PROCESSING AND EXTRACTION OF PHYCOCOLLOIDS

JI MINGHOU

Institute of Oceanology, Academia Sinica
Qingdao, China

Phycocolloids refer to those polysaccharides extracted from both freshwater and marine algae. Until now, only the polysaccharides extracted from marine red and brown algae, such as agar, carrageenan and algin are of economic and commercial significance, since these polysaccharides exhibit high molecular weights, high viscosity and excellent gelling, stabilizing and emulsifying properties. They are also extracted in fairly high amount from the algae. All these polysaccharides are water soluble and could be extracted with hot water or alkaline solution.

The different steps in processing and extraction of phycocolloids used in different factories are essentially based on the same principle. However, each factory usually guards as its own trade secrets the details on equipments used and the manufacturing techniques and process. The common processes adopted in most factories for extracting and processing agar, carrageenan and algin are discussed.

A. Agar

1. Development

Some 300 years ago, presumably in 1658, a Japanese innkeeper Tarozaemon accidentally discovered the method of producing dry agar. In 1919, Matsuoka attempted to make agar in California. Now the biggest agar factory in U.S.A. is the American Agar Company in San Diego, California. During the second world war production of agar in Portugal, Spain, Mexico, South Africa, South America, New Zealand, Australia and France also commenced.

In northern China the agar factories produce strip agar in winter relying on the natural freeze-thaw condition; in other seasons they use the diffusion and press techniques to make agar powder. In South China, all the agar makers use the mechanical freezing process to produce strip agar.

2. Processing of agar

Agar is now primarily produced by two main processes: freeze-thaw and press dehydration. The former is divided into two types: natural and mechanical. Nevertheless, both follow the same fundamental principle of extraction and clarification.

The main steps in processing agar from agarophytes may be summarized as: (1) Extraction of agar with hot water; (2) Filtration to eliminate the weed residues; (3) Concentration and purification (freeze-thaw or gel-press); (4) Drying.

a. Gelidium agar

The flow sheet of Gelidium agar production is shown in Fig. 1.

Fig. 1. Flow sheet of Gelidium agar production

The advantage of the gel-press process is that it omits the freeze-thaw cycle, and the final product is always in powder form. In the Orient, the freeze-thaw process is mainly to produce the square agar or strip agar.

Because agar from Gelidium is always of high grade quality, there is no need for alkali modification as in Gracilaria. In modern factories the hot extraction is performed in a pressure cooker instead of the open steam cooker, favouring the extraction of agar. In Japan and China both types of processing (A and B in Fig. 1) are employed, but in Japan, other species of red seaweeds besides Gelidium are mixed as the subsidiary material while in China, Gelidium is always singly used to produce high quality agar.

b. Gracilaria agar

As the culture of Gracilaria is easier, its growth faster and its price cheaper than that of Gelidium, most of the agar makers nowadays utilize Gracilaria as the main agarophyte. The manufacture of Gracilaria agar is generally similar to that of Gelidium, but since Gracilaria contains a considerable amount of highly sulfated galactan (the precursor of agarose) varying with species, growing season and location, an alkali treatment process for Gracilaria is an extremely important measure to improve the quality of agar product. Fig. 2 shows the plant flow sheet diagram of Gracilaria agar production.

Fig. 2. Flow sheet of Gracilaria agar production

Alkali modification

In 1936 Yanagawa discovered that the alkali treatment of agar from Gracilaria could enhance the gel strength of the product, this method has been extensively used by Gracilaria agar makers. Up to 1961 Rees postulated that the alkali could eliminate the ‘kinks’ (sulfation at C- 6 of 1,4-linked-L-galactose residues) existing in the agar molecules, and 3,6-anhydro rings are formed, resulting in the enhancement of gel strength of the agar produced. The agar makers usually use different concentrations of alkali solution to treat the Gracilaria algae according to the species and the quality of the algae collected. In general, the Gracilaria weeds are treated with 6–7% NaOH solution for 1–2 hours at 70–90 °C (Table 1). For some species, which contain much highly sulfated galactans, a higher concentration of alkali is needed.

Table 1. Alkali treatment for Gracilaria algae.

Location of collection	NaOH concentration used	Temperature of treatment (°C)	Time of treatment (hr)
Argentina	6.0	50–60	1.0
Chile	6.0–7.0	88–90	2.0
Mexico	6.0	90	0.5–1.0
Africa	6.0	70	1.0–1.5
India	20.0	70	1.0
Taiwan	10.0	85–90	1.0
Portugal	4.0–5.0	60	1.0

For Porphyra haitanensis, which is used in considerable amount in Fujian Province, China, due to its high content of the precursor of agarose, a higher concentration of alkali (such as 10–20%) is needed and a higher temperature of treatment (90–100 °C) than for Gracilaria should be used. By such treatment the agar exhibits almost pure agarose skeletal structure shown by both chemical and ¹³C-NMR analysis. The exact techniques of alkali treatment are secrets closely guarded by the makers.

c. Agarose

Agarose, a main product derived from the commercial agar product, recently has been widely used in the biochemical fields. The reason is that its physical and chemical properties approach those of an ideal gel matrix for diffusion and electrokinetic movement of biopolymers. A good number of agarose products has appeared in the market, such as BioProducts SeaKem agarose, SeaPlaque agarose, NaFix glyoxyl agarose, etc, produced by FMC & Co.; Sepharose series of gel filtration medium by Pharmacia Co., and Bio-Gel products by Bio-Rad Laboratories.

There are various methods for manufacturing the agarose, but among these the CPC, DEAE-Cellulose and Polyethylene glycol (PEG) methods are applicable.

CPC method: CPC or hyamine is a cationic polymer, which may react with highly sulfated galactan in agar forming the precipitate. The addition of a small amount of lambda- carrageenan, in this case a highly sulfated anionic polymer, may increase the bulk of the quaternary ammonium salt precipitate, and thus facilitate the removal of sulfated galactan by co-precipitation.
DEAE-Cellulose method: An adequate amount of anionic exchange resin DEAE-cellulose powder are added into the agar solution and the mixture is stirred at 80°C. The sulfate galactan in agar is adsorbed by the anionic resin and removed by filtration. The filtrate is sent to gelling, freeze-thaw, alcohol dehydration, drying and milling to agarose powder.
PEG method: 7–20% PEG (mol. wt. 6,000) and 1% NaCl are added into the boiled agar solution. The agarose is precipitated, isolated and dehydrated by acetone.

B. Carrageenan

1. Development

Although the application of Irish moss extract was reported in 1837, its formal production was initiated only in the 1930's in the United States. Now, the main carrageenan producing countries are U.S.A., Denmark, France, Japan, Spain, China, Korea, Brazil, Philippines, etc.

In China, carrageenan production started in the 1970's in Hainan Island using Eucheuma gelatinae as the raw material; and later Hypnea sp. was utilized as well.

Carrageenan, like agar, is now extensively used in the food industry. Owing to its superior characteristics in dairy food product and biotechnology, its annual production is rapidly increasing and has recently exceeded that of agar. Recent estimates place the annual production of carrageenan over the world at about 15,000 tons, including about 600 tons annually in China.

2. Processing of carrageenan

By the chemical studies on the carrageenan structure it has been known that carrageenan from different species of carrageenophytes essentially may be divided by the structural feature into the Beta, Kappa and Lambda families. Again they are subclassified into 13 types depending upon the position of sulfate groups on (1 → 3) and (1 → 4)-linked D-galactose residues. But among these only kappa, iota and lambda carrageenans are of commercial significance, because they are the main components in carrageenophytes, which have abundant sources, and possess the properties applicable in food, pharmaceutical and other uses. Kappa-carrageenan is the main product, widely used in food industry as a gelling agent. Recently iota-carrageenan has also been used in food and other industries, owing to its peculiar properties which are different from that of Kappa's. Lambda-carrageenan is extensively used in food products as the stabilizer and emulsifier due to its non-gelling and high viscosity properties. With their different properties, their processing and extraction differ somewhat from each other.

a. Kappa-carrageenan

This family contains about 35% 3,6-AG and 25% sulfate, and being more hydrophobic, the freeze-thaw and gel-press processes used in Gracilaria agar production are quite applicable to K-carrageenan processing, as shown in Fig.3.

Fig. 3. Flow sheet of Kappa-carrageenan production

Alkali modification

Usually 5–10% NaOH solution is used to treat Eucheuma sp. or Hypnea sp. at 80–90 °C for a definite time depending on the quality of algae used.

KCl precipitation process

Some carrageenan makers apply KCl precipitation process (Fig. 3 C) for K-carrageenan production from Furcellaria sp. or Eucheuma sp. In this case, the filtrate after hot extraction should be evaporated with the double-effect evaporator to reduce the volume of filtrate and then the filtrate is extruded through spinnerets into a cold 1–1.5% KCl solution. The resulting gelled threads are washed with KCl solution, followed by pressing, drying and milling to K-carrageenan powder.

C. Lambda-carrageenan

Lambda-carrageenan contains about 35% sulfate and 0% 3,6-AG, exhibits no gelling ability at all and is more hydrophilic. Thus it could be dried by using drum dryer or alcohol precipitation process. The former will cause serious degradation of the product, so that the latter process is widely used. Propyl alcohol or ethyl alcohol is used as the dehydrating agent (Fig. 4). Iota-carrageenan has gelling properties in the presence of calcium ions but contains 30% 3,6-AG and 32% sulfate, and thus more hydrophilic than K-carrageenan. It is also difficult to be produced by the freeze-thaw or gell press processes so that it is also produced with alcoholic precipitation as, with lambda-carrageenan.

Fig. 4. Flow sheet of Iota- and Lambda-carrageenan production

Evaporation and alcohol precipitation

After hot extraction the pH of the alkaline liquor is adjusted and subjected to coarse and fine filtration. The filtrate is then concentrated with the double-effect evaporator to reduce the water volume.

D. Semi-refined or semi-processed carrageenan (Fig. 5)

The semi-processed carrageenan is produced simply by alkali treatment of Eucheuma cottonii and drying in the farm on the shelf. A basket of seaweeds is immersed and cooked in hot aqueous KOH at 100 °C and then soaked in fresh water to extract the alkali. The product is dried and ground to powder. By this process the ratio of process water to product is minimized, thereby reducing the cost of the product. This product can usually substitute for extracted carrageenan where a little cloudiness due to the small amount of cellulose present does not interfere.

Fig. 5. Diagram of semi-processed carrageenan production

E. Algin

1. Development

In 1881 Stanford first discovered algin, a polysaccharide from a brown seaweed Laminaria stenophyllum. In 1927, Thornley set up a company to produce alginate in San Diego, U.S.A., which in 1929 was reorganized into the Kelco Company. Alginate production was initiated in U.K. by Alginate Industries Ltd. in 1934–1939. In the 1980's Kelco and Alginate Industries were acquired by Merk & Co. in U.S.A. and now produce about 70% of the world's alginate. The next largest producing country is China, followed by Norway, France, Japan, etc.

In China, algin production was initiated in 1957 in Qingdao from a wild brown seaweed resource, Sargassum pallidum. From the late 1960's Laminaria japonica had replaced Sargassum since the Laminaria was cultivated on a large scale along the coast of China from Dalian to Fujian Province with the maximum annual production of about 270,000 tons dry weight in 1979.

Alginates have a wide application in various industries, but mainly in food industries because of their high viscosity, stabilizing, thickening and gelling abilities. In addition, alginates are extensively used in textile, paper, welding and the pharmaceutical industries.

Recent estimates place the annual production of alginate over the world at about 35,000 tons. China contributes 10,000 tons.

2. Processing of alginate

The rationale of processing and extraction of alginates in various factories are essentially akin to each other, although the exact processing techniques and equipments used are somewhat different and guarded as trade secrets. There are two main processes in the manufacture of alginate: calcification and acidification (Fig. 6). Many algin makers tend to adopt the calcification process because it is easy to dehydrate the product and gives a high yield.

In the extraction and clarification of alginate, alkali and acid are unreasonably and repeatedly used:

Except reaction (4), all other reactions are acidic or alkaline reactions.

Fig. 6. Flow sheet diagram of sodium alginate production

Pretreatment

Among some algin makers, diluted acid is used to pretreat the alginophyte to convert the insoluble calcium and magnesium salts into soluble alginate, Fig 6 (2), facilitating the ion exchange of H⁺ with Na⁺ of Na₂CO₃, and to remove the phenolic compounds which will form brownish products with alkali giving discoloration to the liquor and will cause the loss of viscosity of alginate. Most makers including those in China now use formalin solution instead of acid to pretreat the alginophyte to fix the proteinous matter in algae. The phenolic compounds and other pigments adhere on them, resulting in decolorization of the final alginate product and preservation of high viscosity.

Clarification

Separating the viscous sodium alginate solution from the finely dispersed residues in alginate production is an important step, which closely relates to the quality of the product.

Floatation: This process, widely used by many algin makers, is carried out by bubbling air into dilute liquor (0.2–0.3%) adhering the insoluble residues to the floc and floating onto the surface of the liquor.
Filtration: The quality of alginate to some extent depends on the filtration equipment used. Filter press is used in most alginate plants; a coating of diatomaceous earth as filter aid will effectively eliminate the small suspended residues in liquor. Other algin makers use the Dorr-Oliver vacuum drum filter which is a rotary precoat vacuum filter. The rotary drum is coated with a 2–3 cm layer of precoat material, preferably perlite. After 9–10 hrs, most of the precoat would be removed by the scraper.
Dehydration: The alginic acid gels are dewatered by screw press to 25% or more solids, to which ethyl alcohol and concentrated NaOH solution (40%) and NAOCl solution are introduced. In some makers, the gels are dewatered by hydraulic press to about 20% solids content.

According to various applications the algin makers produce alginic acid and its salts: ammonium alginate, sodium alginate, potassium alginate and calcium alginate. Of these the main product is sodium salts, since it easily dissolve in water and exhibits high stability.

3) Processing of propylene glycol alginate (PGA)

Propylene glycol alginate (PGA) is an important derivative of alginic acid, which is manufactured by reacting alginic acid gels and propylene oxide under pressure (Fig. 7), and could be used in acidic solutions, largely extending the alginate application in food.

Fig. 7. Flow sheet of PGA production

Alginic acid gels with 45–55% moisture content are used to react with gaseous propylene oxide (mole ratio 1:3) in a pressure vessel at 45–60 °C for 8 hrs, giving a final product with about 80% degree of esterification and a pH of 3.8–4.6. A good esterification may be achieved with a low neutralization (0.4%), the solids in an alginic acid gels being as low as 20–34%. The reaction runs generally at 75–85 °C for 2 hrs.

PGA is widely used in acidic drinks or dairy products as stabilizer, and its solution remains unchanged to about pH 3.