· web viewcollected in cagayan, pangasinan, bulacan and sorsogon provinces, respectively....

24 (2016): 1-10

Original Research

Agar yield and quality of Gracilaria spp, Gracilariopsis heteroclada and Hydropuntia edulis

Christoper D. Del Rosario*, Ma. Salvacion R. Ferrer, Editha E. Gamboa, Minerva T. Moises, Josefino Mondragon

National Fisheries Research and Development Institute Corporate 101 Building, Mother Ignacia Street, South Triangle, Quezon City, 1103 Philippines

Received 27 November; Accepted 03 January 2015

Abstract

This study was conceptualized to further provide information on family Gracilariaceae and agar in

Luzon, Philippines. In this study, agar polysaccharides were extracted using 4% NaOH from field

samples of Gracilaria firma, G. manilaensis, Gracilariopsis heteroclada and Hydropuntia edulis

collected in Cagayan, Pangasinan, Bulacan and Sorsogon provinces, respectively. Moisture content

(%) and yield (%) and physico-chemical properties of agar were determined and compared during

dry (February to May) and wet (July to October) seasons. The highest agar yield (17.03%) was

obtained from H. edulis sampled during dry season. Gracilaria manilaensis was more viscous (17.33

cP) with highest gel strength (205.93 g.cm-2) and melting temperature (98 C) within the studied⁰

period. Furthermore, G. firma gel strength had significant variations (p <0.01) between seasons

similar to its gelling point ability. For the melting temperature (°C), only agar extract of H. edulis

showed significant seasonal differences (p <0.01). In chemical properties, the percentage of 3,6-

anhydrogalactose and sulfate contents of agar were statistically different (p <0.01) among the

samples collected in Sorsogon (H. edulis) and Pangasinan (G. manilaensis), respectively. The agar

extracted from G. manilaensis (dry season) was found to have relatively high gel strength and

melting temperature properties in both seasons. Its melting temperature was also found to be

within the food grade agar international standards (≥85°C), similar to Gracilariopsis heteroclada. By

contrast, G. firma was found to be within the bacteriological applications standards (80±5). Lastly,

sulfate contents of the test samples were within the acceptable range (1-4%).

Keywords: Gracilaria firma, Gracilaria manilaensis, Gracilariopsis heteroclada

*Corresponding author E-mail address: [email protected] (Christoper D. Del Rosario)Copyright @ 2015 Del Rosario et al.

Del Rosario et al./The Philippine Journal of Fisheries 24 (2016): 1-10 | 2

1. Introduction

Gracilaria species are principal sources of agar worldwide (Critchley 1993), and approximately 60% of all agar is produced from this alga (Tseng 2001). Agar is a hydrocolloid extracted from cell walls of red-purple marine algae (Class Rhodophyceae). According to the US Pharmacopeia and the Food Chemicals Codex (FCC) its 1.5% aqueous solutions are clear and firms gel between 32 and 39 C which do not melt below 85 C (Murano 1995). At present, agar commands⁰ ⁰ higher price in the international market and has also an edge over the other phycocolloids because of its unique characteristics (low gelling and high melting temperatures) in biotechnology. In addition, 90% of agar consumption is used as a food ingredient while the remaining 10% is used for biotechnological applications. Currently, Gracilaria species is extensively and continually used for food grade agar production merely due to the success of alkaline treatment that really improved its quality.

In the Philippines, Gracilaria is the next most important economic seaweed to Kappaphycus/Eucheuma. About 25 species was reported in the Philippines by Silva et al. (1987), while Trono (1993) had identified 10 species of Gracilaria found in Sorsogon, namely: Gracilaria hysic, G. changii, G. eucheumoides (now renamed Hydropuntia eucheumatoides), G. fastigiata, G. firma, G. fisherii, G. gigas, G. heteroclada (now renamed Gracilariopsis heteroclada), G. salicornia and G. tenuistipitata. Several species of Gracilaria are being consumed as fresh salad or sold by local residents in Northern Luzon except in Buguey, Cagayan where tonnage of dried Gracilaria is being exported for industrial purposes (Ayson and Encarnacion 2008). Gracilaria must be developed to help the emerging industry in many coastal areas in Luzon where this is found abundant.

The development of Gracilaria must include both the production and processing technologies to cater both the local and foreign market. The purpose of this research was to determine the yield and hysic-chemical characteristics of alkali-treated agar extracted from G. edulis (now renamed Hydropuntia edulis (S.G.Gmelin) Gurgel and Fredericq), G. firma Chang and Xia, G. manilaensis Yamamoto and Trono and Gracilariopsis heteroclada J.F.Zhang and B.M.Xia collected in selected coastal areas in Luzon during dry and wet seasons. Moreover, validation of the agar data with the international specifications were assessed for their commercial potential because of the increasing interest in agars from these genera.

2. Materials and Methods

2.1. Sample collection and preparation

Wild stock samples of Gracilaria firma, G. manilaensis, Gracilariopsis heteroclada and H. edulis were collected in Cagayan (18 16.157’N, 121 51.640’E), Pangasinan (16 10.609’N,⁰ ⁰ ⁰ 120 25.077’E), Bulacan (14 45.094’N, 120 48.857’E) and Sorsogon (12 46.017’N, 124 08.351’E),⁰ ⁰ ⁰ ⁰ ⁰ respectively (Fig. 1) during the two pronounced seasons of the country: dry season (February to May) and wet season (July to October). No sample of Gracilariopsis heteroclada was collected in Bulacan during wet season primarily because of the change of environmental factors and strong wave actions experienced during heavy rains and typhoons that affected the growth of


the Gracilariopsis in the area.

Each sample was washed with seawater, sorted and cleaned of extraneous matter (other seaweed species and invertebrates). About two-three kilos of cleaned fresh sample were sundried for about two hours, while few good quality fresh samples were retained and packed in zip lock plastic bag for herbarium purposes and documentation.

Figure. 1 Philippine map showing the sampling sites in Luzon located in the northernmost region of the archipelago.


2.2 Moisture content analysis

In the laboratory, 10 grams of dry seaweed samples in triplicate were weighed and dried further in an oven, set at 60ºC, until constant weight was attained. The difference in weights before and after drying is the weight of the moisture.

2.3 Agar extraction

Extraction of agar from each species was done with the combined methods of Istini et al. (1994) and Santos (1990). Fifty grams (50g) of cleaned and dried seaweed was treated with 1 liter of 4% NaOH solution and heated at 90ºC for 1 hour with constant agitation. After washing thoroughly with running water and until the pH became neutral, the sample was then soaked with 1L of 0.5% acetic acid (CH3COOH) for an hour. It was washed again with running tap water and adjusted the acidity to a pH of 5, chopped into small pieces, homogenized and extracted with 1 liter of distilled water at 90 ºC for about one hour using fabricated seaweed extractor. Later, the filtrate was allowed to gel at room temperature and frozen for 20 hours. Finally, after thawing the gel for about 2 hours at 25 ºC, the agar extract was washed with distilled water, dried in a convection oven at 60 ºC for 24 hours, cooled, and weighed to calculate the percentage of agar yield according to the following formula:

% Agar Yield = Weight of Agar / 50g x 100

2.4 Agar gel analysis

A 1.5% agar solution was prepared using the percent by weight formula to measure the physical properties of agar: viscosity, gel strength, gelling temperature, and melting temperature following the works of Nelson et al. (1983) and Montaño and Pagba (1996). The viscosity (cP) of the solution was measured at 75oC using the spindle 1 of the Brookfield Viscometer (LV DV-II + Pro) and gel strength in g cm-2 with the use of Brookfield CT3 Texture Analyzer. For gelling temperature, 1.5% of agar sol (approximately 20 ml) was poured out into 20mm diameter test tubes with thermometer inserted into it. Glass beads (2.5mm) were dropped one at a time into the tube and the last bead to remain on top of the gel surface was recorded as the gelling temperature of agar. The same gel solution was utilized for melting temperature after equilibrating it overnight. A lead shot (5mm) was placed at the gel surface. Afterwards, the test tube was submerged in a water bath and heated slowly from 30 C. The melting temperature was recorded when the gel started to melt⁰ and the lead shot sank into the solution. In chemical analyses, colorimetric determination of 3,6-anhydrogalactose (3,6-AG) was done by the resorcinol-acetal method using D-fructose as the standard (Craigie and Leigh 1978). Absorbance was read using UV-VIS Spectrophotometer at 555 nm. For sulfate determination, turbidimetric method of Jackson and McCandles (1978) was utilized by reading the absorbance of the solution at 500 nm.


2.5 Environmental parameters

Environmental parameters such as temperature (°C), salinity (ppt) and depth (ft) were monitored within the sampling period using Thermometer, Atago Refractometer and Speedtech Depth Sounder, respectively. The substrate of the sampling areas varied from sandy, muddy to combination of sandy-muddy.

2.6 Data analysis

All extractions, physical and chemical properties were performed in triplicates. One-way analysis of variance (ANOVA) was employed to identify significant variations among treatments. When significant differences were found (p <0.01), Duncan’s Multiple Range Test (DMRT) was applied for comparison of means using the software SPSS Statistics version 21.0. The data were tested for descriptive statistics to obtain the mean and standard error with a level of significance at 95% and homogeneity of variances using the Levene test.

3. Results

Water temperatures in sampling areas ranged from 29°C to 34°C. Salinity of the seawater lowest reading (12 ppt) was recorded in Cagayan during wet season while the highest reading (35 ppt) was recorded both in Pangasinan and Sorsogon during dry season. Depth of the water bodies in both seasons varied from 1.30 to 10.03ft during low tide. Agar was extracted from samples of Gracilaria firma, G. manilaensis, Gracilariopsis heteroclada and H. edulis. The results of ANOVA and Duncan’s test for moisture content and agar yield and physico-chemical properties of the three genera are presented in Fig.2. Results indicated significant differences in all the parameters (p < 0.01) between the seasons except for the viscosity of the agar solution. Moisture content of the four samples ranged from 9.00 to 23.4%, noting that samples of G. manilaensis and H. edulis varied significantly in both seasons (p < 0.01; Fig. 2a). During dry season, the mean values of the agar yields ranged from 8.24 to 17.03%. Gracilariopsis heteroclada obtained the lowest yield while the highest yield was extracted from H. edulis but agar from this species exhibited low gel strength. As for the wet season, mean values of agar yields varied from 7.17 to 11.86%. The lowest and the highest yields were obtained from H. edulis and G. firma, respectively. Considering the two seasons, only the agar yields obtained from H. edulis was statistically different (p <0.01; Fig. 2b). Colors of 1.5% agar solution from G. firma, G. manilaensis, Gracilariopsis heteroclada and H. edulis varied from dark brown, light brown, dark green and light brown, respectively.

Analysis of variance (ANOVA) for the viscosities of agar solutions did not show well-defined variations (Fig. 2c). The highest value (17.33 cP) was recorded during wet season for G. manilaensis and the lowest value (6.90 cP) was obtained from G. firma during dry season. The gel strengths of alkali-treated agars from G. firma were statistically different (p <0.01) between seasons while the other species did not (Fig. 2d). The highest gel strength acquired in the dry (summer) season was


extracted from G. manilaensis (205.93 g.cm-2) while the lowest gel strength was obtained from H. edulis (103.85 g.cm-2). Furthermore, G. manilaensis (195.88 g.cm-2) and H. edulis (102.50 g.cm-2) still obtained the uppermost and undermost gel strength during wet (rainy) season, respectively. The dynamic gelling temperature of agars from G. firma and G. manilaensis varied significantly (p <0.01) among the four species (Fig. 3e). For the melting temperature, only agars extracted from H. edulis revealed significant differences in both seasons (p <0.01; Fig. 3f). The agar extract of G. manilaensis had the highest gelling (52°C) and melting temperatures (98°C) recorded within the studied period. On the other hand, the lowest gelling temperature and melting temperature in summer season were observed from G. firma (39°C) and H. edulis (78°C), respectively. For the rainy season, both G. firma and H. edulis (44°C) had the least gelling temperature while only H. edulis (74°C) had the lowest melting temperature.

Figure 2. Average (+/- SEM) of moisture content (a) agar yield , (b) viscosity, (c) gel strength, (d) gelling temperature contents of agar extracted from G. firma, G. manilaensis, Gracilariopsis

heteroclada and H. edulis.

Analyses of the chemical properties of agar from four species revealed statistical differences (p <0.01) in 3,6-anhydrogalactose contents of G. firma and H. edulis (Fig. 3g) and sulfate contents of G. manilaensis (Fig. 3h). G. firma obtained the highest content of 3,6-anhydrogalactose (44.09%) while H. edulis (28.67%) had the lowest content within the studied period. The sulfate


contents of agar from all samples were low in summer and high in rainy season that varied from 0.78 to 2.28%. G. manilaensis had the lowest reading throughout the duration of the study while H. edulis had the highest. Moreover, the high values in the 3,6-anhydrogalactose content coincided with the low sulfate content among species.

Figure 3. Average (+/- SEM) of moisture content (e) melting temperature, (f) 3,6-anhydrogalactose, (g) and sulfate, and (h) contents of agar extracted from G. firma,

G. manilaensis, Gracilariopsis heteroclada and H. edulis.

4. Discussion

Gracilaria firma, G. manilaensis, Gracilariopsis heteroclada and Hydropuntia edulis collected in selected coastal areas in Luzon, Philippines showed highly significant differences (p <0.01) among the mean yields of the agar in both seasons, similar to other agarophytes (Chirapart and Ohno 1993; Pondevida and Hurtado-Ponce 1996; Freile-Pelegrin and Robledo 1997; as cited by Marinho-Soriano et al. 2003). Considering the means of both seasons in general, the highest agar


yield was observed in summer season coinciding with the highest values of temperature and salinity which confirmed the work of Price and Bielig (1992) and Marinho-Soriano (1999) that high agar yield can be obtained during dry season and lower yield during wet season.

Gel strength is considered as the most important parameter in defining agar quality. The highest mean gel strength (205.93 g.cm-2) value was recorded during dry season at high salinity (35ppt), similar to the results obtained by Lahaye and Yaphe (1988) and Pickering et al. (1990). Based on their studies, higher gel strength in gracilarioid agars has generally been observed during periods of high growth rates, in summer months or at peak plant abundance. Nonetheless, agar extracts show a wide range of gel quality from low grade for traditional domestic use to high grade for industrial use (Villanueva et al. 2009). To estimate agar quality, agars extracted from each species were validated based on the international specifications set by US Pharmacopeia, Food Chemicals Codex (FCC) and American Society of Microbiology (ASM). The gel strengths of the agar extracted from all species of the present study (102.50 g.cm -2 to 205.93 g.cm-2) were below the required specifications in food grade agar (≥600 g.cm-2) and bacteriological agar (>800 g.cm-2) and can only be applicable for traditional domestic use (<600 g.cm -2). The poor quality of gel among samples could possibly be attributed to local environmental conditions where the seaweeds thrive. On the contrary, a much higher gelling temperatures of agar solution were observed during the study. It is known that gelling and melting temperatures are also use to evaluate agar quality. Among the four species studied, the melting point of agar solution extracted from G. manilaensis and Gracilariopsis heteroclada were comparable with food grade agar (≥85), whereas from G. firma was apposite for bacteriological applications (80±5). Almost sulfate contents of the sampled species were within the desired limit of specifications (1-4%).

5. Conclusion

In conclusion, the present results showed that G. manilaensis in Pangasinan collected during dry season had relatively high gel strength and high melting temperature of agar solution. According to US Pharmacopeia and FCC as cited by Murano (1995), the required specifications for commercial agar are gelling temperature between 32-39°C and melting temperature not lower than 85°C. Based on these specifications, melting temperatures of agar extracted from G. firma, G. manilaensis and Gracilariopsis heteroclada were within the range. However, considering the agar properties presented here, G. manilaensis exhibited promising potential for seaweed farming development that could eventually provide better supply of agar worldwide.

6. Acknowledgment

The authors are thankful to the invaluable support of Bureau of Fisheries and Aquatic Resources Region 1, 2, 3 and Bureau of Fisheries and Aquatic Resources-National Seaweed Technology and Development Center during the collection of samples. Also, we would like to express gratitude to the anonymous reviewer of this paper.


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