EFECTO DE LA TEMPERATURA Y EL TIEMPO DE EXTRACCIi)N EN EL PROCESO DE OBTENCI6N DE ALGINATO DE SOD10 A PARTIR DE Macrocystis pyrifera EFFECT OF TEMPERATURE AND EXTRACTION TIME ON THE PROCESS TO OBTAIN SODIUM ALGINATE FROM Macrocystis pyrifera

The alkaline extraction step of the process to obtain sodium alginate from Macrocystu pyrifera was investigated. We compared cold (28°C) and hot (80°C) treatments to determine the effect of extraction temperature on the yield and viscosity of the final product, using samples collected in Bahia Tortugas, Baja California Sur (Mexico), in the summer of 1994. At 80°C we obtained a yield of


INTRODUCTION
Brown algae (phaeophytes) are a potential source of alginates, with properties that vary from one species to another.
Alginate is the generic name given to the salts of alginic acid, which is a polysaccharide built up from a linear polymer based on 1, 4-linked residues of two monomeric units, l3-D mannuronic acid and a-L-guluronic acid (Hirst et al., 1964).

MATERIAL Y MkTODOS
(a) Efecto de la temperatura de extraccidn sobre el rendimiento y la calidad en ttrminos de viscosidad  Arvizu-Higuera et al. (1995). However, there is little information on the extraction stage: Bescond (I 948) mentions in his patent the use of 5-7% sodium carbonate for I2 h for algae breakdown; Lukachyov and Pochkalov (1965) use sodium carbonate and heat to boiling, but do not mention time; Zvered et al. (1969), l-2% sodium carbonate for l-2 h at 27°C; Secconi (1967), basic ammonia solution, 5-7% potassium or sodium for 5-24 h; Baranov et al. (1980) l2-15% sodium carbonate plus 5% sodium phosphate, at 45°C for 30 min; McHugh (I 987) cites the use of sodium carbonate for l-2 h at 50-95°C. The studies generally mention the use of an alkali, such as sodium carbonate, as well as highly variable temperatures and/or times without specifying the yield or viscosity of the final product, using specific parameters or a particular algal species. For this reason, the objective of this study was to determine the effect of temperature and extraction time on the yield and viscosity of sodium alginate, using Macrocystis pyrifera as raw material.

MATERIAL AND METHODS (a) Effect of extraction temperature on yield and quality in terms of viscosity
The method of Hernandez-Carmona et al. (patent pending) was used in the experiments, with 10-g samples of dry algae (M. pyrifera), collected from Bahia Tortugas, Baja California Sur (Mexico), during the summer of 1994 for the experiment of the effect of temperature and in the fall of 1990 for the experiment on treatment time, since this is a high-yield sample. The algae were ground to a 40 mesh size.
The samples were hydrated in a 0.1% formaldehyde solution, at a ratio of nine parts solution to one of algae, for I2 h, to facilitate reactions in the latter stages (McHugh, 1987).
Acid pre-extraction was conducted by washing the algae three times with I50 ml of Las algas fueron molidas hasta un tamario de 40 mallas.
La etapa de extraction alcalina se llevo a cabo en dos formas: para la extraction en caliente se colocaron las muestras en un volumen de veinticinco partes de agua destilada por una de algas y se ajusto el pH a 10 con una solution de carbonato de sodio al 10%. Las muestras se mantuvieron en batio maria a 80°C con agitacion constante de 800 rpm durante 2 h. Para la extraction en frio se realizb el mismo procedimiento, except0 que la muestra se mantuvo en agitation a una temperatura de 28°C por un tiempo de 2 h.
En ambos tratamientos, la pasta obtenida se diluyo con agua caliente y se filtro al vacio, con ayuda de tierra de diatomeas y papel filtro Whatman No. 4. La solution clarificada se precipito con cloruro de calcio al IO%, manteniendo una agitation constante. La muestra precipitada se ftltro y las fibras de alginato de calcio se suspendieron en veinte partes de agua destilada por una parte de algas empleadas; se ajusto el pH a 2 con una solution de Bcido clorhidrico IN y se mantuvo en agitation por I5 min. Posteriormente, la muestra se decant6 y se repitio el lavado dos veces mas, ajustando el pH a I .8, con el fin de convertir el alginato de calcio en acido alginico.
Las fibras de acido alginico se colocaron en una mezcla de alcohol y agua, en una proporcion 1: I, en un volumen de I5 ml por gramo de alga. Se afiadio una solution de carbonato de sodio al 10% hasta obtener un pH de 8. La muestra se mantuvo en agitation durante I h. El alginato de sodio obtenido se filtro, se prenso, distilled water; in each washing, pH was adjusted to 4 with IN hydrochloric acid and the mixture was stirred constantly for I5 min to allow for the transformation of the alginate salts to alginic acid, with the liberation of the calcium ions (Arvizu-Higuera et al., 1995).
Alkaline extraction was carried out in two ways: for the hot extraction, the samples were placed in a volume of twenty-five parts distilled water to one of algae; pH was adjusted to IO with a 10% sodium carbonate solution. The samples were kept in a water bath at 80°C with constant agitation of 800 rpm for 2 h. The same method was used for the cold extraction, except that the sample was kept at 28°C and stirred constantly for 2 h.
In both treatments, the paste obtained was diluted with hot water and vacuum filtered, using diatomaceous earth and Whatman No. 4 filter paper. The clarified solution was precipitated with 10% calcium chloride while being constantly stirred. The precipitated sample was filtered and the calcium alginate fibers suspended in twenty parts distilled water to one part of algae; pH was adjusted to 2 using a IN hydrochloric acid solution, keeping agitation constant for I5 min. The sample was later drained and rinsed two more times in the same manner, adjusting the pH to I.8 in order to convert the calcium alginate to alginic acid.
The alginic acid fibers were placed in a mixture of alcohol and water, at a ratio of 1: I, using 15 ml of mixture per gram of algae. A 10% sodium carbonate solution was added until a pH of 8 was reached. The sample was stirred constantly for I h. The sodium alginate obtained was filtered, pressed and the fibers separated and dried in an oven at 50°C until constant weight. The sodium alginate yields were calculated based on the dry weight of the initial sample.
The viscosity of the alginate obtained was determined for both treatments, A 1% solution was prepared, adjusting the temperature to 25°C and it was measured in a Brookfield LVT viscometer. at a speed of 60 rpm vvith the appropriate spindle. The viscosity was also determined after adding hexametaphosphate sodium to the solution (50% of the alginate weight). in ArviLu-lligucra et a/ : Efccto de tcmperatura y ticmpo de extracci6n en la obtcncihn de alginato se dcsmcnuzaron las iibras y se secaron en un horno a 50°C hasta peso constantc y se calcularon 10s rcndimientos de alginato de sodio con base cn cl peso seco de la muestra initial.
En el primer estudio 10s experimentos se corrieron con cuatro rCplicas y en el Segundo con tres rCplicas. La comparacibn de medias se rcaliz6 mediante una prueba I de Student.
order to sequester the calcium present in the sample and determine the true viscosity of the solution.

(b) Effect of hot extraction (80°C) in terms of treatment time
In order to determine the best extraction time, the alkaline treatment times were varied. from 90 to 165 min, with I5 min intervals.
Four duplicate tests were run in the first study and three in the second. Student's I-test was used to compare the means.

(a) Effect of extraction temperature on yield and quality in terms of viscosity
An average yield of 19.10% was obtained in the hot extraction, whereas an average yield of 15.53% was obtained in the cold extraction. The difference was 3.57% greater in the first treatment and it was also statistically different (a = 0.05) (table I).
The average viscosity of the I% sodium alginate, after adding the calcium sequestering agent, was 398 cps in the hot process and 466 cps in the cold process; in other words, it was 14.59% higher in the first process, but it was not significantly different (u = 0.05) (table I).

(b) Effect of treatment time in the hot extraction
Yield, in terms of treatment time, remained almost constant between the 90 and 165 min treatments, ranging from 26.53% (90 min) to 27.84% (120 min). There were no statistically different changes (c( = 0.05) (fig. I).
The viscosity of the 1% solutions of the different treatment times was also not significantly different (a = 0.05) ranging from 880 cps at 105 min to 746.7 cps at 165 min ( fig. 2). Even though significant differences were not found between the viscosity values obtained, a slight decrease was observed at I05 min, when the highest viscosity value (880 cps) was obtained. However. the reduction in viscosity of the Tabla 1. Efecto de la temperatura en la etapa de extraction en et proceso de production de alginato de sodio. La viscosidad de las soluciones al 1% de 10s diferentes tiempos de tratamiento tampoco 120 min value was only 42.7 cps; this loss can, therefore, be justified, allowing the reaction to continue until 120 min in order to achieve maximum yield.

DISCUSSION
During the process of alginate production in the laboratory, the hot extraction (SOYI) was more effective than the cold extraction, since 3.57% more was obtained in the tirst treatment,    10s 165 min (fig. 2). A pesar de no encontrar diferencia significativa en 10s valores de viscosidad obtenidos, se aprecio un ligero decrement0 a partir de 10s I05 min, cuando se obtuvo el valor m&s alto de viscosidad (880 cps); sin embargo, en el siguiente valor, a 10s 120 min, la reduccibn de viscosidad so10 fue de 42.7 cps, por lo que se puede justiticar esta perdida. permitiendo que continue la reaccion hasta 120 min para lograr el rendimiento maxima.
Ciencias Marinas. Vol. 22, NO. 4. 1996 as well as being significantly different. The quality of the product in terms of viscosity was slightly greater in the cold treatment; however, this difference (67 cps) was not significantly different.
The greater yields obtained in the hot process are due to the more severe extraction conditions, which facilitate the breakdown of the cellular walls of the alga and the solubility of the alginate; whereas in the cold process, some algal particles are not broken down, and part of the alginate remains inside, requiring a greater reaction time (Haug, 1964).
Although hot extraction conditions are more severe and a breaking of the uranic acid chains can occur (Green, 1936) this effect is minimal compared to the cold treatment, due to its short exposure time (2 h), which is not enough to significantly degrade the alginate.
One advantage of the cold extraction is the energy saved compared to the hot extraction; however, the 3.57% decrease in yield does not justify its use, since production costs are lower when the hot treatment is used for 2 h.
According to commercial standards (Kelco, 1986;Protan, l983), the alginates obtained from both the cold and hot processes have a mean viscosity (400 cps) and high viscosity (800 cps) from the fall 1990 sample used to determine extraction time. Hernandez-Carmona et al. (1991) suggested a 2-h, cold extraction method and obtained yields of 24.4% and viscosities of 440 cps (mean), which are similar to those reported in this study.
The alkaline extraction time of the range studied in the hot process (80°C) did not significantly affect the yield and viscosity of the final product, and resulted in a minimum extraction time of 90 min. However, from the chemical point of view, reaction does not end until there are no more increases in yield. If this level is desired, the minimum extraction time is 120 min, with a slight loss in viscosity (43 cps) with respect to the previous time (105 min).
Las altas temperaturas y 10s tiempos de extraction prolongados conducen al rompimiento de las cadenas de acidos uronicos con la consecuente perdida de viscosidad del alginato de sodio (McHugh, 1987). Sin embargo, una vez que se ha encontrado el punto optima de extraction para obtener un product0 de alta calidad y rendimiento, estos factores se pueden emplear para controlar la viscosidad del producto !inal. dcpendiendo del tipo de alginato que se desea obtener. ya que las diferentes industrias que emplean alginatos requieren productos con diferente viscosidad. Por e,jemplo, para la elaboration de algunos productos alimenticios y cosmtticos se emplean alginatos de 800-1.000 cps, para impresiones dentales se emplean viscosidades de 200-300 cps y la industria farmaceutica emplea de 5-10 cps (Protan, 1983). El conocimiento de las variaciones de viscosidad en funcion del tiempo de tratamiento permite tomar decisiones en el proceso de production para lograr la viscosidad deseada. Estas variaciones tambien dependen del tipo de alga que se emplee y la fecha de recoleccion ya que puede presentar la misma tendencia, pero 10s valores de viscosidad en to control the viscosity of the final product. depending on the type of alginatc dcsircd, since the different industries that USC alginatcs require products with different viscosities. For examplc, in the production of some food products and cosmetics, alginates of 800-1,000 cps are used, dental impressions require viscosities of 200-300 cps, and the pharmaceutical industry requires 5-10 cps (Protan. 1983). Knowledge of the variations in viscosity in terms of treatment time will allow for the appropriate steps to be taken during the production process in order to achieve the viscosity desired. These variations can also depend on the type of alga used and the date collected. since the same tendency might occur, but viscosity values, in terms of time, could be different. Thus, each species used in a certain time should be analyzed to obtain their reduction curve.

CONCLUSIONS
During the process of alginate production in the laboratory, hot extraction (80°C) compared to cold extraction (28°C) favors the yield of the sodium alginate obtained in the final product, without significantly affecting quality in terms of viscosity. In this case, the minimum extraction time at 80°C for Macrocystis pyrifera in the range studied is 90 min, which results in an acceptable yield and quality.
English translation by Jennifer Davis, funcion del tiempo son diferentes, por lo que cada especie que se emplee y en una temporada determinada debe ser analizada para obtener su curva de reduction.