Available online at www.sciencedirect.com ScienceDirect

Aquatic Procedia 7 ( 2016 ) 106 – 111

2nd International Symposium on Aquatic Products Processing and Health ISAPPROSH 2015

Extraction and Characterization of Refined K-carrageenan of Red Algae [Kappaphycus alvarezii (Doty ex P.C. Silva, 1996)] Originated from Karimun Jawa Islands

Godras Jati Manuhara*, Danar Praseptiangga, Rachmad Adi Riyanto

Department of Food Science & Technology, Sebelas Maret University, Jl. Ir.Sutami No.36A., 57101 Kentingan, Surakarta, Indonesia

Abstract

The properties of refined κ-carrageenan (RC) extracted from red algae originated from Karimun Jawa Island has never been studied. The objective of this research was determining the effect of KCl on characteristic of refined κ-carrageenan extracted from the red algae [Kappaphycus alvarezii (Doty ex P.C. Silva, 1996)]. The result indicated that higher KCl concentration resulted in the increase of the carrageenan yield, ash and sulphate content, and the decrease of gel strength, moisture and acid- insoluble ash content. The carrageenan viscosity demontrated a fluctuated value due to KCl concentration. This study suggested extraction process by using 2.5 % KCl solution. The carrageenan yield was 34.3 % and the result demonstrated the carrageenan properties as follow: 8.20 cP viscosity, 94.45 g · cm–2 gel strength, 6.3 % moisture content, 59.4 % ash content, 1.78 % acid- insoluble ash, and 7.75 % sulphate content. The absrbance peak at 849 cm–1 detected from infrared spectrscopy indicated D- galactose-4-sulphate which is related with κ-carrageenan.

© 2016 TheThe Authors. Authors. Published Published by by Elsevier Elsevier B.V. B.V. This is an open access article under the CC BY-NC-ND license Peer-review(http://creativecommons.org/licenses/by-nc-nd/4.0/ under responsibility of the Science ).and Editorial Board of ISAPPROSH 2015. Peer-review under responsibility of the science and editorial board of ISAPPROSH 2015 Keywords: Extraction; Kappaphycus alvarezii (Doty ex P.C. Silva, 1996); Karimun Jawa Islands; KCl; refined κ-carrageenan.

* Corresponding author. Tel. : +62 813 2905 2043 E-mail address: [email protected]

2214-241X © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the science and editorial board of ISAPPROSH 2015 doi: 10.1016/j.aqpro.2016.07.014 Godras Jati Manuhara et al. / Aquatic Procedia 7 ( 2016 ) 106 – 111 107

1. Introduction

Carrageenan is polysaccharides extracted from certain red algae and formed by D-galactose and 3,6-anhydro- galactose units which is linked by α-1,3 and β-1,4 glycosidic. The carrageenan contain sulphate ester about 15 % to 40 % and average molecule mass above 100 kDa. It is classified into several types such as λ, κ, ι, ε, μ, which contain 22 % to 35 % of sulphate group. Carrageenan characteristic is affected by amount and position of sulphate ester group of 3,6 anhydro-galactose content. Kappa-carrageenan is used in acetic acid production, cleaning of industrial effluents and also as thickening and gelling agent in food industries (Iglauer et al., 2011; Necas and Bartosikova; 2013; Azevedo et.al., 2013). The availability of raw materials for carrageenan production is assured with the large increasing in seaweed farming areas in Philipines and Indonesia (Thomas, 1999). The properties of semi refined κ-carrageenan (SRC) from red algae [Kappaphycus alvarezii (Doty ex P.C. Silva, 1996)] widely cultivated in Karimun Jawa Islands, Central Java had been studied. The finest structure of SRC was resulted from solar tunnel drier which result in high gel strength and lowest sulphate content. The result demonstrated carrageenan characteristic spectra at absorption band at 848,68 cm–1 that corresponded to sulphation (galactose-4-sulphate level) and indicated that the carrageenan was kappa type (Dewi et al., 2005). However, refined κ-carrageenan (RC) from red algae (Kappaphycus alvarezii) widely cultivated in Karimun Jawa Islands, Central Java has never been studied. In this study, the κ-carrageenan was extracted by hot alkaline solution (Ca(OH)2) and recovered from the solution. The alkaline solution removes some of the sulphate groups from the molecules and increases the formation of 3,6- AG that leads to increased gel strength of carrageenan (Yasita, 2010). The result of previous research indicated that KCl affected the properties of refined carrageenan. The most optimum result was performed by treatments combination as follow 1 % KCl solution, algae and water ratio (1:20), and precipitation temperature at 30 °C. The application of KCl solution increased refined carrageenan gel strength (Arfini, 2011). Therefore, in this study, various concentration of KCl solution (1.5 %, 2.5 %, and 3.5 %) was employed in coagulation process of carrageenan in order to investigate the effect of KCl solution on the carrageenan properties and finally, the best concentration of KCl solution which demonstrated optimum properties of carrageenan would be determined.

2. Materials and methods

2.1. Materials

The red algae (K. alvarezii) was harvested 45 d after planting from Karimun Jawa Islands, Jepara, Central Java. Supporting materials in extracting carrageenan such as water, Ca(OH)2, 96 % alcohol aquades, 1 % HCl, KCl and also materials for analysis were supplied by local distributor.

2.2. Methods

2.2.1. Carrageenan extraction

Dried red algae (K. alvarezii) was washed using flowing water and soaked in 3 L water for 24 h. After soaking, the algae was cut by using scissors and minced by using a blender resulting in the pulp of algae. The pulp was then mixed with the water by comparison 1:80 (v / v). The mixture was conditioned into alkaline condition (pH ± 9) by Ca(OH)2 solution, then the extraction was performed by warming at 90 °C for 2 h by stirring continuously. After the extraction, the waste of algae (solid materials) was separated from the viscous filtrate. Then the filtrate was neutralized with of 1 % HCl solution until pH 7, then reheated at 60 °C for 30 min. The filtrate was coagulated by using KCl solution (1.5 %, 2.5 %, or 3.5 %) with filtrate and KCl solution ratio was 1 : 1 and stirred continuously for 15 min, then the final mixture was filtered to separate carrageenan gel and water. Carrageenan gel was then completely soaked in 96 % alcohol for an hour and stirred continuously. The carrageenan gel was separated from alcohol and water by filtration. The carrageenan was dried by using cabinet dryer at 70 °C for 24 h and milled into 80 mesh size. 108 Godras Jati Manuhara et al. / Aquatic Procedia 7 ( 2016 ) 106 – 111

2.2.2. Analysis

Yield was calculated by dividing carrageenan weight by dried algae weight. Viscosity was measured by viscometer brookfield as follow: carrageenan solution (1.5 %) was heated on a hot plate and stirred regularly until 80 °C and then its viscosity was measured by spindel. Gel strength was determined by preparing carrageenan solution and then the solution was analyzed by Testing Machine MPY (PA-104-30). Moisture, ash, and acid- insoluble ash content were measured by gravimetric method (AOAC, 1990). In order to perform infrared spectroscopy, two milligrams of the RC sample were mixed with 200 mg KBr and pressed into a homogenous pellet and then the pellet was immediately put into the sample holder of FTIR spectrophotometer in which the spectra bands were recorded in the range of 4 000 to 400 cm–1. All treatments and analytical determinations were performed in duplicate. Statistical analysis was performed using SPSS for windows (version 19). The differences of mean ± values among samples varieties was determined using one-way analysis of variance (ANOVA) followed by Duncan Multiple Range Test.

3. Result and discussions

The higher KCl concentration resulted in the increase of the carrageenan yield, ash and sulphate content, and the decrease of gel strength, moisture and acid-insoluble ash. The carrageenan viscosity demontrated a fluctuated value due to KCl concentration (see Table 1).

Table 1. The properties of carrageenan Properties KCl 1.5 % KCl 2.5 % KCl 3.5 % Yield (%)* 19.50 ± 8.76 a 31.32 ± 5.00 ab 43.91 ± 2.88 b Viscosity (cP)* 7.45 ± 0.07 b 8.20 ± 0.14 c 6.10 ± 0.14 a Gel Strength (g · cm–2) * 152.29 ± 13.10 b 94.45 ± 2.76 a 99.07 ± 4.14 a Moisture (%)* 7.78 ± 0.15 b 6.30 ± 0.07 a 5.99 ± 0.02 a Ash (%)* 58.23 ± 0.20 a 59.44 ± 0.07 b 64.16 ± 0.05 c Acid Insoluble Ash (%)* 2.27 ± 0.04 b 1.78 ± 0.13 a 1.82 ± 0.21 ab Sulphate (%)* 6.48 ± 0.07 a 7.75 ± 0.02 b 7.93 ± 0.01 c All treatments and analytical determinations were performed in duplicate. Different letters in a column denote significant differences, P < 0.05. * Mean ± SD

3.1. Yield

The properties of carrageenan extracted by using various concentration of KCl solution were shown in Table 1. The yield of carrageenan in this research was calculated by weighing carrageenan after 80 mesh sifting. This result was close to other study (Arfini, 2011), 21.76 % to 34.02 %. The yield of carrageenan of this research was 19.50 % to 43.91 %. The yield of carrageenan by treatment 2.5 % and 3.5 % of KCl met the requirement of the Department of Commerce of Indonesia (Herliany, 2011), which is minimum 25 % yield. Carrageenan yield resulted from extraction with 2.5 % KCl solution was lower than the other study (Herliany, 2011), but higher than the yield from the same seaweed species cultivated in Vietnam (Ohno et al., 1996). The difference in yield was due to the difference of the extraction method. Herliany (2011) employed extraction method with 0.5 % KOH solution.

3.2. Viscosity

The highest viscosity, 8.20 cP [A centipoise is one millipascal second (mPa · s) in SI units] was performed by carrageenan extracted with 2.5 % KCl solution, and the lowest one, 6.10 cP was performed by carrageenan extracted by 3.5 % KCl solution. The salts decrease viscosity of carrageenan by decreasing electrostatic repulsion Godras Jati Manuhara et al. / Aquatic Procedia 7 ( 2016 ) 106 – 111 109 between sulphate groups (Moirano, 1977). Therefore, the highest concentration of KCl solution resulted in low viscosity. Other research (Arfini, 2011) also found that carrageenan viscosity by using treatment of 1 % KCl was higher than 1.5 % KCl. The highest viscosity of carrageenan resulted in this research was lower than the result from other studies (Dewi et al., 2015; Arfini, 2011; Herliany, 2011; Ohno et al., 1994; Webber et al., 2012). It is important to note that Arfini (2011) employed lower KCl solution (1 %). However, the carrageenan viscosity met the minimum standard (5 cP) set by FAO. The viscosity of food-grade carrageenan should range 5 cP to 800 cP (Necas and Bartosikova, 2013).

3.3. Gel strength

Iglauer et al. (2011) and Nekas and Bartosikova (2013) reported that being different with non-gelling λ- carrageenan, the ĸ-carrageenan gel strength increased as the increase of concentration of K+ and Ca2+ cation when chloride salt was added. However, this result demonstrated different effect of the cation. The highest gel strength was performed by carrageenan extracted with 1.5 % KCl solution. But, it was much lower compared with the results from other studies (Dewi et al., 2015; Arfini, 2011; Herliany, 2011; Ohno et al., 1996), but higher than the result of (Webber et al., 2012). Gelling properties of carrageenan are useful in wide range of water- and milk-based food products (Imeson, 2000).

3.4. Moisture content

The result showed that moisture content of carrageenan was 5.99 % to 7.78 %. The moisture content of all carrageenan samples met the FAO standard. The carrageenan moisture content was lower compared to the results of previous studies (Arfini, 2011; Herliany, 2011).

3.5. Ash content

High concentration of KCl solution might cause high ash content of the carrageenan, so that higher concentration of KCl solution, the higher ash content. High KCl concentration cause high content of kalium. Ariyati et al. (2007) reported that salinity level of the coastal area of Karimunjawa Island is high, about 34 % to 35 % in average. It cause high content of mineral in algae cultivated in Karimunjawa. The ash content of dried red algae (K. alvarezii) harvested in Karimunjawa is 27.54 %. The ash content of the carrageenan (58.23 % to 64.16 %) was out of FAO standard (15 % to 40 %). The ash content in the carrageenan (58.23 % to 64.61 %) was much higher than the ash content of carrageenan in the previous research (Arfini, 2011; Herliany, 2011). The ash content were 35.9 % and 29.59 % respectively.

3.6. Acid-insoluble ash content

The result showed the acid-insoluble ash content of the carrageenan was 1.78 % to 2.27 %. The carrageenan extracted by 2.5 % and 3.5 % KCl solution indicated the acid-insoluble ash content met the maximum standard set by FAO and FCC [16], but the treatment by 1.5 % KCl demonstrated higher acid insoluble ash. Acid-insoluble ash content indicated minerals or metals contamination in a product which is insoluble in acid.

3.7. Sulphate content

The sulphate content of carrageenan in this research was 6.476 % to 7.929 %, lower than quality standard set by FAO and FCC (15 % to 40 %), but higher than previous research (Arfini, 2011; Herliany, 2011) or commercial carrageenan. The content of sulphate caused a repel force between negatively charged sulfate groups, so that the polymer chains get stiff and taut, causing an increase in viscosity. However, this result indicated unclear correlation between the sulphate content and viscocity, since the viscocity did not follow the sulphate content trend. 110 Godras Jati Manuhara et al. / Aquatic Procedia 7 ( 2016 ) 106 – 111

3.8. Infrared spectroscopy

Fig. 1 showed FTIR spectrum of carrageenan extracted by 2.5 % KCl solution. Several absorption peaks observed in this study indicated the presence of sulphate ester (1234 cm–1), glycosidic linkage (1072 cm–1), 3,6- anhydro-d-galactose (926 cm–1), and D-galactose-4-sulphate (849 cm–1). The former peak, which was also observed at 840 cm–1 to 850 cm–1 in other studies (Dewi et al., 2015; Webber et al., 2012), indicated that the carrageenan was κ-type.

Fig. 1. FTIR analysis of refined carrageenan extracted with 2.5 % KCl solution

4. Conclusion

Higher KCl solution concentration resulted in the increase of the carrageenan yield, ash, and sulphate content, and the decrease of gel strength, moisture, and acid-insoluble ash content. This study suggested extraction process by using 2.5 % KCl solution. The carrageenan yield was 34.3 % and the result demonstrated the carrageenan properties as follow: 8.20 cP viscosity, 94.45 g · cm–2 gel strength, 6.3 % moisture content, 59.4 % ash content, 1.78 % acid-insoluble ash, and 7.75 % sulphate content. The carrageenan was confirmed as κ-carrageenan based on the infrared spectrum.

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