J Appl Phycol (2014) 26:923–931 DOI 10.1007/s10811-013-0126-0
Effect of epiphyte infection on physical and chemical properties of carrageenan produced by Kappaphycus alvarezii Doty (Soliericeae, Gigartinales, Rhodophyta) Charles S. Vairappan & Chong Sim Chung & Shigeki Matsunaga
Received: 24 May 2013 / Revised and accepted: 22 August 2013 / Published online: 2 October 2013 # Springer Science+Business Media Dordrecht 2013
Abstract Epiphytism of filamentous red algae is a serious problem in Kappaphycus farms in the Philippines, Indonesia, Malaysia, and Tanzania. The causative organism of epiphyte outbreak has been identified as Neosiphonia apiculata (Hollenberg) Masuda and Kogame, but its actual effect on carrageenan quality has not yet been established. Therefore, yield and quality of carrageenan from healthy and infected specimens were examined. Infected specimens showed 20.5± 2.5 % DW lower carrageenan yield compared with the healthy seaweed (65.5±4.2 % DW). Infected specimens also had a higher phenolic and fatty acid content, compared with healthy specimens. The carrageenan from the infected seaweed showed 74.5±2.8 % lower viscosity, 52.6±3.6 % lower gel strength, 22.9±1.5 % higher syneresis, and 5 °C higher melting temperature as compared with carrageenan from healthy specimens. FTIR and 13C-NMR analysis of carrageenan from infected seaweed did not show any differences in their functionality or carbon atom chemical shift as compared with healthy and standard k -carrageenan. However, size exclusion chromatography showed the infected carrageenan molecular size to be 80 kDa as compared with 800 kDa for the healthy and standard k-carrageenan. These findings prove that infection of Kappaphycus by the filamentous red algae epiphyte, N. apiculata, reduces carrageenan molecular size and affects the physical properties of the carrageenan.
C. S. Vairappan (*) : C. S. Chung Laboratory of Natural Products Chemistry, Institute for Tropical Biology and Conservation, University Malaysia Sabah, 88999 Kota Kinabalu, Sabah, Malaysia e-mail:
[email protected] S. Matsunaga Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Science, The University of Tokyo, 1-1-1 Yayoi( Bunkyo-Ku Tokyo 113-8657, Japan
Keywords Epiphyte . Neosiphonia apiculata . Kappaphycus alvarezii . Carrageenan quality
Introduction The red alga Kappaphycus alvarezii is farmed as raw material for the extraction of carrageenan and is considered an important commercial commodity in the Philippines, Indonesia, Malaysia, and Tanzania (McHugh 2003; Critchley et al. 2004; Vairappan et al. 2008; Hurtado et al. 2008). The increasing demand for carrageenan by the industry because of its diverse product applications makes Kappaphycus an important marine commodity. More than 90 % of the global carrageenophyte culture farms are located in the coastal waters of South East Asian countries (Werner et al. 2004; Vairappan et al. 2001), with Indonesia producing 80 % of the global supply. Kappaphycus farming is a labor-intensive activity, and the yield is often dependent on culture conditions and disease outbreak. Two major problems encountered in high-density commercial farming are “ice–ice” disease and epiphyte outbreaks. Trono (1974) first discovered the emergence of ice–ice disease in the Philippines, since then, involvement of pathogenic microbes and the role of culture environmental conditions have been described in detail by Uyenco et al. (1981) and Largo et al. (1995a, b) as the causative factors for this disease. These findings also led to a better understanding on how stress-induced culture conditions could trigger pathogenesis (Collén et al. 1994; Mtolera et al. 1995, 1996b). The pathogenic agents were identified as part of the Vibrio-Aeromonas and Cytophaga-Flavobacterium complex and their effects were described to be due to lytic enzymatic activities of bacterial exudates (Largo et al. 1995a, 1999; Pedersén et al. 1996b; Vairappan 2006; Gachon et al. 2011). Although an epiphyte outbreak in seaweed farms is not a new phenomenon, similar detailed information on how they occur is
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currently not available. Doty and Alvarez (1975) reported occurrences of epiphytes in Kappaphycus farms, however, the industry was not interested in this problem until lately (Critchley et al. 2004; Hurtado et al. 2006; Vairappan 2006). Recent investigation on epiphyte outbreak revealed Neosiphonia apiculata (Hollenberg) Masuda and Kogame (Masuda et al. 2001) as the causative agent and information is now available on the symptoms, seasonality, and the occurrence of secondary bacterial infection on the epiphyte-infested plants (Vairappan et al. 2008). The occurrence of epiphyte outbreaks in Malaysia, Indonesia, and the Philippines has resulted in a serious reduction of biomass production and decline in carrageenan quality (Vairappan 2006; Hurtado et al. 2006; Vairappan et al. 2008). However, given the lack of concrete evidence on the effect of epiphyte on carrageenan quality, the farmers and fisheries agencies in the major producing countries are not fully aware of the direct effect of epiphyte infection on the quality of the produced carrageenan. This investigation was conducted to show the effect of epiphyte infection on the quantity and quality of carrageenan produced by an infected crop.
Materials and methods Sampling locations Infected and healthy seaweed specimens for carrageenan yield and quality analysis were collected from culture farms in Semporna, Malaysia (6°02′10″ N, 116°01′02″ E). Specimens were cleaned of organic detritus and contaminants, kept in fresh seawater, and then transported to the laboratory under cool conditions (14±2 °C) for 4 h. At the laboratory, specimens were further cleaned using a soft tip brush and a careful selection of healthy and infected materials was made prior to processing and extraction. Plants used for extraction were randomly selected from the healthy and infected piles (n =5). Epiphyte identification and enumeration Epiphyte-infected specimens were cleaned to remove organic contaminations. Thallus of the seaweed were manually immersed in seawater and viewed under a stereo-microscope for thallus degeneration. Presence of "goose-bump"-type symptoms and filamentous algae was classified as infected specimens, whereas the healthy seaweed thallus was void of both. Images were taken using a Nikon digital camera. Epiphyte densities were enumerated visually on 1x1 cm2 sections of seaweed at ×5 magnification. A total of five specimens of infected and healthy seaweed thallus were enumerated. Epiphytes were removed using a pitch stick and forceps, stained with 0.5 % (w/v) cotton blue in lactic acid/ phenol/glycerol/water (1:1:1:1 (v/v)) solution and mounted in 50 % glycerol/seawater on microscope slides. Slides were viewed at ×10 and ×40 magnifications under a compound microscope (Axioskop 40; Carl Zeiss, Germany).
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Total phenolics Total phenolic content in seaweed extracts was determined using the Folin–Ciocalteu assay that is commonly used for plant tissue analysis (Chan et al. 2007). Freeze dried seaweed (1.0 g) from both treatments (n =5) was ground to fine powder and soaked in 10 mL of 80 % (v/v) aqueous methanol for extraction. The mixture was sonicated for 2 min and kept in the dark at 4 °C for 1 h. The resulting solution was filtered and an aliquot of 0.5 mL was mixed with 1.5-mL distilled water and 1.0 mL of 40 % Folin–Ciocalteu phenol reagent and mixed. After 5 min, the mixture was made alkaline with 1.0 mL, 2 N Na2CO3, mixed, and the resulting mixture was kept at 50 °C in the dark for 30 min. Total phenolic contents were determined calorimetrically at 765 nm using a UV–vis spectrophotometer. Gallic acid equivalent (GAE; Sigma-Aldrich) was used as the standard plot curve and total phenolic content was expressed as GAE (in mg g−1). The calibration equation for GAE was y =0.0110x +0.0142 (R 2 =0.99; n =8). Carrageenan extraction and purification Approximately 5.0 g of room temperature (26–28 °C) dried K. alvarezii powder (200 mesh) from each treatment (n =5) was suspended in 500 mL of 4 % NaOH and 0.25 % NaBH4. The mixture was heated in a water bath at 100 °C with constant stirring for 3 h. The resulting solution was allowed to cool, clarified by pressure filtration through a Whatman GF/D glass fiber filter. The solution was then neutralized to a pH 7 with acetic acid. The alkali-modified carrageenan was dialyzed against distilled water using a 12,000 MWCO dialysis membrane (Spectrum, USA) and then freezedried. The same process was conducted for the epiphyteinfected seaweed samples. Melting point determination A total of 6 g (n =5) for both treatments were added to 400 mL of distilled water in a 500-mL beaker to form a 1.5 % carrageenan solution. The solution was then heated in a water bath at 100 °C for 30 min with continuous stirring. The resulting solution was then poured as a thin layer into a Petri dish and left in the refrigerator for 24 h and allowed to set. The gel was then cut into a 1-mm3 cube. Using a Fisher Scientific (USA) melting point apparatus, the temperature was slowly raised at 10 °C min−1 increments until the entire cube was dissolved, at which the temperature was noted. Synaeresis index Synaeresis was carried out to measure the water retaining potential of the carrageenan gel. The gel was prepared by adding 6 g of carrageenan into 400 mL of distilled water to form a 1.5 % carrageenan solution (n =5). The carrageenan solution was then boiled at 100 °C in a water bath for 30 min with continuous stirring. The solution was then poured into a plastic mold in replicates (n =5) until the mold was completely full. The mold was then heat sealed with a plastic cover and left in a refrigerator (4 °C) to set for a period of 24 h. The gel was weighed (A) after wiping the mold's outer surface. Subsequently, the plastic covering was removed and a dry cloth
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was used to wipe off excessive water from the carrageenan gel. The cover and mold, after drying, were also weighed (B). The dry gel was then inserted back into the mold and weighed (C). Synaeresis was calculated according to the following formula: Synaeresisð%Þ ¼
Initial weightðAÞ−Final weightðCÞ 100 Initial weightðAÞ−ðCover and mould weightðBÞÞ
Viscosity Viscosity was tested on two types of carrageenan gel for both the treatments (n =5), potassium chloride (KCl) gel, and water gel. For the KCl gel, 6 g of carrageenan was added into a 500-mL beaker along with 10 mL of KCl solution and 390 mL of distilled water. For the water gel, 6 g of carrageenan was added into a 500-mL beaker with 400 mL of distilled water. Both solutions were then placed into a water bath at 100 °C for 30 min, with continuous stirring. The resulting solution was removed and left to cool at room temperature (26 °C) until it reached a temperature of 75 °C. Sample temperature was maintained by using a water bath. Measurements for viscosity were taken using a Brookfield digital viscometer (Model LVDV-I+) using a number 2 spindle at 60 rpm.
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carrageenan, and infected seaweed carrageenan (n =5) were dissolved in 2.0 mL distilled water by heating in a water bath at 90 °C for 1 h. Films were prepared by drying the solutions on a polyethylene surface at 60 °C, kept in a desiccator until use and analyzed using a Shimadzu Prestige 21 Fourier Transform Infrared spectrophotometer (Shimadzu, Japan). Carrageenan 13C-NMR analysis Approximately 200 mg of the respective carrageenan samples (standard k-carrageenan, healthy seaweed carrageenan, and infected seaweed carrageenan (n =5) were dissolved in 3.0 mL of 1:1 H2O/D2O by a water bath that was heated at 90∼100 °C. The resulting solution was transferred into a 5-mm o.d. NMR tube, and DMSO was added as an internal reference. The 13C-NMR spectra was recorded at 60 °C at 150 MHz using JEOL ECA 600 MHz NMR spectrometer with the following parameters; 45° pulse width=5.8 μs, pulse delay=0.25 s, 5,000 scans, and reference set to DMSO at 39.5 ppm.
Gel strength After the pH had been measured, the solution was used to undergo gel strength measurements for both the treatments (n =5). The solution was poured into twin cylindrical glass molds and left at room temperature (26 °C) for 4 h. The molds were then placed into a refrigerated incubator bath at 10 °C for 1.5 h. The top of the gel was trimmed off and gel strength was tested using a rheometer (Model CR-500DX COMPAC 100) with a 0.75-cm2 spindle. Three readings were taken for each gel mold Carrageenan molecular size determination Molecular size determination was done based on size exclusion chromatography. Approximately 30 mg of freeze-dried standard, healthy, and infected carrageenan (n =5) were suspended in 1 mL of ultrapure water. The solutions were stirred until completely dissolved at 40 °C for 6 h. The samples were then filtered through 0.45 μm PTFE syringe filters and submitted to a size exclusion chromatography reading using Shimadzu HPLC equipped with Waters Ultrahydrogel column (Waters, USA). Elution was monitored by Shimadzu RI detector using 0.1 M NaNO3 as the mobile phase at a flow rate of 1.0 mL min−1, calibrated with SHODEX standards. SHODEX standards used were STANDARD P-82, which consist of eight molecular size markers; (1) P-5 (5,000 Da), (2) P-10 (10,000 Da), (3) P-20 (20,000 Da), (4) P-50 (50,000 Da), (5) P-100 (100,000 Da), (6) P-200 (200,000 Da), (7) P-400 (400,000 Da), and (8) P-800 (800,000 Da). Carrageenan FTIR analysis Approximately 10 mg of respective carrageenans (standard k-carrageenan, healthy seaweed
Fig. 1 K. alvarezii infected with N. apiculata. a Stereo-microscope image of seaweed thallus infected with epiphyte; b scanning electron microscope image of seaweed thallus covered with “goose-bump”-like mounts showing epiphyte intrusion into the thallus
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Statistical analysis All statistical tests were carried out using the statistic package SPSS (version 11.0). All analyses were conducted for a sample size of n =5, hence a nonparametric paired sample t test was conducted. In all tested data, p
