease yang dihasilkan oleh BII-6, BII-4 dan LII berturut-turut mempunyai suhu optimum 65, 60 dan 85 oC, pH optimum 7-8, 8-9 dan stabil sampai 100 menit pada suhu 55, 60, dan 75 oC untuk masing-masing isolat. Kata kunci: protease tahan panas, Bacillus subtillis, Bacillus licheniformis Brevibacillus thermoruber

INTRODUCTION Proteases (EC 3.4.21-24 and 99; peptidyl-peptide hydrolases) are enzyme that hydrolyze proteins via the addition of water across peptide bonds and

catalyze peptide synthesis in organic solvents and in solvents with low water content (Beg et al., 2003). Proteases, which constitute 60% of the total enzyme market (Rao et al., 1998) is the most vital enzyme used industrially and academically. The use of

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protease was estimated reaching to 250 billion US$ by 2010 (Turk, 2006; Mario et al., 2009; Akanbi et al., 2010). They are widely applied in detergent, protein modification, leather, meat, brewing, photographic, dairy, membrane cleansing and waste treatment industries (Kumar et al., 2002; Chu, 2007). The demand of the enzyme will keep increasing due to the need of the enzyme which can withstand on harsh industrial process. Thermostability is the crucial properties of enzyme for industrial application. Thermostable proteases are stable and active above 60-70 oC and withstand to organic solvent, detergent, low and high pH and other denaturing agents (Covan et al., 1985; Covan, 1997; Gupta & Khare, 2006) so that it is particular interest in industrial process. Thermostable proteases has high specific activity due to the protein characteristic as substrate for proteases which unfolded at elevated temperature. They also useful in synthesis of high molecular weight peptide due to their resistance against organic solvents carried out in process with low water content (Sellek & Chaudhuri, 1998; Bruins et al., 2001; Synowiecki, 2008). Commercially, protease are produced by plants, animals and microorganisms. Microorganisms are attractive sources for protease and other enzymes due to their ability to be cultured in large scale in short fermentation time with abundant desire product. To improve the enzyme performance, their genes can be manipulated easier than plants and animals (Tambekar et al., 2009; Ningthoujam & Kshetri, 2010). Enzymologists have special interest to thermophilic microorganisms both at the fundamental and industrial level as natural source of enzymes that are active and stable at elevated temperatures. Hot spring as one of thermophile habitats is considered as promising source for the direct isolation of thermostable enzymes. The microorganisms living in hot spring are not only withstand to elevated temperature but also to the pH of environment and the presence of certain chemical compounds. Some of thermostable protease producing microorganisms were isolated from hot spring as reported by Pakpahan (2009) who isolated three unidentified isolates from Sipolohon hotspring, North Tapanuli, North Sumatera. Wilson & Remigio (2012) also reported novel moderate thermophilic bacterium (EP1001) as thermostable protease producer isolated from an alkaline hot spring, Zimbabwe. The objective of the research was to isolate and to screen the potential isolate which produce thermostable protease from Indonesian hotspring, i.e Padang Cermin (Lampung) and Banyu Wedang(Bali). In this study, screening were conducted in two steps. The first step was carried out using solid media and

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the second step was done using liquid media, followed by proteases characterization. The potential isolates were choosen based on its ability to produce protease which active and stable at highest temperature. MATERIAL AND METHOD Screening of Protease Producing Microorganisms Using Solid Medium Thermostable producing bacteria was isolated from samples collected from hot springs at Padang Cermin, Lampung and Banyu Wedang, Bali using solid medium of Minimal Synthetic Medium (MSM) containing of 0.1% NaCl, 0.1% K 2HPO 4, 0.01% MgSO4, 7H2O, 0.05% yeast extract and supplemented with 1% skim milk. The incubation was carried out at 37, 55 and 70 oC. The clear zone formed around the colony indicated the ability of the isolate to produce protease and designated as the Proteolytics Index (PI). The Proteolitics Index was determined by measuring diameter of clear zone around the colony compare to diameter of the colony. The purified cultures were preserved in 40% glycerol and stored at -70 oC. Phylogenetic Analysis by 16S rRNA Gene Sequencing Bacteria’s DNA was extracted using TIANamp Bacteria DNA Kit. A 16S-rRNA (1.47 kbp) fragment was amplified by PCR using a pair of universal primers (16S rDNA”27F and 16S rDNA”1492R). Fifty ìl of the reaction mixture contained 1 µl chromosomal DNA, 2 µl primer, 22 µl ultrapure water and 25 µl 2xTag PCR Master Mix (TIangen Biotech, China). The PCR reaction was set as: denaturation at 95 ºC for 5 min and 30 cycles of annealing at 55 °C for 30 s, extension at 72 °C for 90 s and denaturation at 95 °C for 60 s. Final extention was carried out after 30 cycle at 72 oC for 10 min. The nucleotide sequences of the fragment were identified using 3730 DNA sequencer (Applied Biosystems, CA, USA) and subjected to a homology search against NCBI DNA database using BLAST (Basic Local Alignment Search Toil) (Altscul et al., 1990) then aligned using Clustal W program (Higgins et al., 1992) available at European Bioinformatics Institute website (http://www.ebi.ac.uk/clustalw/). The sequence retrieve from Gene Bank data base (Benson et al., 2003) available at the NCBI website (http:// www.ncbi.nlm.nih.gov/). Clustal analysis (Sokal & Sneath, 1963) and the neighbor-joining mid point analysis (Saitou & Nei, 1987; Rohlf, 1993) were performed using TREECON for Windows (Version 1.3b) (Van der Peer & De Wachter, 1994). The consistency of each node was estimated by


bootstrapping over markers (Felsenstein, 1985) using 100 pseudoreplications. Srceening in Liquid Medium Three different species (based on 16S-rRNA analysis) of protease producing bacteria isolated from previous screening step were cultured in liquid MSM medium with 1% skim milk and incubated for 35 h at 25-60 oC. The free cell supernatant was measured for protease activity at optimum condition for each isolate. Protease Assay Protease activity was determined by applying a modified method by Takami et al. (1989). The mixture containing of 0.25 ml of 1% casein in 0.025 Tris-Cl buffer pH 7 was incubated with 0.25 ml of enzyme for 10 min. The reaction was stopped by adding 0.5 mL of 0.4 M TCA. The mixture was centrifuged at 10.000 rpm for 10 min. Supernatant (0.5 ml) was mixed with 2.5 ml of 0.4 M Na2CO3 and 0.25 ml of FolinCiocalteu’s Phenol Solution and incubated for 30 min at room temperature. The absorbance of the solutions were read against the blank sample at 660 nm using Spectronic@ 20 GenesysTM. Tyrosin standard solution, in the range of 0-1000 mg/L was prepared in triplicate to obtain a standard curve. One unit (U) of protease was defined as the amount of enzyme that could produce 1 ìg of tyrosine in one minute under the defined assay conditions. Partial Characterization Optimum Temperature and pH. Optimum temperature was determined by measuring enzyme

activity at 40 – 90 oC. Optimum pH for enzyme assay was determined by measuring the enzyme activity using substrate with pH of 4 -10 at optimum temperature. Thermal Stability. The stability of enzyme against thermal was determined by incubating the enzyme in 20 mM buffer Tris-HCl at optimum pH for each isolate at three temperature levels (65, 60 and 55 oC for BII-6, 60, 55 and 50 oC for BII-4, 85, 80 and 75 oC for LII) up to 100 min. The enzyme activity was measured every 10 min and expressed as relative activity against the enzyme without thermal treatment as 100% enzyme activity. RESULT AND DISCUSSION Screening on Solid Medium The sampling site were located at Padang Cermin, Lampung (5o 37’ 59’’ LS; 105o 04’ 20’’ BT) and Banyu Wedang, Bali (8o 10’ 38’’ LS; 114o 35’ 26’’). The properties of samples were presented at Table 1. The screening and isolation of protease producing bacteria were carried out at 55 oC to obtain the thermostable protease producing isolates. There were 6 isolates grown on MSM plate agar containing of 1.5% skim milk and formed clear zone around of the colony after 30 hours incubation at 55 oC (Fig 4.). The code and Proteolitics Index of the isolates were presented at Table 2. Isolates from Banyu Wedang had bigger PI value of 12.5-15.5 mm compared to that from Padang Cermin, which was 4 mm. However, the value of PI was not the only indicator for obtaining a

Table 1. Temperature and pH of in situ Sample Code of Isolates Salinity (o / oo )

pH

Temperature (o C)

LII

4-5

6,9

97

BII

4

8,1

44,8

Source of Isolates Padang Cermin, Lampung Banyu wedang, Bali

Table 2. Proteolitics Index of Protease Producing Microorganisms

Code of Isolates Proteolitics Index P-LII P-BII-1 P-BII-2 P-BII-3 P-BII-4 P-BII-6

4 13,5 12,5 12,5 14,5 15,5

Source of Isolates Padang Cermin, Lampung Banyu Wedang, Bali Banyu Wedang, Bali Banyu Wedang, Bali Banyu Wedang, Bali Banyu Wedang, Bali

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Figure 1. Protease producing microorganisms on agar plate with 1% skim milk. potential protease. The indicator of thermostable protease producing bacteria is the ability of the isolate to produce protease which has optimum temperature and stable at the highest temperature. Therefore, a second step of screening was conducted by growing the potential isolates in liquid medium and measuring the activity at a series of temperature and pH, and investigated their stability against thermal. Phylogenetic Analysis by 16S rRNA Gene Sequencing The analysis of 16S-rRNA sequensing showed that BII-1,BII-2 and BII-6 gave 99% similarity to Bacillus licheniformis, while BII-3 and BII-4 had 99% similarity to Bacillus subtilis and LII had 99% similarity to Brevibacillus thermoruber.

Most of thermostable proteases were reported to be produced by Bacillus sp. such as Bacillus stearothermophilus (Salleh et al., 1977; Razak et al., 1993) Bacillus caldolyticus (Burg et al, 1991), Bacillus subtilis (Ningthoujam & Kshetri, 2010), Bacillus cereus (Jabeen & Qazi, 2011) and Bacillus mojavensis (Haddar, 2009). On the other hand, only few other types of bacteria that were reported to produce thermostable protease such as Aquifex pyrophilus (Choi et al., 1999) and Pseudomonas sp. (Asoodeh & Musaabadi, 2012). Screening on Liquid Medium The first step of screening was carried out at 55oC on solid medium supplemented with 1.5% skim milk resulted 6 isolates forming clear zone around the colony indicating that the isolates had protease activity (Fig 1.). The second screening step was conducted by testing the most potential isolate for

Figure 2. Phylogenetic tree of protease producing microrganisms.

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their ability to produce protease in liquid medium which active and stable at highest temperature. Isolate BII6 and BII-4 were not able to grow at 55oC and the 16S-rRNA identification showed that the isolates had 99% similarity to Bacillus licheniformis and Bacillus subtilis respectively. Hadaar (2009) and Ningthoujam & Kshetri (2010) reported two protease producing isolates that were classified as mesophile and grow optimally at 30–35 oC. Therefore, the three different species isolates (BII-6, BII-4 and LII ) were grown in liquid MSM supplemented with 1% skim milk and incubated up to 34 h at 25-40 oC for BII-6 and BII-4 isolates, and 45-55 oC for LII isolate.

The result showed that the highest protease activity was produced at temperature 35, 30 and 50 oC for BII-6, BII-4 and LII respectively (Fig 3.). The protease was produced by isolates after 22 h incubation. The optimum temperature of the proteases were 65, 60 and 85 oC (Fig 4.), and optimum pH were 7-8, 8, and 9 for BII-6, BII-4 and LII respectively (Fig 5.). The protease produced by LII showed the stability up to 100 min against thermal at highest temperature (75 oC) while protease from BII-2 and BII-6 at 55 and 50 oC respectively (Fig. 6). All isolates produced protease with optimum temperature above the optimum temperature for their growth and enzyme production.

Enzyme activity (U/ml)

90 30°C

80

35°C

A

40°C

70 60 50 40 30 20 10 0 0

5

10

15 20 25 Incubation time (hour)

30

35

Aktvitas Enzim (U/ml) Enzyme activity (U/ml)

90

40

B

80 25°C

70

30°C

35°C

60 50 40 30 20 10 0 0

5

10

15

20

25

30

35

40

Incubation time (hour)

C

45 oC

50 oC

55 oC

Figure 3. Effect of incubation temperatures on protease production (A): BII-6; (B): BII-4; (C): LII.

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A

B

C

Figure 4. Effect of temperature on enzyme activity (A): BII-6; (B): BII-4; (C): LII.

These were in accordance with the previous result as shown by the thermophilic bacterium EP1001 which produced protease at 45 oC with optimum temperature for the enzyme activity of 75 oC (Wilson & Remigio, 2012), and Bacillus subtilis SH1 produced protease at 30 oC with optimum temperature for the enzyme activity of 50 oC (Ningthoujam & Kshetri, 2010). Other researches also reported the similar result (Salleh et

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al., 1977; Burg et al, 1991; Razak et al., 1993; Choi et al., 1999; Haddar, 2009; Jabeen & Qazi, 2011; Asoodeh & Musaabadi, 2012). This study also showed that both Bacillus licheniformis BII-6 and Bacillus subtilis BII-4 isolated from Banyu Wedang hot spring at temperature of 44.8 oC, were mesophile bacteria. The two isolates produced thermostable protease when cultivated at


A

B

C

Figure 5. Effect of pH on enzyme activity (A): BII-6; (B): BII-4; (C): LII.

35 and 30 oC, eventhough both isolates could survive at temperature up to 55 oC and produce clear zones around the colony on agar plate containing skim milk. The investigation showed that the clear zone around the colony or Proteolitics Indeks (PI) on agar plate can only be used to screen protease producing

bacteria but not to compare the ability of isolates to produce the potential protease as reported by Srinivasan et al. (2009); Ningthoujam & Kshetri (2010); and Bayoumi & Bahobil (2011). The characteristics of potential thermostable protease, signed by its ability to withstand against thermal, was showed by further

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A

Time (minute)

Relative activity (%)

B

Time (minute)

C

Figure 6. Effect of temperatures on enzyme stability (A): BII-6; (B): BII-4; (C): LII.

investigation, i.e by growing the isolates in liquid medium. Isolate LII, which only showed barely clear zone on agar plate (Fig. 1 and Table 2), produced thermostable protease with higher activity compare to others (Fig. 3). Protease produced by LII also showed the highest optimum temperature as well as the stability against thermal (Fig. 5 and Fig. 6).

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CONCLUSION There were 6 protease producer isolates (BII-1, BII2, BII-3, BII-4, BII-6 and LII) obtained from screening using solid MSM supplemented with 1.5% skim milk. Based on 16S-rRNA analysis those isolates were identified as Bacillus licheniformis (BII-1, BII-2 and


BII-6), Bacillus subtilis (BII-3 and BII-4) and Brevibacillus thermoruber (LII). Further screening on three different isolates (BII6, BII-4 and LII) in liquid medium revealed that the LII isolate as the most potential protease producer. The enzyme had characteristics of optimum temperature of 85oC, optimum pH of 9 and stable for up to 100 min at 75oC. REFERENCES Akanbi, T.O., Kamaruzaman, A.L., Abu, B.F., Sheikh, A.H. N., Radu, S., Abdul, M.Y., Manap, and Saari, N. 2010. Highly thermostable extracellular lipase-producing Bacillus strain isolated from a Malaysian hotspring and identified using 16S rRNA gene sequencing. Inter. Food Res. J. 17: 45-53. Altscul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403-410. Asoodeh, A. and Mussaabadi, H.M. 2012. Purification and characterization of a thermostable neuthrophilic metalloprotease from Pseudomonas sp. DR89. Iranian J. of Biotech. 10: 120-128. Bayoumi, R.H. and Bahobil, A.S. 2011. Production of thermoalkaliphilic protease by Shewanella putrefaciens-EGKSA21 under optimal conditions for application in biodetergent technology. J. Basic. Appl. Sci. Res. 1: 95-107. Beg, Q.K., Sahai, V., and Gupta, R. 2003. Statistical media optimization and alkaline protease production from Bacillus mojavensis in a bioreactor. Proc. Biochem. 39: 203-210. Benson, D.A., Karsch-Mizrachi, I., Lipman, D.J., Ostell, J., Rapp, B.A., and Wheeler., D.L. 2003. GenBank. Nucleic Acids Res. 31: 23-27. Bruins, M.E., Janssen, A.E.M., and Boom, R.M. 2001. Thermozymes and their applications. Appl. Biochem. Biotechnol. 90: 155-186. Burg, B.V.D., Enequist, H.G., Haar, M.E.V.D., Eijsink, V.G.H., Stulp, B.K., and Venema, G. 1991. A Highly thermostable neutral protease from Bacillus caldolyticus: cloning and expression of the gene in Bacillus subtilis and characterization of the gene Product. J. of Bact. 4107-4115. Choi, I.G., Bang, W.G., Kim, S.H., and Yu, Y.G. 1999. Extremely thermostable serine-type protease from Aquifex pyrophilus. Molecular cloning, expression and characterization. 274: 881–888. Chu, W.H. 2007. Optimization of extracellular alkaline protease production from species of Bacillus. J. Ind. Microbial Biotechnol. 34: 241-245. Covan, D., Daniel, R., and Morgan, H. 1985. Thermophilic proteases: properties and applications. Trends Biotechnol. 3: 68-72. Covan, D.A. 1997. Thermophilic proteins: stability and function in aqueous and organic solvents. Comp.

Biochem. Physiol., Part A: Mol. Integr. Physiol. 118: 429-438. Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 39: 783791. Gupta, A. and Khare, S.K. 2006. A protease stable in organic solvents from solvent tolerant strain Pseudomonas aeruginosa. Biores. Technol. 97: 1788-1793. Hadaar, A. 2009. Two detergent stable serine-protease from Bacillus mojavensis A21: purification, charactecrization, and potential application as a laundry detergent additive. Biores. Technol. 100: 3366-3373. Higgins, D., Blastby, A., Fuchs, R., and Clustal, V. 1992. Improve software for multiple sequence analysis. Comput. Appl. Biosci. 8: 88-192. Jabeen, F. and Qazi, J.I. 2011.Production and characterization detergent compatible thermostable alkaline protease from Bacillus cereus FJ10. J. of Sci. & Indust. Res. 70: 1042-1048. Kumar, D., Gajju, H., and Bhalla, T.C. 2002. Production of a thermostable protease by Bacillus sp. APR-4. Asian J. Microbiol. Biotechnol. Env. Sci. 4: 533-540. Mário, L.T., Florencia, C., Juliana, S., and Adriano, B. 2009. Purification and characterization of a peptide from Bacillus licheniformis showing dual antimicrobial and emulsifying activities. Food Research International. 42(1): 63-68. Ningthoujam, D.S. and Kshetri, P. 2010. A Thermostable Alkaline Protease from a moderately Haloalkalithermotolerant Bacillus Subtilis Strain SH1. Aust J. of Bas. & App. Sci. 10: 5126-5134. Pakpahan, S. 2009. Isolasi Bakteri dan Uji Aktivitas Protease Thermofilik dari Sumeber Air Panas Sipoholon, Tapanuli Utara, Sumatera Utara. Thesis. Sekolah Pascasarjana. Universitas Sumatera Utara. Rao, M.B., Tanksale, A.M., Mohini, S.G., and Deshpande, V.V. 1998. Molecular and biotechnological aspects of microbial proteases. Microbiol. and Mol. Biol. Rev. 62: 597-602. Razak, C., Samad, M., Basri, M., Yunus, W., Ampon, K., and Salleh, A. 1993. Thermostable extracellular protease by B. stearothermophilus. World J. Microbiol. Biotechnol. 10: 260–263. Rohlf, F.J. 1993. NTSYS. PC. Numerical Taxonomy and Multivariated Analysis System, Version 1.8. Applied Biostatistics. New York. NY. Saitou, N. and Nei, M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406–425. Salleh, A.B., Basri, M., and Razak, C. 1977. The effect of temperature on the protease from Bacillus stearothermophilus strain F1. Mal. J. Biochem. Mol. Biol. 2: 37–41. Sellek, G.A. and Chaudhuri, J.B. 1998. Biocatalysis in organic media using enzymes from extremophiles. Enzymes Microb. Technol. 25: 471-482.

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Sokal, R.R. and Sneath, P.H.A. 1963. Principles of Numerical Taxonomy. Freeman. San Francisco. pp. 181-185. Srinivasan, T.R., Soumen Das, Balakrishnan, V., Philip, R., and Kannan, N. 2009. Isolation and characterization of thermostbale protease producing bacteria from tannery industry effluent. Recent Research in Science and Technology. 1: 063–066. Synowiecki, J. 2008. Thermostable enzymes in food processing, in Recent Research Developments in Food Biotechnology. Enzymes as Additives or Processing Aids, Research Signpost, Kerala. Takami, H., Akiba, T., and Horikoshi, K. 1989. Production of extremely thermostable alkaline protease from Bacillus sp. No. AH-101. Applied Microbiology and Biotechnology. 30: 120-124.

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Tambekar, D.H., Kalikar, M.V., Shinde, R.S., Vanjari, L.B., and Pawar, R.G. 2009. Isolation and characterization of multilple enzyme producer Bacillus species from saline belt of Purna river. J. Appl. Sci. Res. 5: 10641066. Turk, B. 2006. Targeting proteases: successes, failures and future prospects. Nat. Rev. Drug Discov. 5: 785798. Van der Peer, Y. and De Wachter, R. 1994. TREECON for Wondows: a software package for construction and drawing of evolutionary trees for Microsoft Windows Environment. Comput. Appl. Biosci. 10: 570-596. Wilson, P. and Remigio, Z. 2012. Production and characterisation of protease enzyme produced by a novel moderate thermophilic bacterium (EP1001) isolated from an alkaline hot spring, Zimbabwe. Af. J. of Microb. Res. 27: 5542-5551.

Screening and Characterization ..................(T. Yulianti, E. Chasanah, and U. S. Friend Tambunan)

SCREENING AND CHARACTERIZATION OF L-GLUTAMINASE PRODUCED BY BACTERIA ISOLATED FROM SANGIHE TALAUD SEA Penapisan dan Karakterisasi L-Glutaminase yang Diproduksi oleh Bakteri dari Perairan Sangihe Talaud Tanti Yulianti3), Ekowati Chasanah1)*, and Usman Sumo Friend Tambunan2) 1

Research and Development Center for Marine and Fisheries Product Processing and Biotechnology 2 Department of Chemistry, Faculty of Mathematics and Science, Universitas Indonesia 3 National Agency of Drug and Food Control * Corresponding author: [email protected]. KS. Tubun Petamburan VI Jakarta Pusat 10260

ABSTRACT L-glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) is a very important enzyme due to its role as flavor enhancer and antileukemic agent. Salt-tolerant L-glutaminase produced by marine bacteria is favorable in food industries. This study describes the screening of Lglutaminase producing marine bacteria from Sangihe-Talaud Sea, North Sulawesi, Indonesia. Screening of L-glutaminase was performed using a liquid medium and identification of selected isolate was performed using molecular-based 16S rDNA. Results showed that there were 7 isolates produced positive results of L-glutaminase, and one of them (II.1 isolate) produced the highest activity, i.e 147.99 U/L, equivalent to the specific activity of 62.32 U/mg. The isolate then selected for further study. Bacterial identification based on 16S rRNA sequencing has revealed that the isolate was 96% similar to Pseudomonas aeruginosa strain CG-T8. Characterization of extracellular L-glutaminase from the II.1 isolate showed that the enzyme worked optimally at temperature of 37-45 °C and pH 7. The enzyme was stable when NaCl solution was added up to 8% and began to decrease on addition of NaCl solution of 16% and 20% with relative activity of 79% and 74%, respectively. The effect of metal ions, e.g Mn2+, Mg2+, and Co2+ in the form of chloride salt, were able to increase enzyme activity, whereas the addition of other metal ions (Zn2+, Fe3+, and Ca2+) decreased the activity. The molecular weights of L-glutaminase was estimated around 42 kDa and 145 kDa. Keywords: L-glutaminase, marine bacteria, 16S rRNA, screening, characterization

ABSTRAK L-glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) merupakan enzim yang sangat penting karena perannya sebagai penghasil flavor dan anti leukimia. L-glutaminase yang tahan garam yang diproduksi oleh bakteri laut sangat diharapkan oleh industri pangan. Penelitian ini bertujuan untuk melakukan penapisan bakteri laut penghasil enzim dari perairan Sangihe-Talaud, Sulawesi Utara, Indonesia. Penapisan dilakukan dengan menggunakan medium cair, dan identifikasi bakteri penghasil enzim dilakukan secara molekuler menggunakan 16S rDNA. Hasil penelitian memperlihatkan bahwa dari 7 isolat yang positif L-glutaminase, 1 isolat (isolat II.1) menghasilkan aktivitas enzim tertinggi yaitu 147,99 U/L, setara dengan aktifitas spesifik 62,32 U/mg. Isolat ini selanjutnya dipelajari lebih lanjut. Berdasarkan identifikasi molekuler, isolat ini memiliki kemiripan 96% dengan Pseudomonas aeruginosa strain CG-T8. Hasil karakterisasi enzim menunjukkan bahwa enzim ini bekerja optimal pada suhu 37-45 °C and pH 7. Enzim stabil ketika dilakukan penambahan NaCl sampai dengan 8% dan mulai berkurang ketika penambahan mencapai 16 dan 20%. Penambahan ion logam Mn2+, Mg2+, and Co2+ dalam bentuk garam klorida mampu meningkatkan kinerja enzim sementara penambahan ion Zn2+, Fe3+, and Ca2+ mengurangi aktivitas enzim. Enzim ini diperkirakan memiliki berat molekul 42 kDa dan 145 kDa Kata kunci: L-glutaminase, bakteri laut, 16S rRNA, penapisan, karakterisasi

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INTRODUCTION L-glutaminase (L-glutamine amidohydrolase, EC.3.5.1.2) is an enzyme that catalyzes the hydrolysis of L-glutamine into L-glutamic acid and ammonia. This enzyme is getting popular due to its important role and its applications in both pharmaceuticals and food industries (Nandakumara et al., 2003). L-glutaminase is proposed as an enzyme therapy for cancer, especially for acute lymphocytic leukemia, in combination or as an alternative to L-asparaginase (Siddalingeshwara et al., 2010; Abdallah et al., 2012). The mechanism of Lglutaminase as anticarcinogenic correlates with glutamine depletion due to inability of the lymphatic tumor cell to synthesize glutamine as the normal cells. Glutaminase is also taking an important role that controls the delicious taste of fermented foods such as soy sauce and in general food products by increasing the glutamic acid content therefore, this enzyme has attracted a great attention in food industries (Moriguchi et al., 1994; Alexandra et al., 2003). Microbes i.e bacteria, fungi and yeast have been reported as potential L-glutaminase sources, and using that microbies as enzyme producers is more preferable due to their simple growth requirements, easy processing and handling as well as cheaper production cost. Reports showed that the majority of microbes producing L-glutaminase have been isolated from soil and aquatic (marine) environment (Nandakumara et al., 2003). Marine environment is expected as a unique L-glutaminase sources such as salt-tolerant and thermo-stable L- glutaminase which is needed by food industries ( Desmond,1997; Prakash et al., 2009). Screening of L-glutaminase from marine bacteria had been reported by Padma & Singhal (2009), from marine Actinomycetes by Balagurunathan et al. (2010), and marine fungi by Sabu (1999). However, information on the L-glutaminase from Indonesia Sea is very rare. The objective of this present study was to isolate, screen and identify marine isolates producing L-glutaminase from Sangihe Talaud Sea, Indonesia. Characterization of the enzyme (crude enzyme) was performed from the best isolate producing the highest activity of L-glutaminase.

during INDEX SATAL 2010 Cruise on R/V Baruna Jaya IV. The sample was kept cool until reaching laboratory. In the laboratory, 1 ml of sample was taken from homogenized samples and initially added in to 20 ml liquid modified Zobell’s media containing 1 g/L yeast extract and 5 g/L peptone dissolved in artificial seawater (ASW). For qualitative screening, 1 ml of enriched media containing the sample were grown in 100 ml flasks containing 20 ml media and put on water bath shaker at 100 rpm maintained at 30 °C for 48 h.The medium contained 5.0 g/L glucosa, 5.0 g/L glutamine, 6.0 g/L Na2HPO4.2H2O, 3.0 g/L KH2PO4, 0.49 g/L MgSO4, 0.05 g/L NaCl, 0.002 g/L CaCl2 and 0.06 ml of 2.5% w/v ethanolic phenol red solution with pH adjusted to 7-8 (Padma & Singhal, 2009). The sample that showed positive result was indicated by the color changes of media, and was further taken up for quantitative screening. Quantitative Screening for L-glutaminase 1 ml of enriched media containing the positive samples from qualitative screening were grown by streaking in media containing 5.0 g/L glucose, 5.0 g/ L glutamine, 1,0 g/Lyeast extract, 6.0 g/L Na2HPO4. 2H2O, 3.0 g/L KH2PO4, 0.49 g/L MgSO4, 0.05 g/L NaCl, and 0.002 g/L CaCl2 with pH adjusted to 7-7.5 for 48 h at 100 rpm at 30 °C. The activity of extracellular L-glutaminase was estimated by spectrophotometer using Nessler reagent as described in analytical determination (Imada et al., 1973). 16S rRNA Sequencing Analysis of Selected Isolate DNA extraction was performed using commercial extraction of DNA. DNA amplification by PCR was performed using F Primer (5’-CAGGCCT AACA CAGGCAAGTC-3’) and R primer (5’-GGGCG GWG TGTACAAGGC-3’). Sequencing of amplification product of 16S rRNA gene was conducted at 1st base life science in Singapore. The sequence obtained was initially analyzed at National Center for Biotechnology Information (NCBI) server (http://www.ncbi.nlm.nih.gov) using Basic Local Alignment Search Tool (Blast) and phylogenetic tree was constructed to identify the isolate (Marchesi et al., 1998). Growth Curve and Enzyme Production

MATERIALS AND METHODS Qualitative Screening and Isolation of L-glutaminase Bacteria Samples (seawater) were collected by water samplers attached to CTD from 4 m until 1500 m depth of Sangihe-Talaud Sea in North Sulawesi, Indonesia

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Growth curve of the isolate producing the enzyme was determined by inoculating 1 loopfull of bacteria in L-glutaminase media, composing of 5.0 g/L glucosa, 1.0 g/L yeast extract, 6.0 g/L Na2HPO4.2H2O, 3.0 g/L KH2PO4, 0.49 g/L MgSO4, 0.05 g/L NaCl, 0.002 g/L CaCl2. Absorbancy at 600 nm was recorded every hour during the first 4 h, followed by every 4 h. To obtain enzyme production profile, measurement of enzyme


activity was conducted following growth curve sampling. Preliminary study on the production of Lglutaminase was conducted by optimizing fermentation condition, i.e cultivation temperature of 25-35 °C, shaker speed of 80 and 100 rpm, and inoculum volume of 5% and 10%. The best result of the preliminary study was inoculum cultivation of 5%, and 10% then used to produce L-glutaminase (Padma & Singhal, 2009).

was 20 to 60 °C. Optimal pH determination was carried out by using buffer solution ranging from 5 to 10, i.e 6-8 using phosphate buffer, 8-9 using Tris-HCl buffer, and 9-10 using carbonate-bicarbonate buffer. Effect of salt on L-glutaminase activity was performed using addition of 0% to 20% NaCl. The effect of metal ions on the L-glutaminase activity on crude extracellular L-glutaminase were measured by similar L-glutaminase assays. SDS-PAGE

Measurement of L-glutaminase Activity For estimation of L-glutaminase activity, a method developed by Imada et al. (1973) was used. The Lglutaminase produced in fermentation broth converts L-glutamine to L-glutamic acid and release ammonia. This ammonia then quantified by spectrophotometry using Nessler reagent. The reaction mixture contained 0.5 ml of crude enzyme extract, 0.5 ml of 0.04 M Lglutamine, 0.5 ml of 0.5 M Tris-Cl buffer pH 8.4, and 0.5 ml distilled water. The reaction, at 37 °C for 30 min, was terminated by adding 0.5 ml of 1.5 M TCA. Then, 0.2 ml Nessler reagent was added to 0.1 ml reaction mixture and made up to 4 ml using distilled water. The absorbance of the mixture was measured at 450 nm after 20 min incubation at room temperature. One unit of L-glutaminase activity was defined as enzyme required for releasing 1.0 ì mol ammonia per min (Imada et al., 1973). The protein concentration was determined by the Bradford method using bovine serum albumin (BSA) as the standard. Characterization of Crude Extracellular L-glutaminase To determine the optimal temperature, the temperature ranges used to react the enzyme mixture

Determination of the molecular weight of Lglutaminase, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed by using 12% polyacrylamide gel and stained with silver staining (Sabu,1999). RESULTS AND DISCUSSION Qualitative Screening and Isolation of L-glutaminase Producers Four samples of water collected from 4 m to 1000 m depth gave positif result of L-glutaminase (Table 1) qualitatively, where as the 3 other samples collected from 1250-1500 m showed negative result. From the four samples, 13 isolates have been succesfully isolated and was taken into further screening quantitatively. The screening study showed that approximately 7 of the 13 isolates gave positive result of L-glutaminase. Among the 7 isolates, the isolate II.1 gave the highest L-glutaminase activity of 147.99 Unit/L or 62.32 Unit/mg of specific activity. This isolate which was taken from 400 m depth of Sangihe-Talaud Sea, was then selected for further study.

Table 1. Screening of L-glutaminase isolates from 4 different depths of Sangihe Talaud Sea

Isolate

Depth (m)

Qualitative

Assay (Unit/L)

I.1 I.2 I.3 I.4 II.1 II.2

4 4 4 4 400 400

+ + + +

36,39 54,59 147,99 81,27

II.3 III.1 III.2 IV.1 IV.2

400 700 700 1000 1000

+ -

N.D -

IV.3 1000 IV.4 1000 N.D : not detected

+ +

42,46 16,98

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16S rRNA Sequencing Analysis of Selected Isolate The sequence homology studies showed the isolate II.1 had 96.0% similar to Pseudomonas aeruginosa strain CG-T8 (P. aeruginosa GU 339295.1). By comparing differences in the nucleic acid sequences of homologous genes from different organisms, a molecular phylogenetic tree was then constructed. The distance between the two sequences is an indicative of their relatedness to each other. Figure 2 showed the phylogenetic tree showing that our isolate had the same distance of relatedness with P. aeruginosa strain CG-T8 (P. aeruginosa GU 339295.1) and Pseudomonas sp. HO 609591.1. Molecular identification based on 16S rDNA sequencing proposed the isolate as P. aeruginosa strain CT-G8 with 96% similarity. Genus Pseudomonas sp. has already known as a source of microbial glutaminase, along with other bacteria, fungi and yeast which are mostly isolated from soil, and it was produced extracellularly. Review on microbial Lglutaminase showed that Pseudomonas specifically P. aeruginosa, P. aurantiaca, P. aureofaciens, P. boreoplois, P. fluorescens, P. ovalis, P. schuylkilliensis and Pseudomonas sp. have secreted L-glutaminase (Nandakumar et al., 2003). Growth Curve of the Isolate II.1 and Production of the Enzyme Figure 3 showed growth curve of the isolate, showing that the isolate reach logaritmic phase after

3 h cultivation up to 10 h cultivation, following stationary phase up to 21 h. L-glutaminase enzyme was produced since 9 h cultivation and attained peak production at 22 h cultivation, which was in stationary phase of Isolate II.1 growth. From this data, this enzyme might be produced as secondary metabolites. Preliminary study on optimizing the cultivation condition of L-glutaminase production was conducted by modified cultivation method of Padma et al. (2009). Figure 4 (A,B,C,D) shows the optimization results demonstrating that the extracellular L-glutaminase from the II.1 isolate was maximally produced when the isolate cultivated at 30 oC, in media having pH 6, using volume inoculum of 5% and at shaking speed of 100 rpm. Based on the growth curve (Figure 3), the inoculum used was the 8-12 h old isolate having 0,650,75 Optical Density (OD) at 600 nm which was equal to 59x107–69x107CFU/ml of the inoculum used. Characteristics of L-glutaminase Enzyme The crude extracellular L-glutaminase produced by Pseudomonas aeruginosa strain CG-T8- II.1. performed optimally at pH 7,0 and stable at 37-45 °C Figure 5. A,B). The enzyme could tolerate NaCl concentration up to 16% and 20%, and loosing the activity by 21% and 25.88%, respectively. The effect of metal ions Mg2+, Co2+ and Mn2+as Cl2 salt increased the activity, while addition of Ca 2+ , Fe 3+ , and Zn2+reduced the activity (Figure 6. A,B). Al Hammed & Jassim (2011) reported that the activity of L-glutaminase from Serratia plymuthica was

Figure 2. Phylogenetic tree of Isolate II.1

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Legend :

(Enzyme Product)

(Growth Curve)

Figure 3. Growth curve of II.1 isolate and it’s enzyme production raised by addition of tetracycline and chloramphenicol with increasing specific activity of glutaminase up to 10.8 U/mg and 15.31 U/mg respectively, in comparison with the control of 3.1 U/mg. Salt tolerance L-glutaminase was important for its application, especially for food industry needs. It was reported that marine isolates M. luteus K-3 and B. subtilis were shown to be highly salt-tolerant up to 16 and 25% of NaCl, respectively. Isolate II.1 produced

quite stable L-glutaminase. However, the relative activity of the enzyme decreased by 21% when added with 16% NaCl solution. Result of SDS-PAGE of the enzyme showed the occurence of two (2) protein bands of 42 kDa and 145 kDa in all samples, i.e crude enzyme, ultrafiltration fraction and amonium sulphate fraction. It was assumed that the bands represent the sub unit and native enzyme from glutaminase class A . As mentioned

A

B

C

D

Figure 4. Production of the enzyme at different treatment: (A) incubation temperature; (B) mixing speed; (C) pH media; (D) 2 (two) different concentration of inoculum.

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B

A

Figure 5. Optimum temperature (A) and pH (B) of L-glutaminase produced by Pseudomonas aeruginosa strain CG-T8- II.1.

A

B

Figure 6. Effect of NaCl (A) and metal ion as chloride salt (B) on the enzyme’s activity.

in the literature glutaminase class A having molecular weight around 35 kDa and 137 kDa (Nandakumara et al., 2003). CONCLUSION L-glutaminase producing marine bacteria was isolated from Sangihe-Talaud Sea, North SulawesiIndonesia from the depth 400 m with the activity of 147.99 Unit/L or 62.32 Unit/mg. Identification using 16S rRNA sequencing revealed the isolate has 96% similarity to Pseudomonas aerginosa strain CT-G8. L-glutaminase has been produced maximally in fermentation condition of 30 °C, 100 rpm, pH of media 6,0, and with starter inoculum of 5%. The crude enzyme can perform optimally at 37- 45 °C, pH 7.0 and retain >50% relative activity when added with NaCl up to 20%. Addition of metal ions Mg2+, Co2+, and Mn2+ in the form of Cl2+ increased the enzyme activity, while addition of other metal ions (Ca2+, Fe3+, and Zn2+) decreased the enzyme activity. Result of SDS-PAGE revealed that the L-glutaminase might approximately had molecular mass of 42 and 145 kDa.

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REFERENCES Abdallah, N.A., Amer S.K., and Habeeb, M.K. 2012. Screening of L-Glutaminase Produced By Actinomycetes Isolated From Different Soils In Egypt. International Journal of ChemTech Research. 4(4): 1451-1460. Alexandra, W.Z., Christiane, G. D., Affolter, M. 2003. Functional Characterization of a Salt-and Thermotolerant Glutaminase from Lactobacillus rhamnosus. Enzyme and Microbial Technology. 32: 862-867. Al Hammed, A.M.A. and Jassim, I.M. 2011. Effect of the antibiotics on Glutaminase purified from Serratia plymuthica isolated from diyala rive. Diyala Agric. Sci. Journal. 3(1): 22-33. Balagurunathan, R., Radhakrisman, M., and Somasundaram, S.T. 2010. L-glutaminase Producing Actinomycetes from Marine SedimentsSelective Isolation, Semi Quantitative Assay and Characterization of Potential Strain. Australian Journal of Basic and Applied Sciences. 4(5): 698-705. David, A. 2004. Significance of Bacterial Identification by Molecular Biology Methods. Endodontic topics. 9:514.


David, R.W. and Craig, B.T. 2010. Glutamine addiction: a new therapeutic target in cancer. USA Trends in Biochemical Sciences. 35: 427–433. Desmond, K. O’toole. 1997. The Role of Microorganisms in Soy Sauce Production. Advances in Applied Microbiology. 45: 87-152. Imada, A.I., Nakahama, K., and Isono, M. 1973. Asparaginase and Glutaminase Activities of Microorganism. Journal of General Microbiology. 76: 8599 Marchesi, J.R., Sato, T., Weigtman, A.J., Martin, T.A., Fry, J.C., Hiom, S.J., Dymock, D., and Wades, W.G. 1998. Design and Evaluation of Useful Bacterium-Specific PCR Primers That Amplify Genes Coding for Bacterial 16S rRNA. Appl. Environ. Microbiol. 64: 795-799. Nandakumara, R., Yoshimune, K., Wakayama, M., and Moriguchi, M. 2003. Review Microbial glutaminase: biochemistry, molecular approaches and applications in the food industry. Journal of Molecular Catalysis B: Enzymatic. 23: 87–100. Origuchi, M., Sakai, K., Tateyama, R., Furuta, Y., and Wakayama, M. 1994. Isolation and Characterization of Salt-Tolerant Glutaminases from Marine

Micrococcus luteus K-3. Journal of Fermentation and Bioengineering. 77(6): 621-625 Padma, V., Iyer, and Rekha, S.S. 2009. Screening and Selection of Marine Isolate for L-Glutaminase Production and Media Optimization Using Response Surface Methodology. Appl. Biochem. Biotechnol. 159: 233-250. Prakash, P.J.E., Poorani, P., Anantharaman, and Balasubramaniam, T. 2009. L-glutaminase Production and the Growth of Marine Bacteria. Research Journal of Microbiol. 4: 168-172. Sathish, T. and Prakasham, R. S. 2010. Isolation an Identification of L-glutaminase an Antileukemic Enzyme Producing Micro-organism from Godavari River Bank Soils in Andhra Pradesh. International Research Journal of Pharmacy. (1): 367-373. Sabu, A. 1999. L-glutaminase Production by Marine Fungi, Thesis. Department of Biotechnology COCHIN University of Science and technology. 682 022 : 168. Siddalingeshware, K.G., Dhatri, D.N., Pramada, T., Vishwanata, T., Sudipta, K.M., and Mohsin, S.M. 2010. Rapid Screening and Confirmation of L-Glutaminase producing novel Aspergillus wentii. International Journal of Chem.Tech. Research. 2(2): 830-833.

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Effect of Chopping Step and Drying......................(S. Wibowo, M. Darmawan, A. R. Hakim, and S. Marsella)

EFFECT OF CHOPPING STEP AND DRYING TECHNIQUE ON THE QUALITY OF ALKALI TREATED COTTONII (ATC) Pengaruh Tahap Pencacahan dan Teknik Pengeringan terhadap Mutu Alkali Treated Cottonii (ATC) Singgih Wibowo1)*, Muhamad Darmawan1), Arif Rahman Hakim1), Seruni Marsella2) 1)

Research and Development Center for Marine and Fisheries Product Processing and Biotechnology 2) Swiss German University, Serpong, Tangerang *Corresponding author: [email protected]. KS. Tubun Petamburan VI, Jakarta Pusat 10260

ABSTRACT A research related to the production of alkali treated cottonii (ATC) had been carried out in order to study the effect of chopping step and drying techniques on the quality of ATC produced. Four treatments were applied in the experiment, namely was copping before sun drying (treatment A); chopping after sun drying (B); chopping before mechanical drying (C); and chopping after mechanical drying (D). The quality parameters of ATC measured were gel strength, moisture content, viscosity, yield, and whiteness. The results showed that the quality of ATC was significantly affected by chopping step and drying technique, especially in ATC gel strength, viscosity and yield. However, the effect of chopping step and drying technique was insignificant to ATC moisture content and whiteness. Chopping seaweed before drying resulted in higher gel strength of the ATC but lower in yield, while chopping after drying tended to result in lower gel strength but higher viscosity and yield. Keywords: drying technique, alkali treated cottonii (ATC), chopping step, quality ABSTRAK Penelitian yang berkaitan dengan produksi alkali treated cottonii (ATC) telah dilakukan untuk mempelajari pengaruh tahapan pencacahan rumput laut dan cara pengeringan terhadap mutu ATC yang dihasilkan. Empat perlakuan digunakan di dalam penelitian ini, yaitu pencacahan sebelum pengeringan dengan penjemuran (perlakuan A); pencacahan setelah pengeringan dengan penjemuran (B); pencacahan sebelum pengeringan dengan pengering mekanis (C); dan pencacahan setelah pengeringan dengan pengering mekanis (D). Parameter mutu yang diamati adalah kekuatan gel, kadar air, viskositas, rendemen, dan derajat keputihan ATC yang dihasilkan. Hasil analisis menunjukkan bahwa mutu ATC dipengaruhi oleh tahapan pencacahan dan cara pengeringan, terutama terhadap kekuatan gel, viskositas, dan rendemen. Pencacahan rumput laut sebelum penjemuran menghasilkan ATC dengan kekuatan gel tinggi tetapi viskositas dan rendemen lebih rendah, sedangkan pencacahan setelah pengeringan menghasilkan kekuatan gel yang lebih rendah tetapi dengan viskositas dan rendemen yang tinggi. Kata Kunci: teknik pengeringan, Alkali Treated Cottonii (ATC), tahap pencacahan, mutu

INTRODUCTION

. Seaweed culture in Indonesia has been well grown

rapidly in accordance with the increasing demand of seaweed from importing countries. Indonesia has targeted to produce 10 million tons of wet seaweed by 2015 (Antara, 2011) equal to approximately 1 million of dry seaweed. Eucheuma cottonii is one of seaweed species cultivated in Indonesia, especially in the eastern part. This type of seaweed is in high demand in the market. The demand for export for E. cottonii in

2002 was very high, reaching 559,888,073 kg which is usually used to produce carrageenan. Carrageenan is a natural linear polysaccharide extracted from red seaweeds (Gigartinales, Rhodophyta) and is extensively used as thickeners, gelling, texturizing, suspending or stabilising agents in various industrial applications ranging from food products to pharmaceutical products and even for chemical industries purposes (Piculell, 1995; Bixler, 1996). In the food industry, carrageenans are widely utilized due to their excellent physical functional

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properties, such as thickening, gelling and stabilizing abilities, and have been used to improve the texture of cottage cheese, to control the viscosity and texture of puddings and dairy desserts, and as binders and stabilizers in the meat-processing industry for the manufacture of patties, sausages and low-fat hamburgers. The food industry accounts for 70–80% of the total world production, estimated at about 45,000 metric tonnes per year, of which about 45% goes to dairy products and 30% to meat and meat derivatives. The total market of carrageenans has been estimated as US $300 million/year (McHugh, 2003). Carrageenans are also used in various non-food products, such as pharmaceutical, cosmetics, printing and textile formulations (Imeson, 2000). Carrageenans stabilize toothpaste preparations, absorb body fluids when formulated in wound dressings and interact with human carotene to give soft skin and silky hair in hand lotions and shampoos, respectively. They have proved to be useful as tableting excipients due to the good compatibility, high robustness and persistent viscoelasticity of the tablet during compression. These interesting properties indicated that carrageenans are suitable excipients for sustained-release formulations (Bhardwaj et al., 2000). E.cottonii can be preserved into alkaline treated cottonii (ATC), or processed into semi refined carrageenan (SRC) and refined carrageenan (RC) which are different quality in term of purity. The purer the quality of carrageenan the higher the market value will be. Marketing of E. cottonii in dried form will result in low revenue while processing it into a purer carrageenan such as RC will produce higher revenue with the consequences of requiring more complicated technology as well as higher cost of production. Processing the seaweed into ATC, a chips of treated cottonii, will require simpler technology and cheaper in cost production compared to RC and is expected more suitable for Indonesia. The solution of potassium hydroxide (KOH) at high temperature is employed to reduce the sulphate content in the seaweed. In such process, cation of K+ dissociated from KOH will affect the gel strength of ATC which is usually used as an important quality parameter. Producing high quality of ATC will also influence by the drying process which is expected to produce 1214% of moisture content. The chopping step, whether performed before or after drying, will probably affect the quality of ATC, especially the gel strength. The aim of this research is to study the effect of chopping step and drying techniques on the quality of ATC produced.

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MATERIALS AND METHODS Seaweed Materials Seaweed used in this research was purchased from the collector located in Bulak Kapal, Tabanan, Bali in April 2011. The seaweed was sorted to remove filth or any non-algal materials such as rope, sand and other materials found in the seaweed. This process is also aim to remove other seaweed species than E. cottonii. The E. cottonii was then packed into plastic bag and transported to the Processing Laboratory of Research and Development Center for Marine and Fisheries Product Processing and Biotechnology in Jakarta. Processing of ATC To produce ATC, sorted E. cottonii was washed and then boiled in KOH solution. The alkali treated seaweed was then dried. Chopping seaweed could be performed before drying or after drying. As much as 50 kg of dried seaweed was washed by using washing machine which was operated at 8 rpm for 20 min. The clean seaweed was drained and boiled in the solution of 8% KOH at 80oC for 2 hours. The treated seaweed was rinsed until the water pH was neutral or rinsed for at least 3 times and drained. The batch of treated seaweed was then equally divided into 4 parts for the experiment. The experiment was designed to apply drying techniques (sun drying and mechanical drying) and chopping steps (chopping before drying and after drying). Based on the design, the treatments employed in this experiment were set as follow. A: Chopping before sun drying B: Chopping after sun drying C: Chopping before mechanical drying D: Chopping after mechanical drying In the treatment A, the drained alkali treated seaweed was chopped into wet chips which were then dried by using sun drying technique. In the treatment B, the drained alkali treated seaweed was dried by using sun drying and then chopped into chips. Treatment C was the same as treatment A, but the chopped seaweed was dried by using mechanical drier. Treatment D was the same as treatment B, but the drying technique used was mechanical drying. Parameter of analysis Several characteristics of raw materials, namely moisture content, acid insoluble ash content, clean anhydrous weight (CAW) and sand content (filth) were


Table 1. Characteristics of seaweed as raw material Parameters

Value

Moisture content (%)

56.83 ± 0.97

Ash content (%)

29.76 ± 1.07

Acid insoluble ash content (%)

0.25 ± 0.10

CAW (%)

36.97 ± 1.75

Sand content (%)

1.09 ± 0.26

evaluated. The quality parameters of ATC evaluated were gel strength (Marine Colloids, 1978), moisture content AOAC, 1995), viscosity (Marine Colloids, 1977), yield (Marine Colloids, 1978), and whiteness (Saito, 2004). In this experiment, the sampling for analysis was taken randomly with 3 replications and the statistical analysis was performed by using SPSS 15. RESULTS AND DISCUSSION Characteristic of Raw Material Results of the analysis on the characteristic of seaweed collected from Bali used as raw material in this experiment are shown in Table 1. According to Table 1, moisture content of seaweed used for the experiment was 56.83%. Considering that standard of moisture content issued by FAO for dried E. cottonii is less than 40% (Anon., 1978), moisture content of the seaweed from Bali was lower. As the lower the moisture content, the smaller the probability of seaweed to be deteriorated and fermented, it means that based on the moisture content, the raw material used in the experiment was good. Meanwhile, the ash content of the seaweed was 29.76%. The ash content in seaweed is commonly originated from sodium salts derived from seawater. Hirao (1971) confirmed that ash content of seaweed is usually 15-40%. The acid insoluble ash of the seaweed, which was quantified as ash that insoluble in hydrochloric acid, was 0.25% that met with the FAO standard of less than 1% (Anon., 1978). Another important parameter for dried seaweed is clean anhydrous weight (CAW) which shows the purity of the seaweed. The CAW of the sample was 36.97% which is higher than FAO standard of less than 30% (Anon., 1978). Meanwhile, the sand content of the seaweed was 1.09%. This high CAW and sand content indicated that the seaweed used in this experiment was dried improperly. Proper drying of seaweed by using a para-para or on the covered ground (Aji et al., 2003) is required to produce a good quality of dried seaweed.

Gel strength The gel strength of ATC in this experiment is presented in Figure 1. Gel strength is one of the very important parameter for ATC which is commonly used as main requisite by ATC industries. The gel strength of ATC resulted from all treatments ranged from 430.58 g/cm2 to 1,136.83 g/cm2. Figure 1 shows that all gel strength results were within the standard of FAO which is required to be larger than 400 g/cm2 (Anon., 1978). Statistically, gel strength of ATC resulted from both drying techniques was significantly different (p < 0.05). The highest value was obtained by treatment A (chopping before sun-drying) and the lowest was obtained by treatment D (chopping after mechanical drying). As shown in Figure 1, it seems that gel strength of ATC resulted from all treatments was very much affected by the chopping step. Chopping the seaweed before sun-drying (treatment A) resulted in higher gel strength of ATC compared to chopping after sun-drying (treatment B). Similarly, chopping before mechanical drying (treatment C) resulted in higher gel strength of ATC compared to chopping after mechanical drying (treatment D). These findings indicate that chopping before drying will be able to result in higher gel strength of ATC. In other word, it can be said that chopping should be performed before drying in order to produce ATC with high gel strength. The gel strength is basically affected by temperature of processing. Meanwhile, chopping could increase the temperature of seaweed, particularly when the seaweed is dry. Chopping the seaweed before drying when the seaweed still is wet might be effective on eliminating the increasing temperature during the chopping. The presence of moisture in the wet seaweed when was chopped might played an important role in compensating the increasing temperature. Consequently, it will produce high gel strength of ATC. Several factors may affect the gel strength value. Gel strength of ATC increases proportionally to the 36-anhydrogalactose content but decreases

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Figure 1. Effect of chopping step and drying technique on the gel strength of ATC. proportionally to the sulphate content (Suryaningrum, 1988). The conversion of C-6 sulphate to 3-6anhydrogalactose is clearly creating new strong component. The 3-6-anhydrogalactose causes the anhydrophilic behavior and increases the formation of double helix which is in turn will yield in high gel strength. Other factors that influence the high gel strength of ATC are raw material condition, age of cultivation, method of extraction and type of chemical used for extraction (Samsuari, 2006). Moisture content The moisture content of ATC resulted in this experiment as presented in Figure 2 was ranged from 11.54% to 15.06%. Figure 2 showed that highest moisture content of ATC was obtained by treatment B (chopping after sun-drying), while lowest moisture

content of ATC was by treatment A (chopping before sun-drying). Statistically, moisture contents of all treatments were significantly different (p < 0.05). However, the moisture content was not significantly affected by chopping step or drying technique. There was no certain pattern that can be drawn from this result. Amongst the results, chopping before sundrying (treatment A) resulted in ATC with moisture content less than the FAO standard of 12% (Anon., 1978). Nonetheless, drying time could be arranged to meet FAO standard for moisture content. Viscosity Other quality parameter of ATC observed in this experiment was viscosity and the result is presented in Figure 3. The viscosity of ATC resulted from the experiment were between 292.50 cPs and 625.50 cPs.

Figure 2. Effect of chopping step and drying technique on moisture content of ATC.

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Figure 3. Effect of chopping step and drying technique on the viscosity of ATC The statistical analysis result showed that there was significant different on viscosity amongst the treatments (p<0.05). As shown in Figure 3, chopping after mechanical drying (treatment D) resulted in ATC with highest viscosity while the lowest viscosity was resulted from chopping before sun-drying (treatment A). Compared to the FAO standard on viscosity requiring viscosity greater than 5 cPs, all ATCs produced were within the standard (Anon., 1978). These viscosities were inversely related to gel strength results (Moirano, 1997) as previously discussed. ATC resulted from treatment A (chopping before sun-drying) showed lowest viscosity (Figure 3) which inversely showed highest gel strength (Figure 1). There are several factors affect the viscosity of hydrocolloids such as ATC. They were the concentration of alkali, extraction temperature, degree of dispersion, sulphate content, treatment given to the

seaweed, and the presence of electrolyte and nonelectrolyte in the system (Saleh et al., 1994). The sulphate groups in the polymers chain have negative charges causing the repulsion along polymer chain and rigidness of polymer chain (Guiseley et al., 1980). The viscosity will affect the gelling point and melting point of the hydrocolloids. High viscosity of carragenan solution, for example, produces rapid melting point in contrast to low viscosity that causes gel formation. Yield Other important parameter of ATC quality observed was the yield as presented in Figure 4. As in Figure 4, the yield of ATC resulted from the treatment were between 31% and 36.10%. Statistically, the result showed that yield of ATC was significantly affected by both chopping steps and drying technique (p<0,05). The Figure shows a tendency that chopping step

Figure 4. Effect of chopping step and drying technique on the yield of ATC.

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affected more to the yield of ATC compare to drying technique. The yield of ATC resulted from sun drying was lower than mechanical drying for both chopping before and after drying. Meanwhile, chopping before drying resulted in lower yield compared to chopping after drying. In the contrary, as shown in Figure1, the gel strength of ATC was higher when the seaweed was chopped before drying. These mean that chopping before drying will result in high gel strength but low in yield. In the contrary, the gel strength of ATC will be lower when seaweed was chopped after drying but the yield was higher. Based on these findings, it can be said that when high gel strength of ATC is an important parameter to be achieved, therefore chopping before drying will be favorable for ATC processing with the consequences of producing low yield of ATC. However, when the gel strength is not the critical requirement for ATC, then, chopping after drying will be more advantageous for ATC processing. Chopping after drying in the ATC processing produces higher yield and consequently will economically be more profitable. Chopping before drying will produce more surface area on the seaweed so then the seaweed has more opportunity to have contact with the alkali in the extraction process. This condition will then increase the melting point of carrageenan allowing it to be extracted out of the cells easily (Widiastuti, 1990). The free carrageenan will then interacted with the sulphate group easily. As a result, a great numbers of 3,6-anhydrogalactose release from the sulphate groups resulting in lower gel strength (Suryaningrum, 1988) and higher yield. However, since yield is also depending on the climate, method of extraction, age or harvesting time and location of cultivation (Chapman & Chapman, 1980), therefore, it is interesting to find out whether the result will be the same for different

seaweed collected from different harvesting time (age) and location of cultivation. Whiteness Other important parameter for ATC quality is degree of whiteness. Figure 6. bellow shows the whiteness result from all treatments. As shown in Figure 6, the highest whiteness of ATC was obtained from chopping before sun drying (58,88%) and the lowest one was from chopping after mechanical drying (50,55%). The result indicated that whiteness of ATC was not significantly affected by the chopping step. The whiteness of ATC was not depending on whether the seaweed chopped before or after drying. Figure 5 showed an indication that the whiteness of the ATC was depending on drying technique used. Sun drying tended to produce whiter ATC compare to mechanical drying did. This finding might related to the role of UV light transmitted by the sun. However, statistically, the different was not significant (p< 0,5). The whiteness of ATC is expected high near to 100% in order to provide more chance to ATC for wider application (Agustine, 2011; Samsuari, 2006). A turbid, vague, blurred or unclear color of ATC will give unattractive color to products. The whiteness of ATC is affected by the raw material, the drying method applied (Agustine, 2011), also heat and concentration of alkali solution used in the process (Saleh, 1994; Antelo et al., 2008). The alkali solution will oxidize the pigment and brighten the seaweed. Chemically, colorant molecules contain double bonds and the process of whitening is an oxidation or reduction process occurred in the double bond (Saleh, 1994). Alteration of this double bond creates a new molecule which does not responsible to color. Moreover, heat treatment as applied in the alkali treatment and drying in the ATC production is

Figure 5. Effect of chopping step and drying technique on the whiteness of ATC.

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able to degrade the pigments in seaweed. Heat which degrades protein chain in the pigment will yield decolorization in seaweed (Antelo et al., 2008). CONCLUSION The quality of ATC was significantly affected by chopping step and drying technique, especially in ATC gel strength, viscosity and yield. The effect of chopping step and drying technique was insignificant to ATC moisture content and whiteness. Chopping before drying (sun or mechanical drying) was favorable for ATC processing producing high gel strength of ATC with the consequences of producing lower yield of ATC. When gel strength of ATC is not the critical requirement for ATC, chopping after drying will economically be more profitable for ATC processing resulting in ATC with higher yield but lower gel strength. REFERENCES BSN. 2006. Cara Pengujian Bakteri TPC. SNI 01-2332.32006. Badan Standardisasi Nasional. Jakarta. Agustine, P. 2011. “Pengaruh Waktu Pemanasan dan Konsentrasi Bahan Pemucat (kaporit) Terhadap Mutu Alkali Treated Cottonii (ATC) dari Rumput Laut.” [thesis]. Faculty of Science and Technology. University of Al Azhar Indonesia, Jakarta. P. 48. Aji, N., Ariyani, F., dan Suryaningrum, T. Pengeringan dan Sortasi. 2003. Teknologi Pemanfaatan Rumput Laut, Pusat Riset Pengolahan Produk dan Sosial Ekonomi Kelautan dan Perikanan. Jakarta. p. 13. Anonymous. 1978. Raw Material Test Laboratory Standard Practice. Marine Colloids Div. Corps. Springfield. USA. pp. 79-92. Antara. 2011. Indonesia produces 10 million tons of seaweed. http://bali.antaranews.com/berita/9613/ indonesia-produksi-rumput-laut-10-juta-ton. accessed june 12. Antelo, F.S., Costa, J.A.V., and Kalil, S.J. 2008. Thermal Degradation Kinetics of the Phycocyanin from Spirulina Plantensis. Biochemistry journal. AOAC. 1995. Official Methods of Analysis of the Association of Official Analitycal Chemist. Inc. Washington DC. pp.185-189. Bhardwaj, T.R., Kanwar, M., Lal, R., and Gupta, A. 2000. Natural gums and modified natural gums as sustained-release carriers. Drug Development and Industrial Pharmacy, 26: pp. 1025–1038. Bixler, H.J. 1996. Recent developments in manufacturing and marketing carrageenan. Hydrobiologia. 326/327: 35–57. Chapman, V.J. and Chapman, D.J. 1980. Seaweeds and Their Uses. Third Edition. Chapman and Hall. London, New York. p. 333.

Guiseley, K.B., Stanley, N.F., and Whitehouse, P.A. 1980. Carrageenan. In: Davids RL (editor). Hand Book of Water Soluble Gums and Resins. Mc Graw Hill Book Company. New York, Toronto, London. pp. 125-142. Hirao, S. 1971. Seaweed in Utilization of Marine Products. In: Osaka, M.; Hirao, S.; Noguchi, E.; Suzuki, T.; and Yokoseki, M. (editors). Overseas Technical Cooperation Agency Goverment of Japan. p. 148. Imeson, A. 2000. Carrageenan. Woodhead Publishing Limited and CRC Press LLC. Marine Colloids. 1977. Carrageenan. Marine Colloid Monograph Number One. Marine Colloids FMC Corp. Springfield, New Jersey. USA. Marine Colloids. 1978. Marine Colloids FMC Corp. “Raw Material Test Laboratory Standart Practise”. Springfield, New Jesey. USA. McHugh, D.J. 2003. A guide to the seaweed industry: FAO fisheries technical paper No. 441 FAO. Rome. pp. 61–72. Moirano AL. 1977. Sulphated Seaweed Polysaccarides. In: Graham HD (editor). Food Colloids the AVI Publishing. Westport. Conn. pp. 347-381. Piculell, L. 1995. Gelling carrageenans. In: A. M. Stephen (editor), Food polysaccharides and their applications. Marcel Dekker. New York. pp. 205–244. Saito, M., Kudo, H., Mandarin, J.M.G., and Benassi, V.T. 2004. Effects of Variety and Cultivating Region on the Color of Soymilk and Other Soybean Processing Foods in Brazil. Japan International Research Center For Agricultural Sciences (JIRCAS) Saleh. M., Herdian, P.D., Suptijah, J., Santoso, dan Indriati, N. 1994. Pengaruh perendaman dalam bahan pemucat terhadap mutu dan rendemen Karaginan dari rumput laut. Jurnal Penelitian Pasca Panen Perikanan. 82: 1-15 Samsuari. 2006. Penelitian Pembuatan Karaginan dari Rumput Laut Eucheuma cottonii di Wilayah Perairan Kabupaten Jeneponto propinsi Sulawesi Selatan. Institut Pertanian Bogor. Sen, O.P. 2005. Total Plate Count. Advances in Fish Processing Technology. ALLIED Publisher. New Delhi. p. 690. Soegiarto, A. and Sulustijo. 1990. Utilization and farming of seaweeds in Indonesia. In: Dogma Jr.I.J.; Trono Jr.G.C.; and Tabbada, R.A. (editors). Culture and use of algae in Southeast Asia: Proceedings of the Symposium on Culture and Utilization of Algae in Southeast Asia, 8-11 December 1981, Tigbauan, Iloilo, Philippines. pp. 9-19. Suryaningrum, T.D. 1988. Kajian sifat-sifat mutu komoditas rumput laut budidaya jenis Eucheuma cottonii dan Eucheuma spinosum. [tesis]. Bogor: Program Pasca Sarjana Institut Pertanian Bogor. p. 181. Widiastuti, H.N. 1990. Pengaruh Konsentrasi NaOH Terhadap Sifat-sifat Karaginan Eucheuma cottonii dari Karimun Jawa dan Madura. Universitas Diponegoro.

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Fucoidan from Brown Seaweed and its Bioactivity...................................(E. Sinurat and E. Marraskuranto)

FUCOIDAN FROM BROWN SEAWEED AND ITS BIOACTIVITY Fukoidan dari Rumput Laut Coklat dan Bioaktifitasnya Ellya Sinurat1)* and Endar Marraskuranto1) Research and Development Center for Marine and Fisheries Product Processing and Biotechnology, Ministry of Marine and Fisheries, KS. Tubun Petamburan VI, Jakarta Pusat 10260* *Corresponding author: [email protected].

ABSTRACT Fucoidan is a polysaccharide which substantially consists of L-fucosa and ester sulphate group and is mainly contained in brown seaweed. For the past ten years, bioactivity studies of fucoidan has been conducted. Recently, fucoidan has been examined for its application in drugs. In a couple of years, fucoidan structure was succesfully identified and its bioactivity was revealed. Fucoidan exhibits various bioactivities such as anticoagulant, antioxidant, anticomplementary, anti-inflamation, gastric protector, and blood lipid level control. This review gives some brief progress in isolation and bioactivity study of fucoidan from brown seaweeds. Key words: fucoidan, brown seaweed, bioactivity, L-fucose ABSTRAK Fukoidan adalah senyawa polisakarida yang secara substansional terdiri atas L-fukosa dan golongan ester sulfat, terutama terdapat pada rumput laut coklat. Dalam jangka waktu sepuluh tahun terakhir, bioaktivitas dari fukoidan telah banyak diteliti. Bahkan belakangan ini telah diteliti aplikasi fukoidan untuk obat. Dalam beberapa tahun terakhir, struktur fukoidan telah berhasil diidentifikasi dan bioaktivitasnya berhasil diketahui. Fukoidan mempunyai banyak bioaktifitas antara lain sebagai antikoagulan, antioksidan, antikomplementari, anti pembengkakan, pelindung lambung, dan pengatur kadar lipid darah. Review ini memberikan ringkasan beberapa kemajuan penelitian isolasi dan bioaktivitas fukoidan dari beberapa jenis rumput laut coklat penghasil fukoidan. Kata kunci: fukoidan, rumput laut coklat, bioaktivitas, L-fukosa

INTRODUCTION Fucoidan is a polysaccharides containing substantial percentage of L-fucose and sulphate ester group which is extracted from brown seaweed and some marine invertebrates (e.g. sea urchin and sea cucumber) (O’Neill, 1954; Patankar, 1993; Duarate, 2001). Polysaccharide, the name is fucoidan, initially come up when it was isolated from brown seaweed by Kylin in 1913. The name of fucoidan was established by IUPAC as fucan, fucosan, or sulphate fucan. In the last decade, fucoidan was isolated from some different species and investigated widely for biological activity, e.g. anticoagulant, antioxidant, anticomplementary properties, antiinflamation, gastroprotector, reducing blood sugar, and antithrombosis. Compared to other sulphate polysaccharides, fucoidan is the most widely distributed and relatively cheap. Recently, fucoidan is also being use as drug and functional food. This paper is a review paper on the recent isolation research of fucoidan and its bioactivity.

Potential of brown seaweed as a fucoidan source has been widely researched, e.g. seaweed Fucus serratus in Murmansk region (Bilan et al., 2004); Undaria pinnatifida, Sporophyll from Korean (Kim et al., 2007); Sargassum polycystum and Sargassum oligocystum from Vietnam (Minh et al., 2005). In Indonesia, there are also several types of brown seaweed which potentially produce fucoidan. One type of brown seaweed species producing fucoidan that has been examined is Sargassum crassifolium (Sinurat et al., 2011). This type of seaweed was obtained from Binuangeun waters, Banten, West Java. FUCOIDAN STRUCTURE AND BIOLOGICAL ACTIVITY Structure of Fucoidan Fucoidan was initially isolated by Kylin (1913) in Janet (2011), the structure from other brown seaweed species was also investigated. Fucoidan isolated from Fucus vesiculosus has a simple chemical

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composition especially consist of fucose and sulphate group, but actually fucoidan has a complex composition. Beside fucose and sulphate group, it also contains other monosaccharide group (e.g. mannose, galactose, glucose, xylose) and uronic acid, even acetyl and protein group. Variations of fucoidan structure depend on source, species of brown seaweed. Its backbone structure was successfully elucidated containing fucose and sulphate group (Li Lu et al., 2008). Recently, fucoidan extracted from Fucus vesiculosus is available commercially in the market. The compositions are fucose 44.1%, sulphate 26.3%, and ash 31.1%, small amount of aminoglucose; its conformation is - D about -123° (Black et al., 1952; Nishino et al., 1992). Based on methylation and alkali treatment, Conchie and O’Neill revealed that its backbone transformed into 1,2- fucose and most of sulphate group was binded to C-4 on fucose unit (Li Rui et al., 2008). Anno et al. (1966) isolated L-fucose 4-sulphate from the same species and its infrared spectrum

Figure 1. Structure model of fucoidan according to Patankar et al. (1993) showed that sulphate group was substituted on atom axial C-4 from L-fucopyranose. Structure model of fucoidan extracted from Fucus vesiculosus and proposed by Conchie had been accepted only after 40 years (Conchie, 1950). In 1993, GC/MS data on fucoidan methylation conducted by Patankar et al. (1993) rebuilt the structure model where its backbone was fucose polymer bonded through -(13) with sulphate group substituted on C-4 in several fucose residues; fucose also bonded on the polymer forming a branch, e.g. one fucose for every 2–3 fucose residues on the backbone. Patankar et al. (1993) explained why his proposed structure is different from Conchie’s. First, the preparation method: Conchie extracted fucoidan using hot water while Patankar using acid which become the standard method for extraction of commercial fucoidan for a couple of years; secondly, the methylation methods: Conchie analyzed his methylation products using chemical and chromatography method while Patankar using GC-EIMS (Anno et al., 1996).

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Bilan et al. (2006) reported that fucoidan extracted from brown seaweed Fucus evanescens, Fucus distichus and Fucus serratus contained fucose, sulphate, and acetate. Fucoidan from F. evanescens had linier backbone containing -L-fucopyranose 2sulphate residue which is connected to 3- and 4- bond position of: (13)--L-Fucp (2SO 3-)-(14)--LFucp(2SO3-)-(14), alternately, where the added sulphate group is located in position 4 at the fucose residue that connected to 3- and 4- position, while the remaining hydroxyl group is acetylated randomly. Fucoidan from F. distichus was arranged in alternating disaccharide unit of: (13)--L-Fucp-(2,4-di-SO3-)(14)--L-Fucp-(2SO3-)-(13). This regular structure might be covered in small amount of randomly acetyl and sulphate unit in this several alternating disaccharide unit. Fucoidan from F. serratus has a branching structure, its main backbone is (13)--LFucp-(14)--L-Fucp-(13), approximately half of its 3-substituted binded residue on C-4 by -L-Fucp(14)--L-Fucp-(13)--L-Fucp-(1trifucosyde) unit. Most of sulphate group occupy C-2 and occasionally C-4, eventhough diglycosilic unit is on position 3,4 and some terminal fucose residue might not has sulphate group. Acetate group occupies C-4 position through 3-fucose bond and C-3 through 4-fucose bond by 7:3 ratio. Fucoidan also contain xylose and galactose in small portion. Fucan from Stoechospermum marginatum has residue backbone of -L-fucopyranosil bound (14) - and (13) - which is substituted at position C-2 and C-3, and most of these residues sulphate fucosyl on C-2 and / or C-4. The bulk structure of fucoidan can be investigated using various electron microscope techniques. Sulphate fucan from Padina gymnospora forms a highly regular structure and exhibits particles in polygonal shape with polycrystal structure pattern. In fact, these particles are consisted of sulphate fucan molecule since these particles are recognised from specific lectin as -L-fucosyl residue. X-ray microanalysis revealed that S is major element according with sulphate group (Silva et al., 2005). Chemical composition of fucoidan from Fucus vesiculosus is relatively more simple than commonly fucoidan which has complex composition. In 1962, Schweiger isolated polysaccharide from Macrocytis pyrifera resulting fucose and galactose ratio of 18:1, and stated for the first time that fucoidan was not pure sulphate fucan but a heteropolymer of fucose, galactose, and small amount of xylose (Li et al., 2008). Furthermore, the other sugars such as mannosa, glucose, xylose, and glucuronic acid were also found in fucoidan from different species of brown seaweed (Table 1) where their existence made them hard to analyze the structure.


The characteristic of fucopyranose 13 bond structure appeared in fucoidan from Ecklonia kurome and Chorda filum. Fucoidan fraction isolated from E. kurome has a branching structure, its main backbone structure is (13)–L-fucosyl and sulphate group which most are bonded to C-4. Fucoidan isolated from Chorda filum comprises of poly--(13)- fucopyranoside backbone with highly branched structure and most of them are -(12)-fucopyranoside single unit. Some of fucopyranose residue contain sulphate on O-4 position (most) and O-2. Some of -(13)-fucose residue are displayed in NMR as acetylated-2-O. Biological Activity of Fucoidan 1. Anticoagulant Fucoidan has a very broad biological activities, however the most widely studied activity is its potential

as anticoagulant. Nishino et al., (1991) conducted anticoagulant activity assay using fucoidan isolated from 9 species of brown seaweed, the parameter are activated partial thromboplastin time (APTT), thromboplastin time (TT), and Xa anti factor activity compared to heparin value (167 unit/mg). All fucoidans showed TT (0-35 unit/mg) and APTT (12-38 unit/mg) activity, while the Xa anti factor activity was not good for several fucoidans. Some of brown seaweed species which were being tested are: E. kurome which showed the highest activity APTT of 38 unit/mg and TT of 35 unit/mg. Hizikia fusiforme with APTT of 25 unit/mg and TT of 22 unit/mg. Anti-thrombin activity of F-4 fraction of fucoidan from Laminaria angustata var. longissima was 200 unit/mg compared to heparin (140 unit/mg). Cumashi et al. (2007) conducted research on anticoagulant properties on fucoidan isolated from 9 brown seaweeds. All extracted fucoidan except

Table 1. Chemical composition of different brown seaweed species Brown seaweed species

Chemical composition

F. vesiculosus

fucose, sulphate

F. evanescens

fucose/sulphate/acetate (1/1.23/0.36)

F. distichus

fucose/sulphate/acetate (1/1.21/0.08)

F. serratus L.

fucose/sulphate/acetate (1/1/0.1)

Lessonia vadosa

fucose/sulphate (1/1.12)

Macrocytis pyrifera

fucose/galactose (18/1), sulphate

Pelvetia wrightii

fucose/galactose (10/1), sulphate

Undaria pinnatifida (Mekabu)

fucose/galactose (1/1.1), sulphate

Ascophyllum nodosum

fucose(49%), xylose(10%), GlcA(11%), sulphate

Himanthalia lorea and Bifurcaria bifurcate

fucose, xylose, GlcA, sulphate

Padina pavonia

fucose, xylose, mannosa, glucose, galactose, sulphate fucose/galactose/sulphate (9/1/9)

Laminaria angustata Eck lonia k urome

fucose, galactose, mannosa, xylose, GlcA, sulphate

Sargassum stenophyllum

fucose, galactose, mannosa, GlcA, glucose, xylose, sulphate

Adenocytis utricularis

fucose, galactose, mannosa, sulphate

Hizik ia fusiforme

fucose, galactose, mannosa, xylose, GlcA, sulphate fucose/xylose/uronic acid/galactose/sulphate (1/0.8/0.7/0.8/0.4) dan (1/0.3/0.4/1.5/1.3)

Dictyota menstrualis Spatoglossum schroederi

fucose/xylose/galactose/sulphate (1/0.5/2/2)

Source: Li lu, 2008.

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Cladosiphon okamuranus, contained a lot of 2-O-D-glucuronopyranosil branches poli--fucopyranoside linear chain which was attached through (13). They showed anticoagulant activity that measured as APTT, whereas only fucoidans from L. saccharina, L. digitata, F. serratus, F. distichus, and F. evanescens showed significant antithrombin activity on platelet aggregation test. Many research showed that anticoagulant activity of fucoidan may be related to the sulphate content and position, molecular weight, and sugar composition. High sulphate content often shows high anticoagulant activity of fucoidan (Ecklonia kurome & Hizikia fusiforme). However, anticoagulant activity increased while antithrombin activity decreased along with theincrement of sulphate content in fucan (Li et al., 2008). Fucoidan which was excessively sulphate and made by reacting natural fucoidan with sulphate also supported this fact. Nishino et al., (1991) made three types of excessively sulphate fucan with different type of sulphate content (ratio of sulphate/sugar, 1.38 -1.98) by means of sulphation reaction of sulphate fucan (sulphate/sugar, 1.28) isolated from E. kurome. Anticoagulant activity of the fucan for APTT and TT respectively were increased 110 -119% and 108-140% from the initial activity. Anticoagulant activity for APTT values (173 unit/mg) for excessively sulphate fucan (ratio of sulphate/sugar, 1.98) was higher than standard heparin (167 unit/mg). Antithrombin activity of cofactor II-mediated heparin of excessively sulphate fucan also increased significantly with the addition of sulphate content. Qiu et al. (2006) reported that excessively sulphate fucoidan showed four times higher in anticoagulant activity than fucoidan in protothrombin doubling time of normal human citrate plasma. Sulphate group position on sugar residue is crucial for anticoagulant activity of fucoidan. This activity is related to sulphate C-2 and disulphate C-2,3. The disulphate 2,3 sugar residues are common structural properties in fucoidan as anticoagulant. Duarate et al. (2001) reported that the anticoagulant properties of fucoidan are determined largely by the sulphate fucose chain, particularly by fucosyl disulphate unit. Silva et al. (2005) reported that sulfate 3-O at C-3 of the 4--L-fucose-1 unit was responsible for the anticoagulant activity of fucoidan from Padina gymnospora. An expected fucoidan is having a long and thrombin bonded conformation sugar chain structure, so it takes a large molecular weight to have anticoagulant activity. Fucoidan (MW 320,000) of Lessonia vadosa (Phaeophyta) showed significant by anticoagulant activity, whereas its radical depolimerized fraction (MW 32,000) showed weak anticoagulant activity. Even

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a slight decrease in molecular size of sulphate fucan reduces its effect significantly on the inactivation of thrombin by heparin cofactor II. Sulphate fucan with 45 units of repeating tetrasaccharide attach to heparin cofactor II but they can not connect efficiently the plasma inhibitor with thrombin. Therefore it is required for at least 100 or more repeating tetrasaccharide units in order to the effect to be occured. In thrombin inactivation process by linear sulphate linear induced by heparin cofactor II, casting mechanism is more dominating than the allosteric effect. Linear sulphate fucan requires longer chain than mamals glycosaminoglycan to obtain anticoagulant activity. Low molecular weight of fucoidan (LMWF) extracted from Ascophyllum nodosum through acid hydrolysis has a repeating structure of [13)--L-Fuc(2SO3-) (14)--L-Fuc (2,3 diSO3-) - (1]n and molecular weight of 3090 Da and also has in vitro anticoagulant activity. It means that the anticoagulant activity is not only based on molecular weight, but also determined by the branched structure. Sulphation level of LMWF has three sulphate for every disaccharide, the same as heparin main repeating unit [4)--L-IdoA (2 SO3-) (14)--D-Glc-(NSO3-, 6 SO3-) -13]n. Several studies showed that sugar composition (fucose, galactose, etc.) of fucoidan is associated with anticoagulant activity. But it is presumably that it is not the sugar but sulphate which has the effect on anticoagulant activity. Pereira et al. (2002) demonstrated that sulphate 2,3 linked to -Lgalactan, but not to -L-fucan is a potential thrombin inhibitor via antithrombin or heparin cofactor II media. Uronic acid was not required for anticoagulant activity, but it can increase the anticoagulant activity by increasing the flexibility of the sugar chain. Clearly, many fucoidans could be prolong their APTT, but slightly delay their TT. This stated that the anticoagulant activity is mainly due to coagulant endogenesis approach which is restrained by fucoidan (Li et al., 2008; Zhao et al., 2005). However, fucoidan derived from fermented brown seaweed Sargassum fulvellum can inhibit intrinsic and extrinsic pathway of blood coagulation (Zheng et al., 2002). Thrombin plays an important role in thrombosis, so thrombin inhibitor is the core of the antithrombotic drug research. Many studies showed that most of the anticoagulant activity of fucoidan is mediated via antithrombin inhibition by heparin cofactor II. Its activity also accelerates thrombin inhibition and Xa factor by antithrombin but at lower potential. However, Zoysa et al. (2008) reported that the anticoagulant properties of fucoidan from F. vesiculosus was determined by thrombin inhibition mediated via in vitro and in vivo


plasma antithrombin-III trials, has a similar anticoagulant activity with heparin. Mourao (2004) summarized antithrombosis and anticoagulant activity of sulphate fucan. Sulphate fucan derived from algae and invertebrate had strong anticoagulant activity, mediated by antithrombin and/ or heparin cofactor II. This aspect was clarified by research on invertebrate derived polysaccharides. These results clearly established that the structure of linear and regulated sulphate -L-fucan and sulphate D L--galactan showed anticoagulant activity, suggesting not only a function of charge density, but also on the composition pattern of sulphate and monosaccharide. Melo et al. ( 2004) conducted a study on the mechanism of anticoagulant activity of sulphate galactan. Anticoagulant activity of sulphate polysaccharide was achieved mainly via cofactor plasma charge which is a natural inhibitor of coagulation protease. Their results showed the following: 1) structural interaction requirement between sulphate galactan with coagulation inhibitor and targeted protease was not just a consequence of the sulphate galactan charge density, 2) basic structure of this interaction was complex because it is usually involved a heterogeneous polysaccharide but it depend on the distribution of sulphate group and monosaccharide composition, 3) it was needed a longer chain of sulphate galactan than heparin to reach the anticoagulant activity, 4) it was possible that sulphate galactan structure was large, not small component such as heparin, however its interaction with antithrombin was the decisive factor, 5) sulphate galactan of molecular weight 15-45 kDa binded antithrombin but it can not reach the plasma and thrombin inhibitors. This last effect required molecular size of 45 kDa, 6) sulphate galactan and heparin binded to a different binding site on antithrombin, 7) compared to heparin, sulphate galactan was less effective in promoting antithrombin activation via structural conformation. Overall, those observations suggested that different mechanisms dominated on antithrombin activation via structural conformation to ensure anticoagulant activity of sulphate galactan mediated by antithrombin. It was possible that sulphate galactan linked both with antithrombin and thrombin, and left protease in its inactive form. Antithrombin activation via structural conformation and covalent complex formation with thrombin seem less important in anticoagulant activity of sulphate galactan than that of heparin. Results showed that interaction paradigm between heparin and antithrombin can not be applied to other sulphate polysaccharides. Any type of polysaccharides can be form a specific complex with plasma inhibitors and targeted protease.

2. Antioxidant Activity Several studies showed that fucoidan had significant in vitro antioxidant activity. Fucoidan is a natural antioxidant and has the potential to prevent diseases caused by free radicals. Fucoidan from brown seaweed L. japonica clearly can prevent the increase of lipid peroxides in the blood serum, liver and spleen of mice with diabetes. However, there was no inhibitory effect either on spontaneously lipid peroxidation homogenate or induced in vitro by Cys / FeSO4 (Cumashi et al., 2007). Fucoidan had strong scavenging effect on superoxide radicals, its effect on hydroxyl radical was weak and less effect on DPPH. This fucoidan effectively inhibited H2O2 induced hemolysis in mice erythrocytes and showed a significantly protective effect on lipid peroxidation in liver homogenate of mice induced by FeSO4-ascorbic acid combination (Qiu et al., 2006). Micheline et al., (2007) reported that fucoidan homofucan from F. vesiculosus and heterofucan from Padina gymnospora had an inhibition effect on the formation of hydroxyl and superoxide radicals. Fucan showed lower antioxidant activity than fucoidan. The antioxidant activity of fucoidan is related to molecular weight and fucoidan sulphate content. Fucoidan fraction of L. japonica has a very good scavenging capacity on superoxide radicals and hipochloric acid, except for L-B sulphate fraction. In LDL oxidation system, L-A and L-B low molecular weight fraction showed a significant inhibitor effect on LDL oxidation induced by Cu2+, but F-A and F-B had a weak inhibitory effect in the system because of their large molecular weight. Both molecular weight and fucoidan sulphate content have a very important role on these effects on azo 2-2'-azobis (2-amidinopropan) dihydrochloride (AAPH) radical induced by LDL oxidation. Correlation between sulphate content and superoxide radical scavenging ability is positive, ratio of sulphate content and fucose is an effective indicator of antioxidant activity in the sample (Zhao et al., 2005). 3. Anticomplementary Activity Complement system is a major component in immunity and primarily is involved in innate and humoral responses. The system also provides a link between innate immunity and adaptation responsed self-defense. Uncontrolled activities in the system resulted in harm to the host organism, such as those observed in ischemic and anaphylactic shock or xenograft rejection (Tissot & Daniel, 2003). Algal fucoidan of Ascophyllum nodosum was first reported as a anticomplementer molecule by Blondin et al. (1996). In addition, other fucoidan of fucales (F. evanescens) and other brown algae of Laminariale also reported to have complement inhibitor properties.

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Tissot & Daniel (2003) summarized research on anticomplementer activity of fucoidan. 4. Anti-inflamation All fucoidan derived from 9 species of brown algae inhibited leucocytes mobilization using inflammatory model in mice, and both fucose and sulphate content as well as other structural properties of their polisaccharide chain did not exhibited evident effect of fucoidan in the model. Mekabu fucoidan can eliminate inflammation in lung (pulmonary) with response to down-regulated Th2-dominated, which may be useful for treating inflammation caused by allergies (Maruyamaa et al., 2005). Yang et al. (2006) investigated the effects of fucoidan on inducible nitric oxide synthase (iNOS) expression in a macrophage cell line, RAW264.7. Fucoidan in low concentration range (10 ì g/ml) increased alkaline iNOS expression levels in stationary macrophage. Yang et al. (2006) discovered for the first time that fucoidan inhibited nitric oxide (NO) release in RAW264.7 cells stimulated with lipopolysaccharide (LPS). Inhibitory effect on activator protein 1 (AP-1) activated by fucoidan may be related to NO closing and anti-inflammatory effect. 5. Gastric Protector Fucoidan from Cladosiphon okamuranus Tokida is a safe material and has potential as a gastric protector. It is available an antiulcer drug and adhesion inhibitor for Helicobacter pyroli containing fucoidan as active ingredient. The new drug is effective in treating and preventing gastritis and inhibiting adhesion of Helicobacter pyroli in the stomach (Shibata et al., 2000; Silva et al., 2005). Fucoidan from C. okamuranus showed cancer cell growth inhibition activity in the stomach but did not show any effects on normal cells. The sulphate content and molecular weight of the fucoidan were 9.8% (w/w) and approximately 3.2 million, respectively (Tissot & Daniel, 2003). 6. Lowering Blood Lipid Level Fucoidan is among active agents similar to sialic acid and may increase negative charge of cell surface such as causing effect on the aggregation of cholesterol in blood, then lowering cholesterol level in serum. Fucoidan of L. japonica can actually lower total cholesterol, triglycerides, and LDL-C and raising HDLC in serum of mice suffering from hypercholesterolemia and of rat with hyperlipidemia, and efficiently prevented the formation of hipercholesterolemia via experiments in mice. The fucoidan also can actually lowered cholesterol and triglyceride levels in the serum of

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patients with hyperlipidemia, without the side effects that damage liver and kidneys (Wang et al., 2008). Sulphate fucan of low molecular weight (molecular weight approximately of 8000 Da) from L. japonica clearly lowered blood lipid in rats with hyperlipidemia. Oligosaccharide fucoidans showed good antihypertensive effect in renovascular hypertensive rats and one possible mechanism was that fucoidan might inhibit the production of angiotensin II plasma (Zoysa et al., 2008). CONCLUSION Fucoidan extracted from brown seaweed has a complex and heterogeneous structure, that’s why the structure has not been clearly described until now. However, fucoidan has interesting biological activity so that many researches are conducted to investigate their structure and bioactivity every year. Since bioactivity tests is conducted using crude fucoidan, it is not easy to determine the relationship between their structure and activity. But, at least, we learned that these bioactivities not only depend on their high charge density, but also their structure. The upcoming research on the conformational structure of fucan should focus on deeper understanding on the biological properties of fucoidan. Brown algae are abundant throughout the world and some species (e.g. H. fusiforme, L. japonica) has been cultivated on a large scale. Currently most of the brown algae are consumed as food or additives in food, while some of them have developed into new drugs and functional foods. Through chemical modification, such as sulphation process in certain position and methylation, some activities of fucoidan increase significantly. By focusing research on the structure of fucoidan and investigate the relationship between its activity and structure can provide a basic theory in resource development and utilization of brown algae. REFFERENCES Anno, K., Terahata, H., and Hayashi, Y. 1966. Isolation and purification of fucoidan from brown seaweed Pelvetia wrightii. Agri. Biol. Chem. 30: 495-499. Bilan, M.I., Grachev, A.A., Ustuzhanina, N.E., Shashkov, A.S., Nifantiev, N.E., and Usov, A.I. 2004. A highly regular fraction of a fucoidan from the brown seaweed Fucus distichus L. Carbohydr. Res. 339: 511-517. Bilan, M.I., Grachev, A.A., Shashkov, A.S., Nifantiev, N.E., and Usov, A.I. 2006. Structure of a fucoidan from the brown seaweed Fucus serratus L. Carbohydr. Res. 341: 238-245. Black, W.A.P., Dewar, E.T., and Woodward, F.N. 1952. Manufacture of algal chemicals. IV. -Laboratory-scale


isolation of fucoidan from brown marine algae. J. Sci. Food Agric. 3: 122-129. Blondin, C., Chaubet F., Nardela, A., Singuin, C., and Jozefonvicz, J. 1996. Relationships between chemical characteristics and anticomplementary activity of fucans. Biomaterials. 17: 597-603. Conchie, J. and Percival, E.G.V. 1950. Fucoidan part II. The hydrolysis of a methylated fucoidin prepared from Fucus vesiculosus. J. Chem. Soc. 827-833. Cumashi, A., Ushakova, N.A., Preobrazhenskaya, M.E., D’Incecco, A., Piccoli, A., Totani, L., Tinari, N., Morozevich, G.E., Berman, A.E., Bilan, M.I.,Usov, A.I., Nadezhda E., Grachev, A.A., Sanderson, C.J., Kelly, M., Rabinovich, G.A., and Iacobelli, S. 2007. A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds. Glycobiology. 17: 541-552. Duarate, M., Cardoso, M., and Noseda, M. 2001. Structural studies on fucoidans from the brown seaweed Sargassum stenophyllum. Carbohydr. Res. 333: 281293. Janet Helen Fitton. 2011. Therapies from Fucoidan; Multifunctional Marine Polymers., Mar. Drugs. 9(10): 1731–1760. Kim, W.J., Kim, S.M., Kim, H.G., Oh, H.R., Lee, K.B., Lee, Y.K., Park, Y.P. (2007). Purification and Anticoagulant Activity of a Fucoidan from Korean Undaria pinnatifida Sporophyll. Algae. 22: 247-252. Li, B., Lu, F., Wei, X., and Zhao, R. 2008. Fucoidan: Structure and Bioactivity, Molecules. 13: 1671-1695. Li, B., Rui, X.Z., and Xin, J.W. 2008. Anticoagulant activity of fucoidan from Hizikia fusiforme. Agro Food Ind. Hitech. 19: 22-24. Maruyamaa, H., Tamauchib, H., Hashimotoc, M., and Nakano, T. 2005. Suppression of Th2 immune responses by Mekabu fucoidan from Undaria pinnatifida Sporophylls. Int. Arch. Allergy Immunol. 137: 289-294. Melo, F.R., Pereira, M.S., Foguel, D., and Mourao, P.A.S. 2004. Antithrombin-mediated Anticoagulant Activity of Sulfated Polysaccharides. J. Biol. Chem. 279: 2082420835. Micheline, R.S., Cybelle, M., Celina, G.D., Fernando, F.S., Hugo, O.R., and Edda, L. 2007. Antioxidant activities of sulfated polysaccharides from brown and red seaweeds. J. Appl. Phycol. 19: 153-160. Minh Bui, L., Ngo Quoc, B., Nguyen D., Pham Duc, T., and Tran T. 2005. Studies on fucoidan and its production from vietnamese brown seaweeds. AJSTD. 22(4): 371-380.

Mourao, P.A.S. 2004. Use of sulfated fucans as anticoagulant and antithrombotic agents: future perspectives. Curr. Pharmaceut. Des. 10: 967-981. Nishino, T. and Nagumo, T. 1992. Anticoagulant and antithrombin activities of oversulfated fucans. Carbohydr. Res. 229: 355-362. Nishino, T., Nagumo, T., and Kiyohara, H. 1991. Structure characterization of a new anticoagulant fucan sulfate from the brown seaweed Ecklonia kurome. Carbohydr. Res. 211: 77-90. O’Neill, A.N. 1954. Degradative studies on fucoidan. J. Amer. Chem. Soc. 76: 5074-5076. Patankar, M. S., Oehninger, S., and Barnett, T. 1993. A revised structure for fucoidan may explain some of its biological activities. J. Biol. Chem. 268: 2177021776. Pereira, M.S., Vilela-Silva, A. E. S., Valente, A., and Mourao, P. A . S. 2002.  2-sulfated, 3-linked ±-Lgalactan is an anticoagulant polysaccharide. Carbohydr. Res. 337: 2231-2238. Qiu, X. D., Amarasekara, A., and Doctor, V. 2006. Effect of oversulfateion on the chemical and biological properties of fucoidan. Carbohydrate Polymers. 63: 224-228. Shibata, H., Kimura-Takagi, I., Nagaoka, M., Hashimoto, S., Aiyama, R., Iha, M., Ueyama, S., and Yokokura, T. 2000. Properties of fucoidan from Cladosiphon okamuranus tokida in gastric mucosal protection. BioFactors. 11: 235-245. Silva, T. M. A., Alves, L. G., Queiroz, K. C. S., Santos, M. G. L., Marques, C. T., Chavante, S. F., Rocha, H. A. O., and Leite, E. L. 2005. Partial characterization and anticoagulant activity of a heterofucan from the brown seaweed Padina gymnospora. Braz. J. Med. Biol. Res. 38: 523-533. Sinurat, E., Rosmawaty, P., dan Saepudin, E. 2011. Ekstraksi dan Uji Aktivitas Fukoidan dari Rumput Laut Coklat (Sargassum crassifolium) sebagai Antikoagulant. Jurnal Pascapanen dan Bioteknologi Kelautan dan Perikanan. 6(2): 131-138. Tissot, B. and Daniel, R. 2003. Biological properties of sulfated fucans: the potent inhibiting activity of algal fucoidan against the human complement system. Glycobiology. 13: 29G-30G. Wang, J., Zhang, Q., Zhang, Z., and Li, Z. 2008. Antioxidant activity of sulfated polysaccharide fractions extracted from Laminaria japonica. Int. J. Biol. Macromol. 42: 127-132. Yang, J.W., Se, Y.Y., Soo, J.O., Sang, K.K., and Keon, W.K. 2006. Bifunctional effects of fucoidan on the expression of inducible nitric oxide synthase. Biochem. Biophys. Res. 346: 345-350. Zhao, X., Xue, C.H., Cai, Y.P., Wang, D.F., and Fang, Y. 2005. The study of antioxidant activities of fucoidan from Laminaria japonica. High Tech. Lett. 11: 91-94. Zheng, J., Wang, Y., and Qian, J.J. 2002. Isolation, purification and the anticoagulant activities of fucoidan. J. Mol. Sci. 18: 109-112. Zoysa, M., Nikapitiya, C., Jeon, Y.J., Jee, Y., and Lee, J. 2008. Anticoagulant activity of sulfated polysaccharide isolated from fermented brown seaweed Sargassum fulvellum. J. Appl. Phycol. 20: 67-74.

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ISSN 2089-5690

SQUALEN BULETIN PASCAPANEN DAN BIOTEKNOLOGI KELAUTAN DAN PERIKANAN (SQUALEN: BULLETIN OF MARINE AND FISHERIES POSTHARVEST AND BIOTECHNOLOGY)  Vol. 7  No. 1  May 2012

CURRENT CONTENT/LEMBAR ABSTRAK SYNTHESIS OF POLYVINYL ALCOHOL-CHITOSAN HYDROGEL AND STUDY OF ITS SWELLING AND ANTIBACTERIAL PROPERTIES SINTESIS HIDROGEL POLIVINIL ALKOHOL-KITOSAN DAN STUDI SIFAT MENGEMBANG DAN ANTIBAKTERINYA Thamrin Wikanta1), Erizal2), Tjahyono2) and Sugiyono1) 1)

Research and Development Center for Marine and Fisheries Product Processing and Biotechnology, KS. Tubun Petamburan VI Jakarta Pusat 10260 2) Center for the Application Isotopes and Radiation Technology, Jl. Lebak Bulus Raya No.49. Jakarta Selatan *Correspondence Author: [email protected]

ABSTRACT The aim of this research was to synthesize a hydrogel for wound dressing by mixing of polyvinyl alcohol (PVA) and chitosan (CTS) and processed by combination technique of freezing-thawing and irradiation by gamma ray, and to study of its properties. PVA aqueous solution 10% (w/v) was mixed with 2% (w/v) chitosan (CTS) solution and homogenized. The PVA-CTS mixture was processed by freezing-thawing up to 3 cycles, and then irradiated by gamma rays at the dose ranged of 20-50 kGy (dose rate was 10 kGy/hour). Result showed that PVA-CTS hydrogel with the gel fraction of 83%, 87%, 90%, and 83% were obtained at the irradiation dose of 20 kGy, 30 kGy, 40 kGy, and 50 kGy, respectively. Increasing of irradiation dose caused increasing of water absorption of hydrogel, i.e. 1.700 %, 1.715 %, 1.913 %, and 2.036 %, respectively, and the hydrogel reached the equilibrium in 25 hours. The hydrogel showed very slow water evaporation rate (~ 2%) at the initial time (1 hour) and then increased very fast (up ~50 %) at 24 h, i.e. 43%, 39.13%, 44%, and 53%, respectively. The elongation at break of hydrogels were obtained 245%, 322%, 322%, and 205% with the maximum value were obtained at irradiation dose ranged of 30-40 kGy. The presence of chitosan in the PVA hydrogel made it having higher antibacterial properties with the inhibition zone value of 8 mm at irradiation dose of 30-40 kGy compared to PVA hydrogel as a negative control (6 mm) and to chloramphenicol as a positive control (8 mm). Keywords: hydrogel, polyvinyl alcohol (PVA), Chitosan (CTS), freezing-thawing, gamma irradiation ABSTRAK Tujuan dari penelitian ini adalah mensintesis hidrogel untuk pembalut luka dengan mencampurkan polivinil alkohol (PVA) dan kitosan (CTS) dan diproses dengan kombinasi teknik beku-leleh dan irradiasi sinar gamma, serta mempelajari sifat-sifatnya. Larutan PVA 10% (b/v) dicampur dengan 2% (b/v) larutan kitosan (CTS) dan dihomogenkan. Campuran PVA-CTS diproses dengan beku-leleh hingga 3 siklus, dan kemudian diirradiasi dengan sinar gamma pada kisaran dosis 20-50 kGy (laju dosis adalah 10 kGy/jam). Hasil penelitian menunjukkan bahwa hidrogel PVA-CTS dengan fraksi gel 83%, 87%, 90%, dan 83% didapatkan pada dosis irradiasi masingmasing 20 kGy, 30 kGy, 40 kGy, and 50 kGy. Meningkatnya dosis irradiasi mengakibatkan meningkatnya absorpsi air oleh hidrogel, yaitu masing-masing 1.700 %, 1.715 %, 1.913 %, dan 2.036 %, dan hidrogel mencapai kondisi keseimbangan dalam 25 jam. Hidrogel menunjukkan laju penguapan air sangat lambat (~ 2%) pada awal waktu (1 jam) dan selanjutnya meningkat dengan sangat cepat (hingga ~50 %) ~ pada 24 jam, yaitu masing-masing 43%, 39.13%, 44%, dan 53%. Nilai perpanjangan putus dari hidrogel didapatkan 245%, 322%, 322%, dan 205% dengan nilai maksimum didapatkan pada dosis irradiasi berkisar 30-40 kGy. Adanya kitosan dalam hidrogel PVA menjadikannya memiliki sifat antibakteri lebih tinggi dengan nilai zona inhibisi 8 mm pada dosis irradiasi 30-40 kGy dibandingkan dengan hidrogel PVA sebagai kontrol negatif (6 mm) dan dengan kloramfenikol sebagai kontrol positif (8 mm). Kata Kunci: hidrogel, polivinil alkohol (PVA), kitosan (CTS), beku-leleh, irradiasi gamma

Squalen Vol 7 No 1, May 2012

IDENTIFICATION AND PARTIAL CHARACTERIZATION OF CRUDE EXTRACELLULAR ENZYMES FROM BACTERIA ISOLATED FROM SHRIMP WASTE PROCESSING Identifikasi dan Karakterisasi Parsial Enzim Kasar Ekstraseluler dari Bakteri yang Diisolasi dari Pengolahan Limbah Udang Ekowati Chasanah1), Mahrus Ali2), and Miftahul Ilmi3) 1) Research and Development Center for Marine and Fisheries Product Processing and Biotechnology Dept. of Fisheries Faculty of Agriculture, University of Lampung; 3)Faculty of Biology, Gadjah Mada University, Yogyakarta *Correspondence Author: Ekowati Chasanah, KS.Tubun Petamburan VI Jakarta Pusat 10260, E-mail: [email protected] 2)

ABSTRACT Attention on chitin degrading enzymes has been growing since their ability to reduce the waste of shrimp/other crustaceans processing industries and converting them into value added products such as biologically active chitin and chitosan oligomer. Previous experiment found that KLU 11.16 isolate was one of the potential bacteria isolated from shrimp waste producing chitinolytic enzymes including chitosanases. A study on the identification of KLU 11.16 extracellular crude enzyme was carried out by cultivating the bacteria on chitin medium. Due to the wide application of chitosanase, the characterization of the crude chitosanase was carried out after an identification of the enzymes secreted. Based on assessment using zymogram technique, this bacteria secreted a mixed extracellular chitinolytic enzyme and other hydrolytic enzyme. The crude chitinolytic enzyme degraded 85% deacetylated (DA) better than 100% DA chitosan, and slightly degraded glycol chitin, indicating that KLU 11.16 secreted chitosanases and chitinases enzyme. In addition to the chitinolytic enzyme, the bacteria also secreted protein and carbohydrate degrading enzymes when running at SDS-PAGE enriched with casein, soluble starch and CMC substrates. Crude chitosanases enzyme was performed well at pH 6 and temperature of 300C, and the activity can be increased by addition of 1 mM Fe2+ in form of chloride salt. Addition of detergent, i.e 1mM of Triton X-100 and SDS slightly decreased the activity. Future application of the crude chitosanase from KLU 11.16 was on producing chitosan derivative such as chitosan oligomer using substrate of 85% DA chitosan, which is more digestable by other enzymes secreted by KLU 11.16 Keywords: extracellular enzyme, KLU 11.16, identification ABSTRAK Perhatian terhadap enzim pendegradasi kitin semakin berkembang sejak diketahui bahwa enzim ini mampu mengubah limbah pengolahan udang dan krustase lain menjadi produk yang memiliki nilai tambah yang besar seperti oligomer kitin dan kitosan yang memiliki aktivitas biologi. Riset terdahulu mendapatkan isolat KLU 11.16, yang diisolasi dari limbah udang, sebagai salah satu isolat yang berpotensi memproduksi enzim pendegradasi kitin (kitinolitik), termasuk enzim kitosanase. Penelitian tentang identifikasi enzim kasar yang disekresi oleh KLU 11.16 telah dilakukan dengan mengkultivasi isolat bakteri tersebut di medium kitin. Selanjutnya, karakterisasi hanya dilakukan terhadap kitosanase dari enzim kasar yang disekresi tersebut mengingat luasnya aplikasi kitosanase. Dengan menggunakan teknik zimogram, hasil menunjukkan bahwa isolat bakteri ini mensekresikan campuran enzim ekstraseluler pendegradasi kitin dan hidrolitik lain. Enzim kasar kitinolitik pendegradasi kitin dapat menghidrolisis 85% terdeasetilasi (DA) 85% kitosan lebih baik dari pada kitosan DA 100%, dan sedikit menghidrolisis glikol kitosan, mengindikasikan bahwa KLU 11.16 mensekresikan kitosanase dan kitinase. Selain enzim kitinolitik, bakteri tersebut juga mensekresikan enzim pendegradasi protein dan karbohidrat. Kitosanase yang ada dalam campuran enzim kasar tersebut beraktivitas secara baik pada pH 6 dan suhu 300C, dan akan meningkat aktivitasnya apabila ditambah dengan 1 mM Fe 2+ dalam bentuk garam klorida. Penambahan detergen Triton X-100 dan SDS sebesar 1mM sedikit mengurangi aktivitas enzim. Aplikasi kedepan enzim kasar kitosanase dari KLU 11.16 ini adalah untuk memproduksi oligomer kitosan dengan menggunakan substrat kitosan DA 85%, yang lebih mampu dihidrolisis oleh enzim-enzim yang disekresi oleh KLU 11.16. Kata Kunci: enzim ekstraseluler, KLU 11.16, identifikasi


BACTERIAL DIVERSITY OF THE DEEP SEA OF SANGIHE TALAUD, SULAWESI Keanekaragaman Bakteri Laut Dalam Sangihe Talaud, Sulawesi Gintung Patantis1), Ekowati Chasanah1), Dewi Seswita Zilda1), and Ikhsan B. Waluyo2) 1)

Research and Development Center for Marine and Fisheries Product Processing and Biotechnology The Agency for The Assessment and Application of Technology (BPPT), Jl. MH. Thamrin 8 Jakarta *Correspondence author: Gintung Patantis, KS. Tubun Petamburan VI Jakarta Pusat 10260, E-mail: [email protected] 2)

ABSTRACT Deep sea is an extreme environment characterized by cold temperature, high pressure, lack of light and nutrients. Microorganisms live in these habitat are unique microorganisms and known to have tremendous source of potential agents for biotechnology processes. Indonesia as an archipelagic country has a vast deep ocean. This study aims to see the diversity of bacteria in Sangihe Talaud Deep Sea, Sulawesi. Analysis of bacterial diversity was carried out by cultured and uncultured method. Terminal Restriction Fragment Length Polymorphism (T-RFLP) technique was used for uncultured analysis of the microorganisms biodiversity, while cultured one was done by plating the samples of water onto Zobell media. The results showed that, there were 21 isolates obtained by cultured method. The identification which based on 16S rDNA by PCR method showed the genus of Pseudomonas, Pseudoalteromonas, Alteromonas, Vibrio, Shewanella and Uncultured bacterium were identified. However, 14 classes of bacteria were obtained by using TRFLP method i.e Acetobacteraceae class, Actinobacteria, á-proteobacteria, -proteobacteria, äproteobacteria, ã-proteobacteria, Bacili, Bacteroidetes, Chlorobi, Chroococcales, Clostridia, Erysipelotrichi, Synergistia, and Zetaproteobacteria. There were also unclassified bacteria and uncultured bacterium found in the samples. Keywords: deep sea, diversity, bacteria, PCR, T-RFLP, Sangihe Talaud

ABSTRAK Laut dalam merupakan lingkungan ekstrim yang dicirikan oleh suhu dingin, tekanan tinggi, cahaya dan nutrien yang kurang. Mikroorganisme yang hidup pada habitat tersebut merupakan mikroorganisme yang unik dan diketahui mempunyai potensi bioteknologi yang sangat besar. Indonesia sebagai negara kepulauan mempunyai laut dalam yang luas. Penelitian ini bertujuan untuk melihat keanekaragaman bakteri yang ada di laut dalam Sangihe Talaud, Sulawesi. Analisis keragaman bakteri dilakukan dengan metode kultur dan uncultured. Teknik Terminal Restriction Fragment Length Polymorphism (T-RFLP) digunakan untuk analisis keragaman mikroorganisme secara uncultured, sedangkan analisis kultur dilakukan dengan menumbuhkan sampel air pada media Zobell. Hasil penelitian menunjukkan bahwa dengan teknik kultur didapatkan 21 isolat. Identifikasi dengan PCR 16S rDNA menunjukkan genus Pseudomonas, Pseudoalteromonas, Alteromonas, Vibrio, Shewanella dan Uncultured bacterium. Sebanyak 14 kelas didapatkan dengan teknik T-RFLP, yaitu : kelas Acetobacteraceae, Actinobacteria, áproteobacteria, -proteobacteria, ä-proteobacteria, ã-proteobacteria, Bacili, Bacteroidetes, Chlorobi, Chroococcales, Clostridia, Erysipelotrichi, Synergistia, dan Zetaproteobacteria. Hasil identifikasi juga menemukan bakteri yang tidak terklasifikasi dan bakteri yang tidak dapat dikultur. Kata Kunci: laut dalam, keanekaragaman, bakteri, PCR, T-RFLP, Sangihe Talaud


THE POTENTIAL OF HETEROTROPHIC MICROALGAE (Schizochytrium sp.) AS A SOURCE OF DHA Potensi Mikroalga Heterotroph (Schizochytrium sp.) sebagai Sumber DHA Arif Rahman Hakim 1)

Research Institute for Fisheries Post-harvest Mechanization;

*Correspondence author: Arif Rahman Hakim, KS. Tubun Petamburan VI Jakarta Pusat 10260, E-mail: [email protected]

ABSTRACT Docosahexanoic acid (DHA) is commercially obtained from marine fish. With an increasing human population, the supplies of DHA are still not sufficient to meet the world’s need of DHA as food supplement. The objective of this review is to discuss Schizochytrium sp., one of microalgae which is rich in DHA, as one of the best candidate as producer of sustainable and affordable DHA. Heterotrophic microalgae, especially genus Schizochytrium, produces omega-3 fatty acids up to 40% of total unsaturated fatty acids. Cultivation of the microalgae is easy as it does not require sunlight as source of energy. Previous publication reported that several local strains of Schizochytrium have been isolated from mangrove area in Indonesia. We expect that those strains can be cultivated in mass production as producer of DHA. Keywords: docosahexanoic acids, fatty acids, Schizochytrium sp. ABSTRAK Docosahexanoic acid (DHA) komersial sebagian besar diperoleh dari ikan laut. Dengan meningkatnya populasi penduduk dunia, sumber ini tidak akan mencukupi permintaan dari masyarakat terhadap DHA sebagai salah satu makanan kesehatan. Tujuan dari review ini adalah untuk mendiskusikan Schizochytrium sebagai salah satu mikroalga yang kaya DHA, sebagai kandidat mikroalga yang mampu memproduksi DHA secara berkelanjutan dan terjangkau. Jenis mikroalga yang bersifat heterotrof terutama genus Schizochytrium mampu menghasilkan asam lemak omega 3 mencapai 40% dari total asam lemak tak jenuhnya. Kultivasi mikroalga ini juga lebih mudah karena tidak membutuhkan sinar matahari sebagai sumber energinya. Publikasi sebelumnya menunjukkan bahwa beberapa strain lokal telah di isolasi dari daerah mangrove di Indonesia. Kami berharap strain tersebut bisa dikembangkan ke produksi DHA secara masal. Kata Kunci: asam docosaheksanoat, asam lemak, Schizochytrium sp.


APPLICATION OF MODIFIED ATMOSPHERE PACKAGING (MAP) ON FRESH FISH

Aplikasi Pengemasan dengan Atmosfir Termodifikasi (MAP) pada Ikan Segar Putri Wullandari and Diini Fithriani1) 1) Research Institute for Fisheries Post-harvest Mechanization *Correspondence author: Putri Wullandari, KS. Tubun Petamburan VI Jakarta Pusat 10260, E-mail: [email protected]

ABSTRACT Packaging has many functions, e.g. as containment, to protect products from physical damage, and from H2O, O2, and CO2 exposure, as well as to attract the consumers. Based on the technology, packaging can be divided into 3 types i.e. passive packaging, active packaging, and smart packaging. Modified Atmosphere Packaging (MAP) is the most common active packaging found in the market. MAP has been used to extend the shelf life of several fish such as raw whiting (cod family), mackerel, salmon fillet, cod fillet, fresh bluefin tuna fillet, etc. MAP which was combined with freeze-chilling has been proved to extend the shelf life of raw whiting, mackerel, and salmon fillet. MAP also increased cod fillet’s shelf life up to 20 days, whiting fillets packed in 100% CO2 and stored at 4°C temperature up to 15 days. MAP combined with antioxidant on fresh bluefin tuna fillets stored at 3°C for 18 days was able to extend products shelf life from 2 days (control) to 18 days, meanwhile 100% N2 in packaging has protective effect on haemoglobin and lipid oxidation. Packaging innovations and ingenuity will continuously develop MAP that is oriented for consumer, enhance the product, environmentally responsive, friendly, and cost effective. Keywords: active packaging, modified atmosphere packaging, fresh fish product

ABSTRAK Pengemasan memiliki banyak fungsi, yaitu sebagai wadah, melindungi produk dari kerusakan fisik, paparan H 2O, O 2, dan CO 2 , dan untuk menarik konsumen. Berdasarkan teknologi pengemasannya, kemasan dapat dibagi menjadi 3, yaitu kemasan pasif, kemasan aktif, dan kemasan pintar. Salah satu contoh pengemasan aktif yang sudah dikenal di masyarakat adalah Modified Atmosphere Packaging (MAP). MAP telah banyak digunakan untuk memperpanjang umur simpan beberapa jenis ikan segar, seperti whiting (sejenis cod), mackerel, filet salmon, filet cod, filet tuna sirip biru, dan yang lainnya. Penggunaan MAP yang dikombinasikan dengan penyimpanan beku dapat memperpanjang umur simpan whiting, mackerel, dan fillet salmon. Filet ikan cod yang dikemas dengan MAP memiliki umur simpan 20 hari sedangkan umur simpan filet whiting yang dikemas dalam 100% CO2 dan disimpan pada suhu 4°C bisa mencapai 15 hari. MAP yang dikombinasikan dengan antioksidan pada filet tuna sirip biru yang disimpan pada suhu 3°C selama 18 hari dapat memperpanjang umur simpan dari 2 hari (kontrol) menjadi 18 hari, dan kandungan 100% N2 dalam kemasan memiliki efek protektif terhadap haemoglobin dan oksidasi lemak. Inovasi dan kreativitas dalam pengembangan teknologi pengemasan akan terus menyediakan MAP yang berorientasi pada keinginan konsumen, pengembangan produk, ramah lingkungan, dan hemat biaya. Kata Kunci: pengemasan aktif, pengemasan atmosfir termodifikasi, produk ikan segar

SQUALEN BULETIN PASCAPANEN DAN BIOTEKNOLOGI KELAUTAN DAN PERIKANAN (BULLETIN OF MARINE AND FISHERIES POSTHARVEST AND BIOTECHNOLOGY)  Vol. 7  No. 2  August 2012

ISSN 2089-5690

LEMBAR ABSTRAK / CURRENT CONTENT

DEVELOPMENT OF REAL TIME POLYMERASE CHAIN REACTION FOR DETECTION OF Salmonella typhimurium AND Salmonella enteritidis IN FISH Pengembangan Real Time PCR untuk Mendeteksi Keberadaan Salmonella tyhpimurium dan Salmonella enteritidis pada Ikan Tuti Hartati Siregar1)*, Jennifer Elliman2) and Leigh Owens2) 1)

Research and Development Centre for Marine and Fisheries Product Processing and Biotechnology, Indonesia 2) James Cook University, Australia *Correspondence Author: [email protected]. KS. Tubun Petamburan VI Jakarta Pusat 10260

Abstract Previously designed endpoint PCR has been adapted for use with real time PCR to detect the presence of Salmonella typhimurium and Salmonella enteritidis in fish. Optimization of a standard curve in the presence of herring sperm DNA as background matrix indicated that the real time PCR highly efficient with the Pearson coefficient of determination (R2) value = 0.99937 and slope (M) value = -3.44. An enrichment method (overnight culture) significantly increased (p<0.05) the sensitivity of real time PCR. Comparison of real time PCR and the conventional isolation method based on biochemical tests has been conducted. In terms of their sensitivity, real time PCR and the conventional methods are not significantly different in the level of confidence 95%. Both real time PCR with enrichment method and conventional biochemical method can detect the presence of Salmonella spp. in spiked sample. However the direct extraction method was only detecting the presence of Salmonella in higher concentration. While the sensitivity both conventional and real time PCR are similar, the real time PCR has an advantage to detect the pathogen qualitatively and quantitatively depending on processing method. Keywords : real time PCR, Salmonella contamination, fish Abstrak Desain PCR terdahulu yang dimodifikasi telah digunakan untuk mengembangkan metode real time PCR untuk mendeteksi keberadaan Salmonella typhimurium dan Salmonella enteritidis pada ikan. Optimasi kurva standar real time PCR dengan menggunakan DNA sperma ikan herring sebagai latar belakang matriks menunjukkan kondisi real time PCR yang telah didisain mempunyai efisiensi yang tinggi dengan nilai R2 = 0.99937 dan M = -3.44. Metode pengayaan kultur dengan inkubasi selama 24 jam dapat meningkatkan sensitivitas real time PCR secara signifikan (p<0,05). Perbandingan antara metode real time PCR dengan metode konvensional berdasarkan reaksi biokimia juga telah dilakukan. Berdasarkan hasil analisis statistik dengan tingkat kepercayaan 95%, sensitivitas antara real time PCR dengan metode konvensional terlihat tidak berbeda nyata. Baik metode konvensional maupun metode molekular dengan cara pengayaan kultur dapat mendeteksi keberadaan Salmonella sp. Sementara metode real time PCR tanpa pengayaan kultur hanya dapat mendeteksi keberadaan Salmonella pada konsentrasi yang cukup tinggi. Walaupun sensitivitas tidak berbeda nyata secara statistik, real time PCR mempunyai keunggulan dibanding metode konvensional karena mampu melakukan kuantitasi terhadap sampel sementara metode konvensional hanya mampu melakukan uji kualitatif. Kata Kunci : real time PCR, kontaminasi Salmonella, ikan

Squalen Vol 7 No 2, August 2012

IDENTIFICATION AND CULTIVATION OF MFW 23-08 ISOLATED FROM MARINE SPONGES FOR BIOACTIVE COMPOUND PRODUCTION Identifikasi dan Kultivasi Kapang MFW 23-08 yang Diisolasi dari Spons Laut untuk Produksi Komponen Bioaktif Ekowati Chasanah1)*, Asri Pratitis1), and Wibowo Mangunwardoyo2) 1)

Research and Development Center for Marine and Fisheries Product Processing and Biotechnology 2) Departement of Microbiology, Faculty of Mathematic and Science, University of Indonesia *Correspondence Author: [email protected]. KS. Tubun Petamburan VI Jakarta Pusat 10260.

ABSTRACT Production of marine bioactive compound for commercial usage has been hampered due to the problem of raw material supply. To overcome this, marine microbes especially those associated with the bioactive-compound producer biota, has been explored as bioactive sources, with several advantages such as shorter production time, cheaper production cost and avoiding over exploitation of marine biota sources. Previous research showed that fungi MFW 23-08 was one of the potential isolates from Wakatobi sponges which produced bioactive compounds that was active against breast cancer cell line and as antioxidant. This study was intended to identify MFW 23-08 and optimize the production of its bioactive compound through optimization of MFW 23-08 culture. Culture optimization was conducted using 3 liquid media, i.e. malt extract broth (MEB), glucose peptone yeast (GPY), and minimal fungal media (MFM), and cultivation periods, i.e. 2, 4, 6, 8, and 10 wk. Results revealed that MFW 23-08 crude extract of 2 wk-MFM cultivation, at the concentration of 30 ì g/ml, was able to retard 87% breast cancer (T47D) cell growth. While at concentration of 100 ì g/ml, the 6 wk. MEB cultivated extract was able to hamper free radicals (56%). However, the crude extract from MFM media cultivation, in the concentration of 50 and 100 ì g/ml was not able to inhibit Escherichia coli and Staphylococcus aureus growth. Based on molecular identification using ITS1-ITS4 primers, MFW 23-08 isolate was 99% similar to Penicillium citrinum, P. griseofolvum and Penicillium sp. Keywords: bioactive, fungi, sponge, MFW 23-08, breast cancer cell ABSTRAK Produksi komponen bioaktif dari laut secara komersial dibatasi oleh permasalahan pasokan bahan baku. Untuk mengatasi hal ini, mikroba terutama yang berasosiasi dengan biota laut penghasil senyawa aktif telah dieksplorasi sebagai sumber senyawa aktif, dengan beberapa keuntungan seperti waktu produksi yang menjadi lebih pendek, produksi dengan biaya lebih rendah dan menghindari eksploitasi berlebih terhadap biota sumber senyawa bioaktif laut. Penelitian terdahulu menghasilkan kapang MFW 23-08 sebagai salah satu kapang yang berpotensi menghasilkan senyawa bioaktif antikanker payudara dan sebagai sumber antioksidan. Penelitian ini bertujuan untuk melakukan identifikasi MFW 23-08 dan optimasi produksi senyawa bioaktif yang dihasilkan oleh kapang MFW 23-08. Optimasi kultivasi dilakukan dengan menggunakan 3 medium cair yaitu malt extract broth (MEB), glucose peptone yeast (GPY), dan minimal fungal media (MFM), serta waktu kultivasi, yaitu 2, 4, 6, 8, and 10 minggu. Hasil penelitian memperlihatkan bahwa 30 ì g/ml ekstrak kasar MFW 23-08 yang dikultivasi dalam MFM selama 2 minggu mampu menghambat 87% pertumbuhan sel kanker payudara (T47D). Ekstrak kasar MFW 23-08 yang dikultivasi pada MEB selama 6 minggu, pada konsentrasi 100 ì g/ml, mampu menghambat radikal bebas 56%. Namun demikian, ekstrak yang diproduksi pada media MFM, pada konsentrasi 50 and 100 ì g/ml tidak mampu menghambat pertumbuhan Escherichia coli dan Staphylococcus aureus. Identifikasi molekuler menggunakan primer ITS1-ITS4 memperlihatkan bahwa kapang MFW 23-08 memiliki kemiripan sebesar 99% dengan Penicillium citrinum, P. griseofolvum and Penicillium sp. Kata Kunci: bioaktif, kapang, spons, `MFW 23-08, sel kanker payudara


MICROBIOLOGICAL RISK ASSESSMENT OF FISHERY PRODUCT IN INDONESIA: A PROPOSED MODEL FOR THE RISK OF Vibrio parahaemolyticus IN SHRIMP Kajian Resiko Mikrobiologi terhadap Produk Perikanan di Indonesia: Rancangan Model untuk Resiko Vibrio parahaemolyticus pada Udang Novalia Rachmawati*) and Radestya Triwibowo Research and Development Center for Marine and Fisheries Product Processing and Biotechnology *Correspondence Author: [email protected]. KS. Tubun Petamburan VI Jakarta Pusat 10260

ABSTRACT Increasing fish consumption value should be supported by enhancing the safety and quality of fish products. Microbiological level is most of importance since every food contains microorganisms which could multiplicate due to temperature abuse and time delay during handling and processing. Risk assessment is a structurally and scientifically based approach aimed to protect consumer from risk (hazard) particularly microbiological hazard when consuming certain food. Microbiological risk assessment of fishery products have not been structurally developed in Indonesia, eventhough several initial data on hazard identification have been available. As an attempt to build an integrated risk assessment, a model for microbiological risk of pathogenic Vibrio parahaemolyticus in shrimp will be proposed in this article. The available data could be used as a starting point while other required data could be collected in collaboration with other related institutions. Keywords: microbiological risk assessment, Vibrio parahaemolyticus, shrimp ABSTRAK Peningkatan nilai konsumsi produk perikanan sudah seharusnya didukung dengan peningkatan keamanan dan kualitas produk perikanan. Keamanan mikrobiologi menjadi sangat penting mengingat setiap makanan mengandung mikroorganisme yang dapat berkembang dengan cepat apabila tidak ditangani dan diproses dengan baik dan benar (pada suhu dan waktu yang tepat). Kajian resiko merupakan suatu pendekatan yang terstruktur dan berdasarkan pada kaidah ilmiah yang bertujuan untuk memberikan perlindungan kepada konsumen dari resiko (bahaya), dalam hal ini bahaya mikrobiologi, yang muncul apabila mengkonsumsi suatu makanan. Kajian resiko mikrobiologi produk perikanan belum terlaksana dengan baik di Indonesia, meskipun beberapa data awal yang mendukung kajian tersebut telah dipublikasikan. Sebagai salah satu upaya untuk membangun kajian resiko mikrobiologi yang terintegrasi, dalam artikel ini akan dipaparkan tentang rancangan model untuk kajian resiko pada Vibrio parahaemolyticus patogen pada udang. Data yang sudah ada dapat dijadikan sebagai data awal dalam kajian ini, sedangkan data lain yang masih dibutuhkan dapat diperoleh bersama-sama dengan instansi lain yang terkait. Kata Kunci: kajian resiko mikrobiologis, Vibrio parahaemolyticus, udang


THE USE OF CHEMICAL ADDITIVES FOR FISHERIES PRODUCT PRESERVATION

Penggunaan Bahan Tambahan Kimia untuk Pengawetan Produk Perikanan Syamdidi Research and Development Center for Marine and Fisheries Product Processing and Biotechnology Correspondence Author: [email protected]. KS. Tubun Petamburan VI Jakarta Pusat 10260

ABSTRACT Preservation is a common practice in processed food products including fisheries product. The purpose of preservation in food is not only maintain the quality of food but also to prolong the shelf life of food itself. Preservatives can be divided into two groups i.e. natural and chemical preservatives. The chemical preservatives potentially used in fishery industry are nitrite, sulfur dioxide, benzoic acid and, sorbic acid. These preservatives have their own characteristics on inhibition of microorganisms. Food characteristic such as pH, and aw are the key factors on the activity of antimicrobial agent. Keywords: preservatives, chemicals, antimicrobial, food, fisheries product ABSTRAK Penggunaan bahan pengawet sudah lazim dilakukan untuk produk olahan pangan termasuk produk perikanan. Bahan pengawet termasuk bahan tambahan makanan yang bertujuan untuk menjaga kualitas dan memperpanjang daya simpan produk. Bahan pengawet dapat dikelompokkan menjadi bahan pengawet alami dan bahan pengawet buatan atau yang dikenal dengan bahan pengawet kimia. Beberapa bahan pengawet kimia yang potensial digunakan dalam produk perikanan adalah nitrit, asam benzoat, asam askorbat, sulfur dioksida dan pengawet dengan cara disemprot. Bahan-bahan pengawet tersebut memiliki karakteristik dan daya hambat yang berbeda terhadap mikroorganisme. Hal ini dipengaruhi oleh beberapa faktor antara lain pH, aw dan karakteristik produk makanan itu sendiri. Kata Kunci: pengawet, kimia, antimikroba, pangan, produk perikanan


AN EMERGING MARINE BIOTECHNOLOGY: MARINE DRUG DISCOVERY Perkembangan Bioteknologi Kelautan: Upaya Menemukan Obat dari Laut Nurrahmi Dewi Fajarningsih Research and Development Center for Marine and Fisheries Product Processing and Biotechnology Corresponding author: [email protected]. KS. Tubun Petamburan VI Jakarta Pusat 10260

ABSTRACT Marine natural resources offer an opportunity to discover a novel chemical diversity with interesting pharmacologically active compounds to treat many diseases such as cancer, inflammation, bacterial and parasitic infections, and many other diseases. Marine drug discovery is a rising area in marine biotechnology. Several hits of marine-derived drug compounds were approved; two of them are Ziconotide and Trabectedin. In 2004, Ziconotide was approved as pain treatment drugs in the United States and Europe. Then, in 2007, Trabectedin was also approved as anticancer drug in Europe. The main problem in marine drug discovery research is material supply problem. Up till now, strategies to overcome the problem are “Pharmaceutical aquaculture” of biologically active marine biota and chemical synthesis approach. Chemical synthesis approach is feasible solution to be used, especially when working with less complex structure of compounds. However, when working with structurally complex compounds where total or even semi synthesis was very difficult to be provided, aquaculture can be a solution. Currently, the use of microbiology, biochemistry, genetic, bioinformatics, genomic and meta-genomic has been intensifying in order to have a better result in marine natural product drug discovery. As chemical synthesis needs an expensive investment of advanced technology and highly skilled human resources, thus pharmaceutical aquaculture is more practicable to overcome the material supply insufficiency in Indonesia. Up till now, many Indonesian marine bioprospectors have been working with culturable marine microorganism to produce bioactive compounds and some others starting to work with genomic and metagenomic-based drug discovery. Keywords: Marine biotechnology, marine drug discovery, aquaculture, chemical synthesis ABSTRAK Kekayaan hayati laut menjadi sumber yang menjanjikan dalam kegiatan pencarian senyawa kimia baru dengan aktifitas farmakologi yang unggul sebagai kandidat obat kanker, peradangan, infeksi bakteri dan parasit, serta berbagai penyakit lainnya. Penemuan senyawa obat baru dari laut merupakan salah satu bidang penting dalam bioteknologi kelautan. Hingga saat ini, beberapa obat yang dikembangkan dari senyawa bahan alam laut telah disetujui penggunaannya; dua diantaranya adalah Ziconotide dan Trabectedin. Pada tahun 2004, Ziconotide telah disetujui penggunaannya sebagai obat pereda sakit di Amerika Serikat dan Eropa. Kemudian, pada tahun 2007, Trabectedin juga disetujui untuk digunakan sebagai obat antikanker di Eropa. Masalah utama dalam pengembangan senyawa obat dari laut adalah masalah pasokan bahan baku. Dua strategi yang saat ini digunakan untuk mengatasi masalah tersebut adalah dengan budidaya biota laut penghasil senyawa kimia aktif dan dengan pendekatan sintesis kimia. Ketika bekerja dengan struktur senyawa kimia yang tidak terlalu kompleks, pendekatan sintesis kimia dapat menjadi solusi untuk produksi senyawa kimia tersebut. Namun, ketika bekerja dengan senyawa kimia kompleks yang sulit untuk dilakukan sintesis total atau semi sintesis, maka budidaya dapat menjadi solusi. Saat ini, berbagai bidang ilmu seperti mikrobiologi, biokimia, genetika, bioinformatika, genomika dan metagenomika semakin intensif dikembangkan untuk mendukung usaha penemuan senyawa obat baru dari laut. Karena sintesis kimia membutuhkan investasi yang mahal terutama untuk teknologi canggih dan sumber daya manusia yang ahli, maka budidaya biota laut penghasil bahan farmasi menjadi solusi yang lebih praktis untuk mengatasi masalah pasokan bahan farmasi tersebut. Saat ini, banyak peneliti bioprospeksi kelautan Indonesia bekerja dengan mikroorganisme laut yang dapat dikultur untuk menghasilkan senyawa bioaktif dan sebagian kecil telah memulai penelitian genomik dan metagenomik sebagai usaha untuk menemukan bahan obat dari laut. Kata kunci: bioteknologi kelautan, pencarian obat dari laut, budidaya, sintesis kimia

SQUALEN BULETIN PASCAPANEN DAN BIOTEKNOLOGI KELAUTAN DAN PERIKANAN (BULLETIN OF MARINE AND FISHERIES POSTHARVEST AND BIOTECHNOLOGY)  Vol. 7  No. 3  Desember 2012

ISSN 2089-5690

LEMBAR ABSTRAK / CURRENT CONTENT

STUDY ON THE EFFECT OF POLLUTANTS ON THE PRODUCTION OF AAPTAMINES AND THE CYTOTOXICITY OF CRUDE EXTRACT FROM Aaptos suberitoides Studi Pengaruh Polutan terhadap Produksi Aaptamin dan Sitotoksisitas Ekstrak Pekat dari Aaptos suberitoides Ariyanti Suhita Dewi1)*, Tri Aryono Hadi2), Hedi Indra Januar1), Asri Pratitis1) and Ekowati Chasanah1) 1)

Research and Development Center for Marine and Fisheries Product Processing and Biotechnology, Ministry of Marine and Fisheries, Petamburan VI, Jakarta, Indonesia 10260 2) Research Center of Oceanography, Indonesian Institute of Sciences, Pasir Putih, Jakarta, Indonesia 14430 *) Corresponding author: [email protected]

ABSTRACT This experiment was conducted to study the effects of anthropogenic stressor on the spatial variability of secondary metabolites from marine sponge Aaptos suberitoides. Samplings were conducted at 7 sites in Marine National Park of Thousand Islands that are extended within 30 km off Jakarta bay on late February 2011. Sponges were collected and quantified by means of liquid chromatography coupled with photo-diode array detection, whereas, cytotoxicity of sponges extracts was determined against T47D (breast) cancer cell lines. Results showed that the spatial production of aaptamine and isoaaptamine did not correlate with the quality of their surrounding habitat, despite nitrite and nitrate levels significantly affected the bioactivity of crude extracts. Keywords: A. suberitoides, aaptamines, spatial production, cytotoxicity, pollutants ABSTRAK Penelitian ini dilakukan untuk mempelajari efek tekanan antropogenik terhadap variabilitas spasial metabolit sekunder dari spons laut A. suberitoides. Kegiatan sampling dilakukan di tujuh titik yang berada di area Taman Nasional Kepulauan Seribu yang tersebar sepanjang 30 km dari wilayah Teluk Jakarta pada bulan Februari 2011. Spons dikoleksi dan dikuantifikasi menggunakan kromatografi cair yang dilengkapi deteksi photo-diode array sedangkan sitotoksisitas ekstrak spons diukur terhadap sel kanker T47D (payudara). Hasil penelitian menunjukkan bahwa produksi spasial dari aaptamin dan isoaaptamin tidak berkorelasi dengan kualitas dari habitat disekitarnya, meskipun kadar nitrit dan nitrat mempengaruhi bioaktivitas ekstrak pekat secara signifikan. Keywords: A. suberitoides, aaptamine, produksi spasial, sitotoksisitas, polutan

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Squalen Vol 7 No 3, December 2012

SCREENING OF THERMOSTABLE PROTEASE PRODUCING MICROORGANISMS ISOLATED FROM INDONESIAN HOTSPRING Penapisan Mikroorganisme Penghasil Protease Tahan Panas yang Diiisolasi dari Sumber Air Panas Indonesia Dewi Seswita Zilda1)*, Eni Harmayani2), Jaka Widada2), Widya Asmara2), Hari Eko Irianto1), Gintung Patantis1) and Yusro Nuri Fawzya1) 1)

Research and Development Center for Marine and Fishery Product Processing and Biotechnology 2) Biotechnology Study Program, Gadjah Mada University * Corresponding author: [email protected]

ABSTRACT Although many proteases had been studied and characterized, only a few of them are commercially available. Protease thermostability is one of the crucial properties for industrial application. This research aimed to isolate and to screen the potential isolate which produce thermostable protease. There were 6 isolates (BII-1, BII-2, BII-3, BII-4, BII-6 and LII), isolated using solid Minimal Synthetic Medium (MSM) supplemented with 1.5% skim milk, that have, protease activity. Based on the 16S-rRNA gene sequencing analysis, isolates BII-1, BII-2 and BII6 were identified as Bacillus licheniformis, isolates BII-3 and BII-4 were identified as Bacillus subtilis, while isolate LII was identified as Brevibacillus thermoruber. Three isolates (BII-6, BII-4 and LII) were then further investigated for the second screening step using liquid MSM supplemented with 1% skim milk. The isolates (BII-6, BII-4 and LII) optimally produced protease when they were cultivated at 35, 30 and 50 oC respectively after 22 h of incubation. Protease produced by BII-6, BII-4 and LII had optimum temperature of 65, 60 and 85 oC, optimum pH at 78, 8 and 9 and stable up to 100 min at 55, 60 and 75 oC respectively. Keywords: thermostable protease, Bacillus subtillis, Bacillus licheniformis, Brevibacillus thermoruber ABSTRAK Meskipun banyak protease yang sudah dipelajari dan dikarakterisasi, hanya beberapa yang tersedia secara komersial. Ketahanan protease terhadap panas merupakan salah satu sifat penting untuk diaplikasikan di industri. Penelitian ini bertujuan untuk mengisolasi dan menapis isolat potensial yang menghasilkan protease yang tahan terhadap panas. Ada 6 isolat bakteri (BII-1, BII-2, BII-3, BII-4, BII-6 dan LII) yang diisolasi dengan menggunakan media padat Minimal Synthetic Medium (MSM) dengan penambahan 1,5% skim milk mampu menghasilkan protease. Analisis susunan gen 16S rRNA terhadap keenam isolat menunjukkan bahwa hanya ada 3 isolat berbeda yang teridentifikasi sebagai Bacillus licheniformis (BII-1, BII-2 dan BII-6), Bacillus subtilis (BII-3 dan BII-4) dan Brevibacillus thermoruber(LII). Tiga isolat (BII-6, BII-4, dan LII) selanjutnya diteliti lebih lanjut untuk penapisan kedua menggunakan medium cair MSM dengan penambahan 1% skim milk. Isolat-isolat ini menghasilkan protease secara optimal ketika dikultivasi pada suhu 35, 30, dan 50 oC untuk masing-masing isolat setelah inkubasi selama 22 jam. Protease yang dihasilkan oleh BII-6, BII-4 dan LII berturut-turut mempunyai suhu optimum 65, 60 dan 85 oC, pH optimum 7-8, 8-9 dan stabil sampai 100 menit pada suhu 55, 60, dan 75 oC untuk masing-masing isolat. Kata kunci: protease tahan panas, Bacillus subtillis, Bacillus licheniformis Brevibacillus thermoruber


SCREENING AND CHARACTERIZATION OF L-GLUTAMINASE PRODUCED BY BACTERIA ISOLATED FROM SANGIHE TALAUD SEA Penapisan dan Karakterisasi L-Glutaminase yang Diproduksi oleh Bakteri dari Perairan Sangihe Talaud Tanti Yulianti3), Ekowati Chasanah1)*, and Usman Sumo Friend Tambunan2) 1

Research and Development Center for Marine and Fisheries Product Processing and Biotechnology 2 Department of Chemistry, Faculty of Mathematics and Science, Universitas Indonesia 3 National Agency of Drug and Food Control * Corresponding author: [email protected]. KS. Tubun Petamburan VI Jakarta Pusat 10260

ABSTRACT L-glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) is a very important enzyme due to its role as flavor enhancer and antileukemic agent. Salt-tolerant L-glutaminase produced by marine bacteria is favorable in food industries. This study describes the screening of Lglutaminase producing marine bacteria from Sangihe-Talaud Sea, North Sulawesi, Indonesia. Screening of L-glutaminase was performed using a liquid medium and identification of selected isolate was performed using molecular-based 16S rDNA. Results showed that there were 7 isolates produced positive results of L-glutaminase, and one of them (II.1 isolate) produced the highest activity, i.e 147.99 U/L, equivalent to the specific activity of 62.32 U/mg. The isolate then selected for further study. Bacterial identification based on 16S rRNA sequencing has revealed that the isolate was 96% similar to Pseudomonas aeruginosa strain CG-T8. Characterization of extracellular L-glutaminase from the II.1 isolate showed that the enzyme worked optimally at temperature of 37-45 °C and pH 7. The enzyme was stable when NaCl solution was added up to 8% and began to decrease on addition of NaCl solution of 16% and 20% with relative activity of 79% and 74%, respectively. The effect of metal ions, e.g Mn2+, Mg2+, and Co2+ in the form of chloride salt, were able to increase enzyme activity, whereas the addition of other metal ions (Zn2+, Fe3+, and Ca2+) decreased the activity. The molecular weights of L-glutaminase was estimated around 42 kDa and 145 kDa. Keywords: L-glutaminase, marine bacteria, 16S rRNA, screening, characterization

ABSTRAK L-glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) merupakan enzim yang sangat penting karena perannya sebagai penghasil flavor dan anti leukimia. L-glutaminase yang tahan garam yang diproduksi oleh bakteri laut sangat diharapkan oleh industri pangan. Penelitian ini bertujuan untuk melakukan penapisan bakteri laut penghasil enzim dari perairan Sangihe-Talaud, Sulawesi Utara, Indonesia. Penapisan dilakukan dengan menggunakan medium cair, dan identifikasi bakteri penghasil enzim dilakukan secara molekuler menggunakan 16S rDNA. Hasil penelitian memperlihatkan bahwa dari 7 isolat yang positif L-glutaminase, 1 (isolat II.1) isolat menghasilkan aktivitas enzim tertinggi yaitu 147,99 U/L, setara dengan aktifitas spesifik 62,32 U/mg. Isolat ini selanjutnya dipelajari lebih lanjut. Berdasarkan identifikasi molekuler, isolat ini memiliki kemiripan 96% dengan Pseudomonas aeruginosa strain CG-T8. Hasil karakterisasi enzim menunjukkan bahwa enzim ini bekerja optimal pada suhu 37-45 °C and pH 7. Enzim stabil ketika dilakukan penambahan NaCl sampai dengan 8% dan mulai berkurang ketika penambahan mencapai 16 dan 20%. Penambahan ion logam Mn2+, Mg2+, and Co2+ dalam bentuk garam klorida mampu meningkatkan kinerja enzim sementara penambahan ion Zn2+, Fe3+, and Ca2+ mengurangi aktivitas enzim. Enzim ini diperkirakan memiliki berat molekul 42 kDa dan 145 kDa Kata kunci: L-glutaminase, bakteri laut, 16S rRNA, penapisan, karakterisasi


EFFECT OF CHOPPING STEP AND DRYING TECHNIQUE ON THE QUALITY OF ALKALI TREATED COTTONII (ATC) Pengaruh Tahap Pencacahan dan Teknik Pengeringan terhadap Mutu Alkali Treated Cottonii (ATC) Singgih Wibowo1)*, Muhamad Darmawan1), Arif Rahman Hakim1), Seruni Marsella2) 1)

Research and Development Center for Marine and Fisheries Product Processing and Biotechnology 2) Swiss German University, Serpong, Tangerang *Corresponding author: [email protected]. KS. Tubun Petamburan VI, Jakarta Pusat 10260

ABSTRACT A research related to the production of alkali treated cottonii (ATC) had been carried out in order to study the effect of chopping step and drying techniques on the quality of ATC produced. Four treatments were applied in the experiment, namely was copping before sun drying (treatment A); chopping after sun drying (B); chopping before mechanical drying (C); and chopping after mechanical drying (D). The quality parameters of ATC measured were gel strength, moisture content, viscosity, yield, and whiteness. The results showed that the quality of ATC was significantly affected by chopping step and drying technique, especially in ATC gel strength, viscosity and yield. However, the effect of chopping step and drying technique was insignificant to ATC moisture content and whiteness. Chopping seaweed before drying resulted in higher gel strength of the ATC but lower in yield, while chopping after drying tended to result in lower gel strength but higher viscosity and yield. Keywords: drying technique, alkali treated cottonii (ATC), chopping step, quality ABSTRAK Penelitian yang berkaitan dengan produksi alkali treated cottonii (ATC) telah dilakukan untuk mempelajari pengaruh tahapan pencacahan rumput laut dan cara pengeringan terhadap mutu ATC yang dihasilkan. Empat perlakuan digunakan di dalam penelitian ini, yaitu pencacahan sebelum pengeringan dengan penjemuran (perlakuan A); pencacahan setelah pengeringan dengan penjemuran (B); pencacahan sebelum pengeringan dengan pengering mekanis (C); dan pencacahan setelah pengeringan dengan pengering mekanis (D). Parameter mutu yang diamati adalah kekuatan gel, kadar air, viskositas, rendemen, dan derajat keputihan ATC yang dihasilkan. Hasil analisis menunjukkan bahwa mutu ATC dipengaruhi oleh tahapan pencacahan dan cara pengeringan, terutama terhadap kekuatan gel, viskositas, dan rendemen. Pencacahan rumput laut sebelum penjemuran menghasilkan ATC dengan kekuatan gel tinggi tetapi viskositas dan rendemen lebih rendah, sedangkan pencacahan setelah pengeringan menghasilkan kekuatan gel yang lebih rendah tetapi dengan viskositas dan rendemen yang tinggi. Kata Kunci: teknik pengeringan, Alkali Treated Cottonii (ATC), tahap pencacahan, mutu

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FUCOIDAN FROM BROWN SEAWEED AND ITS BIOACTIVITY Fukoidan dari Rumput Laut Coklat dan Bioaktifitasnya Ellya Sinurat1)* and Endar Marraskuranto1) Research and Development Center for Marine and Fisheries Product Processing and Biotechnology, Ministry of Marine and Fisheries, KS. Tubun Petamburan VI, Jakarta Pusat 10260* Corresponding author: [email protected].

ABSTRACT Fucoidan is a polysaccharide which substantially consists of L-fucosa and ester sulphate group and is mainly contained in brown seaweed. For the past ten years, bioactivity studies of fucoidan has been conducted. Recently, fucoidan has been examined for its application in drugs. In a couple of years, fucoidan structure was succesfully identified and its bioactivity was revealed. Fucoidan exhibits various bioactivities such as anticoagulant, antioxidant, anticomplementary, anti-inflamation, gastric protector, and blood lipid level control. This review gives some brief progress in isolation and bioactivity study of fucoidan from brown seaweeds. Key words: fucoidan, brown seaweed, bioactivity, L-fucose ABSTRAK Fukoidan adalah senyawa polisakarida yang secara substansional terdiri atas L-fukosa dan golongan ester sulfat, terutama terdapat pada rumput laut coklat. Dalam jangka waktu sepuluh tahun terakhir, bioaktivitas dari fukoidan telah banyak diteliti. Bahkan belakangan ini telah diteliti aplikasi fukoidan untuk obat. Dalam beberapa tahun terakhir, struktur fukoidan telah berhasil diidentifikasi dan bioaktivitasnya berhasil diketahui. Fukoidan mempunyai banyak bioaktifitas antara lain sebagai antikoagulan, antioksidan, antikomplementari, anti pembengkakan, pelindung lambung, dan pengatur kadar lipid darah. Review ini memberikan ringkasan beberapa kemajuan penelitian isolasi dan bioaktivitas fukoidan dari beberapa jenis rumput laut coklat penghasil fukoidan. Kata kunci: fukoidan, rumput laut coklat, bioaktivitas, L-fukosa

KEYWORDS INDEX/INDEKS KATA KUNCI

A A. suberitoides aaptamines active packaging antimicrobial aquaculture B Bacillus licheniformis Bacillus subtillis bacteria bioactive bioactivity breast cancer cell Brevibacillus thermoruber brown seaweed C characterization chemical shynthesis chemicals Chitosan (CTS) cytotoxicity D deep sea diversity docosahexanoic acids E extracellular enzyme F fatty acids fish fisheries product food freezing-thawing fresh fish product fucoidan fungi G gamma irradiation

97 97 39 79 89 105 105 19 59 131 59 105 131 115 89 79 1 97 19 19 29 11 29 51 79 79 1 39 131 59 1

H hydrogel I identification K KLU 11.16 L L-fucose L-glutaminase M marine bacteria Marine biotechnology marine drug discovery MFW 23-08 microbiological risk assesment modified atmosphere packaging P PCR pollutants polyvinyl alcohol (PV A) preservatives R real time PCR 16S rRNA S Salmonella contamination Sangihe Talaud Schizochytrium sp. screening shrimp spatial production sponge T thermostable protease T-RFLP V Vibrio parahaemolyticus

1 11 11 131 115 115 89 89 59 67 39 19 97 1 79 51 115 51 19 29 115 67 97 59 105 19 67

Squalen Vol.7 No.3 Page Jakarta, December 2012 ISSN

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