CC BY
31
2009
Известия ТИНРО
Том 157
УДК 668.393.51:582.26
M.L. Bui1, T.V. Tran Thi1, N.H. Le1, Q.C. Nguyen1, A.V. Podkorytova2
1 Nha Trang Institute of Technology and Research Application, 02 Hung Vuong, Nha Trang, Khanh Hoa, Vietnam; 2 VNIRO, 17, V. Krasnoselskaya, 107140, Moscow, Russia
SEASONAL VARIATION OF AGAR PHYSICAL PROPERTIES AND BIOMASS OF GRACILARIA TENUISTIPITATA FROM THE NHATRANG BAY, CENTRAL VIETNAM
Seasonal variation of Gracilaria tenuistipitata biomass and physical properties of agar from this red alga growing in the Song Cai River estuary (Nhatrang Bay) was monitored from January to December 2005. The biomass increased from January to March (in spring) to the maximum wet weight 475 g/m2 and then declined gradually. The yield and gel strength of agar had a prominent variation, the range of agar yield fluctuation in the period from April to August was 11-28 % of dry weight. Alkaline treatment lowered the agar yield to 9.3-23.8 %, while sulfate concentration had 70.0-72.4 % decreasing, the content of 3,6-anhydrogalactose had 22.0-59.9 % increasing, and the content of 6-O-methyl galactose did not change. Gel strength increased to the maximum value 923 g/cm2 (for 1 % agar solution) after the 2-hour alkaline treatment by 6 % NaOH at 70-80 oC (for the specimens collected in March before the peak of biomass and agar yield). Thus, the algae G. tenuistipitata from the coastal zone of Nhatrang Bay has the content and quality of agar sufficient to be considered as raw material for food-grade agar production.
Key words: Gracilaria, agar, physical and chemical properties of alga, alga biomass, seasonal cycle of alga, environmental parameters.
Буй Мин Ли, Тран Тай Танх Ван, Ли Нху Хо, Нгуен Кук Кванг, Подкорытова А.В.* Сезонные изменения физических свойств агара и биомассы Gracilaria tenuistipitata из залива Ньячанг, Центральный Вьетнам // Изв. ТИНРО. — 2009. — Т. 157. — С. 274-280.
Изучены сезонные изменения биомассы и физических свойств агара из красных водорослей Gracilaria tenuistipitata, выращенных в устье реки Song Cai, зал. Nhatrang, в период с января по декабрь 2005 г. Результаты показывают увеличение биомассы с января до марта (весенний сезон) при достижении максимального веса 475 г сырой массы на 1 м2 в марте и затем ее постепенное снижение. Изменения выхода агара и прочности гелей показали сезонную зависимость. Выход агара варьирует от 11 до 28 % в расчете на сухой вес в период с августа до апреля. После щелочной обработки выход агара варьирует от 9,3 до 23,8 %,
* Буй Мин Ли, кандидат химических наук, директор института, e-mail: bminhly@dng.vnn.vn; Тран Тай Танх Ван, кандидат химических наук, руководитель лаборатории аналитической химии и технологии; Ли Нху Хо, руководитель отдела органических материалов из морских ресурсов; Нгуен Кук Кванг, научный сотрудник; Подкорытова Антонина Владимировна, доктор технических наук, профессор, заведующая лабораторией, е-mail: podkor@vniro.ru.
концентрация сульфата снизилась на 70,0-72,4 %, а содержание 3,6-ангидрога-лактозы увеличилось на 22,0-59,9 % от исходного, в то время как содержание 6-O-метил галактозы в агаре не изменилось. Прочность геля увеличилась и достигла максимального показателя 923 г/см2 (1 %-ный раствор агара) при обработке водорослей 6 %-ным раствором NaOH в течение 2 ч, при температуре 70-80 0C для водорослей, собранных в марте, за месяц до того, как они достигли самой высокой продуктивности по биомассе и выходу агара. Таким образом, G. tenui-stipitata, растущая в прибрежной зоне Ньячанга, по содержанию и качеству агара является сырьем, пригодным для производства пищевого агара.
Ключевые слова: Gracilaria, агар, физико-химические свойства, биомасса, сезонность, параметры окружающей среды.
Introduction
Agar is a polysaccharide extracted principally from the orders of Gelidiaceae and Gracilariaceae. It has numerous applications in food industry and in biotechnologies (Armisen, Galatas, 1987). Although the species of Gracilaria generally produce agar with low gel strength, they are considered as the most important source of commercially valuable agar (Armisen, 1995) and have been the object of numerous studies on phycocolloid characterization.
Several studies have shown that agar yield depends upon species, season and environmental parameters (Lignell, Pedersen, 1989; Marinho-Soriano et al., 1999; Villanueva et al., 1999). The gel properties of agar also can vary according to certain parameters. These include thallus nitrogen content, plant growth (Lahaye, Yaphe, 1988).
In Vietnam, several agarophytes of commercial value are recorded (Ohno et al., 1997). However, the main species is Gracilaria tenuistipitata found along the coast of Vietnam, from Quang Ninh to Baria Vung Tau province. At present, this species is cultivated in ponds, lagoons and harvested about 7,000 ton dry wt year-1. Although some studies on seaweeds of Gracilaria have been done in tropical and sub tropical areas (Yang et al., 1981; Ohno et al., 1997; Villanueva et al., 1999), little is known about the seasonal variation of biomass and agar yield from these macro alga. The object of this study is to determine the seasonality of biomass, agar yield and physical properties of agar from Gracilaria tenuistipitata. The influence of environmental parameters on agar is also examined.
Materials and methods
The algal samples were collected in the estuary of Song Cai River (12011 '45'' N, 109012'30" E), Nhatrang Bay from January to December 2005. There are two remarkable seasons in this area, the rainy season from September to December and the dry season during the rest of the year.
The algae were sampler during low water. Every month the algae were sampled randomly (triplicate) with a square metallic frame (0.25 m2) within a permanent quadrat. The collected plants were transported to the laboratory, washed with tap water, cleaned of epiphytes and oven dried at 60 0C prior to agar extraction.
Environmental parameters were measured simultaneously. Salinity was measured using a hand-held temperature compensated AO refractometer, pH with an orion portable pH meter (Model SA 250) and temperature using an ordinary mercury quick reading thermometer.
Agar extraction. Extraction of native polysaccharides: Samples of air-dried seaweed (40 g) were acid treated in 2 l solution of 0.11-0.18 % H2SO4 for 1 h, and washed in running tap water for another 2 h. The samples were then boiled for 1 h with 1.5 l of distilled water (pH 7-8) using a Bunsen burner in a 2 l Erlenmeyer flask equipped with a reflux condenser. The agar extracts were dehydrated by freezing and pressing.
Preparation and extraction of alkali-modified agar: Samples of air-dried seaweed (40 g) were treated in a 2 l solution of 6 % NaOH at 70-80 0C for 3 h, and washed in running tap water for 30 min. The seaweeds samples were then extracted as described above for native polysaccharides.
Gel properties and chemical analysis. The following agar qualities were determined: viscosity 1 % agar solution at 80 0C using Brookfield viscosimeter, gel strength of 1 % agar solution set over night using MCO gel tester, gelling and melting temperatures of 1 % agar solution modified from Whyte, Englar (1980).
Constituent sugar analysis: Alditol acetate derivatives were prepared by the double hydrolysis-reduction procedure and analyzed by gas liquid chromatography (GLC) according to the method of Falshaw and Furneux (Stevenson, Furneaux, 1991).
Data were analyzed statistically using analysis of variance (ANOVA) in Statistical Analysis Software version 6.12 (SAS Institute Inc., USA) Means, standard deviations, ranges and correlations were calculated using the software STATISTICA version 6.0 (Statistic Inc., USA).
Results and discussion
Environmental parameters. Water temperature showed a typical seasonal pattern of tropical areas with increasing average temperature in the dry season, and decreasing one in the rainy season, ranging from 24.5-28.5 0C in rainy season to 30.0-32.0 0C in dry season. Salinity was relatively constant (28 %%) with the maximum observed during the dry season (34 %%) (Fig. 1).
Fig. 1. Seasonal variation of water temperature and salinity
Рис. 1. Сезонные изменения температуры и солености воды
Biomass. The mean monthly biomass of alga was abruptly increased at the end of the rainy season and reached the highest value in March (475 g/m2). And then, there was a gradual increase of the biomass. The lowest biomass value coincided with the period of rainy season (Fig. 2).
600 и Cai river, Nha Trang
сч jh 500 - ■
400 -
Biomass 300 - i
200 - 1
100 - 0 -Month
1 2 4 5 6 7 8 9 10 11 12 ■ Biomass
Fig. 2. Seasonal variation of the Gracilaria tenuistipitata biomass
Рис. 2. Сезонные изменения биомассы Gracilaria tenuistipitata
Agar composition. Composition of native agar from G. tenuistipitata was characterized by low contents of 2-O-methyl galactose — LA2 (about 3.5 mol%), 6-O-methyl galactose — DG6 (about 16.0 mol%), and rather high concentration of 3,6-anhydrogalactose — LA (about 24.0 mol%) and galactose (about 56.0 mol%).
The sulfate content ranged between 3.8 and 5.9 % (Fig. 3).
The sulfate content of G. tenuis-tipitata from native agar was low in Summer (3.6 %) and high in autumn (6.3 %) with the mean value 4.67 ± ± 1.32 % during the studied period. This parameter correlated negatively with water temperature and salinity and gel strength.
There was an inverse relationship between the galactose and anhydro-galactose content which indicated that part of the galactose was probably presented in the L-form if an ideal repeating structure of agar is assumed.
Fig. 3. Monthly variation of the main constituents of agar from Gracilaria tenuistipitata (molar percentage of total sugar content) (DG: Galactose + DG6): N — native agar; A — agar after alkaline treatment
Рис. 3. Ежемесячные изменения главных структурных элементов агара из Gracilaria tenuistipitata (моль% от общего содержания Сахаров) (DG: Галактоза + DG6): A — агар после щелочной обработкой, N — природный агар
Agar yield and physical characteristics. The yield of native agar varied significantly between seasons (P < 0.01). The highest agar yield was found in the end of spring (April) (28 %) and the lowest in Autumn (August) (11 %). The agar yield with alkali treatment was low (5 to 10 %) in compare to that of native agar (Fig. 4).
Fig. 4. Seasonal variation in the yield of native and alkaline processed agar
Рис. 4. Сезонные изменения выхода природного агара и обработанного щелочью
J ЧЙ - Cai гг. er, Nha Trang г 35
Ф 20 - А* O""
— ..С ■ Ф
го 1 ■-' % 10 - 4-fi ■ 1 1 Native1;
Month .345 6 7 8 9 10 11 12
—■ - Alkali yie Id—»—Native у ¡eld
Variation of agar yield correlated positively with water temperature and salinity and negatively with the gel strength.
Viscosity of 1.0 % agar solution varied from 7.25 to 15.20 CP, gelling temperature varied between 40-42 0C, and melting temperature varied between 85.793.5 0C. The gel strength of the native agar extracted from G. tenuistipitata taken from the studied sites was very low: from 33.0 g/cm2 in September to 102.9 g/cm2 in April (Fig. 5). However, after alkali pretreatment of the alga, the gel strength of agar increased to 923 g/cm2 for the sample harvested in March.
The gel strength is the maximum in the end of Spring (900 g/cm2), decreased during the Summer growth period to the minimum value of 194 g/cm2 in May. From May, the gel strength increased again to 336.4-352.5 g/cm2 in Autumn.
Seasonal fluctuation of the gel strength showed highly significant differences (P < 0.01). Positive correlation was found between the gel strength and agar yield (r = 0.3), while this parameter correlated negatively with water temperature, salinity and nutrients.
Fig. 5. Seasonal variation of viscosity and gel strength of 1 % agar solution: N — native agar; A — agar after alkaline treatment
Рис. 5. Сезонное изменение вязкости и прочности геля
1 %-ного раствора агара: A —
агар после щелочной обработ-
ки, N — природный агар
The melting and gelling temperatures were relatively stable over the studied
period, except a small decrease in Spring. The mean gelling temperature about 40.0-42.0 0C and the mean melting temperature about 85.7-93.5 0C from the alkali-treated algae were in good agreement with the high 6-O-methyl galactose content of this agar as indicated in the reference (Guiseley, 1970).
The highest agar yield was observed in the dry season with high salinity (33 %%) and temperature (23-28 0C). These results suggest that these two parameters can influence on the agar biosynthesis. The yield of the alkali treated agar ranged between
11 to 28 %. The agar loss by diffusion during alkali treatment was higher in the plants collected during the rainy season than in the plants collected during the dry season. The agar yield from G. tenuistipitata was considerably lower than reported in literature either for native or alkali treated agar. The maximum recorded value was 43.4 % (Chirapart, Ohno, 1993).
1000
900
800
ST 700 E
600 § 500
J 400 (ff
* 300
/ \
/
' / \
/ \
A
200 100
0
Monti)
20 18 16 14
12 i
1 ^ s
о О
10 t 8 о
P
6 —
4
2 0
345 Gel strength
7
89 viscosity
1000
900
800
700
_ 600 еч
Ц 500 is
j. 400
1Я
s 300
S 200
o> О
100
A
Л
■ —♦
♦
20
18
16
14
12 £ о о
10 i У 8 о и
(Л
6 —
4 2 0
Month 1
2345 -■— Gel strength
6 7 8 9 10
—ф - viscosity
A
2
6
N
0
Perhaps the most remarkable season effect in agar yield was observed for G. tenuistipitata during the rainy season. A relationship possibly exists between the yield and rainfalls since there is a reduction in salinity from 33 %o in dry season to 26 % in rainy season. Bird (1988) has proposed that in low salinity the agar deposition between cell walls might provide additional structural support for turgid cells. Similar results were obtained for G. edulis (Gemelin) Silva from Thonithurai in the Gulf of Mannar, India (Mal, Subbaramaiah, 1989), and G. chorda from Japan (Chirapart, Ohno, 1993).
In the present study, the biomass of G. tenuistipitata showed remarkable seasonal variation with the minimum value in rainy period and the maximum in dry season coinciding with the seasonal variation of agar yield. The peak was recorded in March (475 g/m2) while low value (~ 0) was recorded in August. The peak of biomass recorded in dry season is similar to Gracilariopsis bailinae (Pondevida, Hurtado-Ponce, 1996). Agar yield from G. tenuistipitata was high in the period of increasing biomass at relatively low water temperature (23.0-28.5 0C), and was low in the period of low biomass at relatively high water temperature (over 30 0C). These data coincided with the results of Bird, Ryther (1990) and Christiaen et al. (1987) where the agar yield declined with a high water temperature in Summer.
Seasonal variations of the percentage (% molar of total sugar) of each monosaccharide presented in native agar were very low (Fig. 4). The 3,6-anhydrogalactose (22.3-24.7 %) and galactose (60.0-63.0 %) were the main components of agarose. Substituted sugars such as 6-O-methyl D-galactose (15.8-16.5 %) and 2-O-Methyl anhydrogalactose (3.4-3.6 %) were also detected.
The sulfate content was 4.67 ± 1.32 % during the studied period. After alkaline treatment, the content of sulfate decreased to 1.1-1.4 %. This diminution of sulfates indicated that more than a half of sulfate groups corresponded to precursor of 3,6-anhydrogalactose.
The higher gel strengths have previously been reported from the plants harvested at the time of peak plant abundance (Whyte et al., 1981). These results are in agreement with the fact that the growth in the Nhatrang Bay occurs during dry season when environmental conditions are more favorable. Lahaye (Lahaye, Yaphe, 1988) proposed that actively growing algae synthesized high concentrations of young agar enriched with L-galactose-6-sulfate which could form strong gels only after alkali modification due to its conversion to 3,6-anhydrogalactose.
There if shown in this study that alkaline treatment plays an important role for agar qualities. After alkaline pretreatment of algae, the gel strength of agar increased to 923 g/cm2 for the G. tenuistipitata harvested in March (in the end of Spring). This increasing was caused by the changes of concentrations of sulfate and 3,6-anhydrogalactose, while sulfate concentration decreased in 72.4 %, 3,6-anhydro-galactose content increased in 59.9 %, and 6-O-methyl galactose content did not vary during the alkaline treatment process. After the alkaline treatment of algal materials harvested from January to March (growth period of young fronds), the 3,6-anhydrogalactose content was satisfactorily increased to the highest value (46.5 %) in March.
Conclusion
The dry and warm season is the best environmental condition to obtain a good raw material of G. tenuistipitata for producing a high quality agar with the gel strength higher than 900 g/cm2 after alkaline treatment, suitable for industrial applications and export. The best harvest period of the alga is defined as early Summer when the biomass, agar content and gel strength are close to their maximum.
References
Armisen R. World-wide use and importance of Gracilaria // J. Appl. Phycol. — 1995. — № 7. — P. 231-243.
Armisen R., Galatas F. Production, properties and uses of agar // Production and utilization of products from commercial seaweed / ed D.J. McHugh. — 1987. — FAO Technical paper. Vol. 288. — P. 1-57.
Bird K.T. Agar production and quality from Gracilaria sp. strain G-16: Effects of environmental factors // Bot. Mar. — 1988. — Vol. 31. — P. 291-295.
Bird K.T., Ryther J.H. Cultivation of Gracilaria sp. strain G-16: Effects of environmental factors // Bot. Mar. — 1990. — Vol. 31. — P. 33-39.
Chirapart A., Ohno M. Seasonal variation in the physical properties of agar and biomass of Gracilaria sp. (chorda type) from Tosa Bay, southern Japan // Hydrobiologia. — 1993. — Vol. 260-262. — P. 541-547.
Christiaen D., Stadler T., Ondarza M., Verdus M.C. Structures and functions of the polysaccharides from the cell wall of Gracilaria verrucosa (Rhodophyceae, Gigartina-les) // Proc. Int. Seaweed Symp. — 1987. — № 12. — P. 139-146.
Guiseley K.B. The relation between methoxyl content and gelling temperature of agarose // Carbohydr. Res. — 1970. — Vol. 13. — P. 247-256.
Lahaye M., Yaphe W. Effects of season on the chemical structure and gel strength of Gracilaria pseudoverrucosa agar (Gracilariaceae, Rhodophyta) // Carbohydr. Polymer. — 1988. — № 8. — P. 285-301.
Lignell A., Pedersen M. Agar composition as a function of morphology and growth rate. Studies on some morphological strain Gracilaria secundata and Gracilaria verrucosa (Rhodophyta) // Bot. Mar. — 1989. — Vol. 32. — P. 219-227.
Mal K.T., Subbaramaiah K. Yield and physical properties of agar in the reproductive phases of Gracilaria edulis (Gmel.) // Silva. Phykos. — 1989. — Vol. 28. — P. 260-266.
Marinho-Soriano E., Bourret E., DeCasabianca M.I., Maury L. Agar from the reproductive and vegetative stages of Gracilaria bursa-pastoris // Bioresour. Tech. — 1999. — Vol. 67. — P. 1-5.
Ohno M., Huynh Q.N., Hilave S. Biology and agar quality of cultivated Gracilaria from Viet Nam // Bull. Mar. Sci. Fish. Kochi Univ. — 1997. — Vol. 17. — P. 15-21.
Pondevida H.B., Hurtado-Ponce A.Q. Assessment of some agarophytes from the coastal areas of Iloilo, Philippines 1. Seasonal variations in the biomass of Gracilaria change, Gracilaria manilaensis and Gracilariopsis bailinae (Gracilariales, Rhodophyta) // Bot. Mar. — 1996. — Vol. 39. — P. 117-122.
Stevenson T.T., Furneaux R.H. Chemical methods for analysis of sulfated galactans from red algae // Carbohydr. Res. — 1991. — Vol. 210. — P. 277-298.
Villanueva R.D., Motana N.E., Romero J.B. et al. Seasonal variation in the yield, gelling properties, and chemical composition of agars from Gracilaria euchemoides and Gelidiella acerosa (Rhodophyta) from Philipines // Bot. Mar. — 1999. — Vol. 42. — P. 175-182.
Whyte J.N.C., Englar J R. Chemical composition and quality of agar in the morphotypes of Gracilaria from British Columbia // Bot. Mar. — 1980. — Vol. 23. — P. 277-283.
Whyte J.N.C., Englar J.R., Saunders R.G., Lindsay J.C. Seasonal variations in biomass, quantity and quality of agar from the reproductive and vegetative stages of Gracilaria (verrucosa type) // Bot. Mar. — 1981. — Vol. 24. — P. 493-501.
Yang S.S., Wang C.Y., Wang H.H. Season variation of agar-agar produced in Taiwan area // Proc. Int. Seaweed Symp. — 1981. — № 10. — P. 737-742.
Поступила в редакцию 17.03.09 г.
