CHAPTER III: PROPERTIES, MANUFACTURE AND APPLICATION OF SEAWEED POLYSACCHARIDES - AGAR, CARRAGEENAN AND ALGIN

PROPERTIES, MANUFACTURE AND APPLICATION OF SEAWEED POLYSACCHARIDES AGAR, CARRAGEENAN AND ALGIN

1) AGAR -- A RED SEAWEED POLYSACCHARIDE

1. INTRODUCTION

A. History

Agar was the first phycocolloid discovered and prepared as a purified extract. According to a Japanese legend the original manufacturing method of agar was discovered in the middle part of the 17th century, presumably in 1658. “A Japanese officer in the winter of that year arrived at a little inn. The innkeeper Minoya Tarozaemon ceremoniously received him and offered a traditional seaweed jelly dish as dinner, which was prepared by cooking Gelidium sp. with water. After dinner the surplus jelly was thrown outdoors by the innkeeper. The jelly was frozen during the night, and thawed and dried in the sun, leaving on several days a white, porous and dried substance. Tarozaemon found this soft substance and boiled it in water. On setting, he obtained a whiter jelly than the original one.” The method of agar manufacture was accidentally discovered!

Agar in the form of a sweetened and flavored gel has been known in the Orient for ages. It is known in Japan as “Kanten” meaning “cold weather,” in China it is “Dongfen” or “frozen powder.” The word “agar” is Malayan and is used in the double form agar-agar, originally referred to jellies of certain seaweeds especially Eucheuma muricatum of the East Indies. It was said that Chinese migrants to the East Indies imported the Japanese kanten for their own use. They also called it agar-agar. The Europeans in the East Indies learned to use this Japanese product for making fruit jellies, and subsequently introduced it to Europe. Thus, a Malayan term became attached to a Japanese product.

Agar was very little known when Koch first used it as culture medium. Hesse first succeeded in using agar as the culture medium in cultivating gelatin. This trial was communicated to Robert Koch. In 1882, Koch announced the use of agar as a culture medium in his famous experiments on tuberculosis bacteria. Thus, agar, a curious seaweed product, worked its way into science and industry until it reached its present stage of prominence among the colloids.

Agar production by modern industrial freezing techniques was initiated in 1921 in California, U.S.A. by a Japanese named Matsuoka. Now the biggest agar factory in the U.S.A. is the American Agar Company in San Diego, California. During the second world war the production of agar commenced in Portugal and Spain as well.

In Japan, some two-thirds of the agar makers still rely on the natural winter weather to produce strip agar and square agar. The rest have modern equipped factories using the mechanical freeze-thaw process.

In China the agar factories in the North make agar in winter relying on the natural freezing conditions. In other seasons they use diffusion and press techniques to produce agar powder. In the South all agar makers use the mechanical freezing process.

Even in today's modern agar factories the fundamental principle of extraction and purification (freeze-thaw) of agar is similar to that found by Tarozaemon more than three hundred years ago.

B. Definition of Agar

In 1945 Dr. Tseng defined agar as “the dried amorphous, gelatin-like, non-nitrogenous extract from Gelidium and other agarophytes, being the sulfuric acid ester of a linear galactan, insoluble in cold but soluble in hot water, a one per cent neutral solution of which sets at 35°C to 50°C to a firm gel, melting at 80°C to 100°C.”

The United States Pharmacopoeia (1980) defined agar as “the dried hydrophilic colloid extract obtained from Gelidium cartilagineum, Gracilaria confervoides, and related algae of the class Rhodophyceae”.

Araki (1966) referred to agar as a gel-forming substance obtainable from certain species of red seaweeds called “agarophytes” composed of neutral gelling molecules, agarose, and to a lesser extent acidic non-gelling molecules, agaropectin. He also noted that agaropectin is closely related to agarose as it has a similar backbone structure. Araki's definition of agar involved the chemical structure of the polymers, but it was an over-simplication of the complex continuum between neutral and highly charged polymers existing in algae.

The American Society for Microbiology (1981) in the “Manual of Methods for General Bacteriology” defined agar according to Araki as “an extract from certain red marine algae consisting of two polysaccharides, agarose and agaropectin, with the former comprising about 70% of the mixture”. It is not enzymatically degraded by most bacterial species: agar gels are stable up to 65°C or higher, yet molten agar does not gel until cooled to about 40°C, and agar gels have a high degree of transparency. Rees (1969), from chemical structure, defined the agar family of polysaccharides as the polymers sharing a common backbone structure: 1,4-linked- 3,6 -anhydro-α-L-galactopyranose alternating with 1,3-linked-B-D-galactopyranose (Fig.3), which may be “masked” to a varying extent by different sugar residues. He pointed out that agar belongs to the family of polysaccharides and that the component having the greatest gelling tendency is agarose.

2. Agarophytes and agar production

Agarophytes, the red seaweeds used as the raw material for manufacturing agar, are mainly the genera Gelidium (Gelidiaceae), Gracilaria (Gracilariaceae), Pterocladia (Gelidiaceae), and Ahnfeltia (Phyllophoraceae) in different countries. Gelidium yields the best quality of agar, but its cultivation is difficult and its natural resource is less than Gracilaria, which is being cultivated in several countries and regions in commercial scale. Pterocladia and Ahnfeltia grow only in a few regions and are utilized only in New Zealand and U.S.S.R., respectively. Fig. 1 and Table 1 show the distribution of agarophytes and carrageenophytes over the world.

Table 2 indicates the estimated world production of dried agarophytes (by 1980's information).

Gracilaria, the most abundant and promising resource of agar production, has probably more than 150 species, distributed mainly in the temperate and subtropical zones. Some of them are cultivated on large scale in Chile, Taiwan, Vietnam, and to some extent, Thailand.

Fig.1 Distribution map of agarophytes and carrageenophytes

In China there are about 20 species of Gracilaria growing mainly along the coast of Guangxi, Guangdong and Hainan Provinces. Among them Gracilaria asiatica (formerly, G. verrucosa), G. tenuis-tipitata, G. tenuistipitata var. liui, G. blodgettii etc. are used as the raw material for agar production in South China. In addition, Gelidiella acerosa (Gelidiaceae), Laurencia sp. (Rhodome-laceae) etc. are also used in this region.

* In India and Vietnam there exists a small agar production.
** Include few other red seaweeds.

Recently Porphyra haitanensis (Bangiaceae) has been used in considerable amounts for agar production in Fujian Province.

As shown in Table 3 Japan is the world's largest producer of agar, having about 170 agar factories in 1980 and an annual production of more than 2,000 tons. Next are Spain and Chile. Apparently, Gracilaria became an important source for agar production because it is easily harvested and cultivated. Recent estimates place the annual production of agar over the world at about 8,000 tons.

Some countries that have abundant agarophyte resources, such as Chile, The Philippines, Brazil, Portugal, Indonesia, other countries export the agarophytes to other regions. Japan imports a large amount of Gracilaria every year from these regions. Table 4 indicates the quantity of agarophytes imported by Japan in 1984.

Nowadays, about 60% of agarophytes collected for agar production are attributed to Gracilaria spp., 35% to Gelidium sp. and 3% to others.

3. Chemistry of agar

The chemical components of agar had been analyzed from 1859 to 1938 by many scientists, and verified to consist of D-galactose, 3, 6-anhydro-L-galactose and sulfate. From the 1940's to 1950's the substituted galactose such as methylated, sulfated and pyruvated galactoses were proved to be the constituents of the agar molecule as well.

Araki (1956) offered the evidence proving the heterogeneity of the agar by separating the agar into two different polysaccharides named agarose and aparopectin using the acetylation method. The agarose is a virtually neutral polymer, while the agaropectin is an acidic polymer. Later, Araki et al and other scientists -- by acid hydrolysis and enzymic degradation of agar -- isolated the agarobiose and neoagarobiose, respectively, and revealed that the agarose is composed of agarobiose repeating disaccharide units alternating with 1, 3-linked-β-D-galactopyranose and 1, 4-linked-3, 6-anhydro-α-L-galactopyranose (Fig. 2). The agaropectin seems to have the same backbone as the agarose, but contains considerable amount of acid groups such as sulfate, pyruvate and glucuronate groups.

From the 1960's to the 1980's, application of the new techniques in the study of agar such as fractionation, in exchange chromatography, enzymic degradation and especially ¹³C-NMR spectroscopy permitted a more precise study of the basic chemical structure and distribution of the repeating units in various agars.

Recent fractionation studies by Yaphe et al (1971) on DEAE-Sephadex A-50 column indicated that agar is not made up only of one neutral and one charged polysaccharide but is composed of a complex series of related polysaccharides which range from a virtually neutral molecule to a highly charged (sulfated) galactan. The neutral polysaccharide has gelling ability and approaches the structure of an ideal agarose, which still contains a trace of sulfate (0.1 to 0.5%) and pyruvic acid (0.02%).

More recently, ¹³C-NMR spectroscopy was verified to be a powerful tool to elucidate the various disaccharide repeating units of agarose present in different agar molecules. By the assignment of the chemical shifts in ¹³C-NMR spectra of the carbon atoms in agaroses contained in agars isolated from different species of Gracilaria, the structural feature of va- rious forms of alternating disaccharides could be easily ascertained. Fig. 3 illustrates the structure of agarobiose and masked agarobiose repeating units and the precursor of agarobiose isolated from different species of Gracilaria, confirmed by chemical and ¹³C-NMR spectroscopic methods.

Fig. 4A illustrates the ¹³C-NMR spectra of agars from Gracilaria spp., consisting of various types of agarobioses (Ji et al., 1986, 1988).

Agar from Gracilaria asiatica (formerly G. verrucosa) is mainly composed of 6-OCH₃-agarobiose disaccharide repeating unit, that of G. eucheumoides is of 2-OCH₃-agarobiose, and that of G. textorii is of agarobiose 6-sulfate. All these agarobiose exhibit the specific chemical shifts of some carbon atoms.

Fig. 4B shows the ¹³C-NMR spectra of agars from Gelidiella, Laurencia and Porphyra sp. (Ji et al, 1986). The former two alga were revealed to contain the agar composed mainly of 6-OCH₃-agarobiose with high content of methoxyl group and high gel strength, while Porphyra haitanensis has a higher content of biogenetic precursor of agarose, 1,3-link ed β-D-galactopyranose alternating with 1,4-linked α-L-galactopy-ranose 6-sulfate, which has no gelling ability. But after alkali modification (see below) the spectrum of the polymer (Fig. 4B b) exhibits the signals of 12 carbons typical to agarose, thus the polymer possess higher gel strength.

Fig. 3 Disacchariue repeat units of agar

A. Biogenetic precursor of agarobiose (Forphyra haitanensis Gracilaria Gjeostedtii)

B. Various agarobioses

Fig. 4A ¹³C-NMR spectra of agar-type polysaccharides

1. Agar of Gracilaria asiatica (G. verrucosa)
2. Agar of Gracilaria eucheumoides
3. Agar of Gracilaria textcrii

Fig. 4B ¹³C-NMR spectra of agar-type polysaccharices

1. Agar of Gelidiella acerosa, b: Agar of Laurencia flexilis var. tropica
2. Sulfate galactan of Porphyra haitanensis
3. Alkali-troatec agar of Pornhyra haitaneneis

4) Properties of agar

A. Gelation

Agar has the ability to form gels upon cooling of a hot solution to 30 – 40°C and to melt to sols upon heating to 90 – 95°C. The mechanism of gelation of agar may be shown as in Fig.5. At temperatures above the melting point of the gel, thermal agitation overcomes the tendency to form helices and the polymer exists in solution as a random coil. On cooling, a three-dimensional network builds up in which double helices form the junction points of the polymer chains. (Gel I). Further cooling leads to aggregation of these junction points (Gel II). The presence of the sulfate at C6 of the 1,4-linked-L-galactose residues such as in the precursor of agarose, acts as a ‘Kink’ to prevent the double helix from forming. Closure of the ring to form the 3,6-anhydrode, and elimination of the C-6 sulfate group makes the chain straighten and leads to great regularity in the polymer, resulting in enhancing gel strength due to increased capability of forming a double helix (Rees, 1969).

Fig. 5 Gelling mechanism of agar

Generally speaking, the gel strength of agar is conditioned by the content of agarose, a higher gelling component, in it. The agar sample with higher content of 3,6-AG and lower sulfate content always give higher gel strength. The idealized agarose is free of sulfate group, but actually there exists a trace (0.1 – 0.5%) of it, since no pure agarose molecules are present in the nature. Table 5 indicates that the yield, physical parameters and the content of chemical components in agars from Chinese Gracilaria spp. vary with the species and location harvested.

B. Mechanism of alkali modification of agar

Some Gracilaria sp. yield agar with very weak gel strength which was used in Japan in the past as supplementary raw material in combination with Gelidium spp. to produce the agar.

In 1936 Yanagawa offered the method of treating the agar solution or algae with alkali to improve the quality of the agar of Gracilaria, but he did not understand the reason why the quality was enhanced. Later the alkali treatment method was widely applied in the agar industry.

In 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 (Fig. 6). Then subsequent increase of 3, 6-AG and the decrease of sulfate made the agar produced exhibit a high gel strength. This change of conformation from C1 to 1C also takes place in the same manner in vivo by an enzyme, ‘dekinkase’ with the maturity of the thallus. Table 6 shows the changes in the chemical composition of the agars from different species of Gracilaria before and after alkali treatment (Yaphe et al, 1971).

The molar ratio of D-galactose plus 6-OCH₃-D-galactose to 3, 6-anhydro-L-galactose plus sulfate is ca. 1:1. The content of 6-OCH₃-D-galactose varies from 2.3% for the polysaccharide from G. compressa to 21.0% for that of G. foliifera. After alkali treatment, there is an increase in 3,6-AG content, decrease in sulfate content, and no significant change in the content of 6-OCH₃-D-galactose. The pyruvic acid content is not affected by alkali treatment. The higher content of pyruvic acid occurs in agars having 3.5 – 4.5% sulfate content. Before alkali treatment, only the agar from G. debilis (sulfate 3.4%) had a high gel strength. After alkali modification the gel strength of all polysaccharides increased.

Fig.6 Transformation of the precursor of agarose to the agarose

C. Gel strength

The gel strength of an agar is determined for its 1% gel using a gel tester. Usually 1% of Gelidium agars give a gel strength ranging from 300 –500 g/cm². With Gracilaria agars, the gel strength ranges from 50 to 300 g/cm² and it could reach 500 g/cm² or more after alkali modification. The gel strength of the agar varies with the concentration used, the species and growing location of the agarophytes and the production process.

Table 7 shows the seasonal variation of the yield and gel strength of the agar from G. asiatica collected in Qingdao Bay.

Table 7. Seasonal variation of yield and gel strength of the agar from Gracilaria asiatica

The yield of agar is high (23–26%) from June to August and the gel strength also seems to increase with the maturity of the plant.

D. Gelling and melting temperatures

Gelling temperatures of agars from Gelidium spp. range from 28 to 31°C and melting temperatures from 80°C to 90°C, the differences between two temperatures being 51–60°C; for agars from Gracilaria spp. the gelling and melting temperatures range from 29– 42°C and 76–92°C, respectively, the differences between two temperatures being 23–59°C. The difference between melting and gelling temperatures is called as ‘hysteresis’. The gelling temperature of agar correlates with the methoxyl content. The higher methoxyl content in Gracilaria agarose exhibits a higher gelling temperature. For example, when the methoxyl content in agarose increases from 0.5% to 5%, the gelling temperature will increase from 35°C to 45°C.

^a I apressed as a percentage of the polysaccharide. All the sulphate is assumed to be on the L residues for the purpose of this calculation.

E. Viscosity and molecular weight

The viscosity of an agar solution at constant temperature and concentration is a direct function of the average molecular weight. The viscosity rarely exceeds 10–15 cp. at 1% concentration at 60–90°C. Usually the viscosity is lower as the gel strength is greater for the agar solution. The average molecular weight of agar ranges from 8,000 to greater than 100,000.

Fig. 7 Gel strength of agar + locust bean gum

F. Compatibility

Agar is usually compatible with most other polysaccharides and with proteins in near neutral conditions. No precipitation or distinct degradation occurs. But from Fig. 7 it can be seen that the gel strength of Gelidium agar increases when substituting part of the agar by locust bean gum, reaching its maximum strength at an approximate concentration of 1.33% of agar and 0.17% of locust bean gum. On the other hand Gracilaria agar used in high sugar concentration solutions (above 50%) increases its gel strength much more than Gelidium agar does.

5. Manufacture of agar

A. Gelidium agar

Gelidium spp. usually contains excellent quality of agar having high gel strength and low sulfate content so that pretreatment with alkali is not needed as in the case of Gracilaria spp. Fig. 8 is a plant flow sheet diagram of Gelidium agar.

Fig. 8 Plant flow sheet diagram of Gelidium agar production

Sun bleaching: The seaweeds are washed and spread on the cement ground in the sun. Spray with fresh water until the thalli are thoroughly bleached. In a modern agar factory, the seaweeds are bleached in the extraction process or for the agar jellies (see below).

Hot extraction: The seaweeds are heated in a pressure cooker with steam pressure at 1 kg/cm² (ca. 121°C) with an amount of water 15–20 times the weight of dry seaweed for 1–1.5 hr. If necessary, a suitable amount of bleaching agent such as sodium hypochrite or calcium hypochlorite is added to bleach the seaweeds during extraction.

Filtration: The agar sol is discharged from the extractor and is hot filtered through the vacuum filter or filter press with filter aid to eliminate the small seaweed residues thus obtaining a clear filtrate.

Gelling: The filtrate is introduced to the gelling boxes and cooled at room temperature to gel. The jellies are cut into slender sticks or square bars.

Freezing: The jelly sticks or square bars are arranged on reed mats and naturally frozen outdoors in the cold nights in winter at about -10°C. In a modern processing plant the jellies are placed in the freezing chamber to be frozen.

Thawing and drying: The frozen jellies are thawed and dried in the sun for about half a month. If the freezing chamber is used, the frozen jellies are thawed with fresh water, centrifuged to dehydrate and placed in the drying chamber or driedunder the sun, becoming dry strip agar.

Hydraulic press: The jelly sticks are washed in water tanks to diffuse out the soluble matter. Sometimes in this case a suitable amount of bleaching agent is added to bleach the jellies. These are then subjected to the hydraulic press to dehydrate.

Drying and milling: The dehydrated jellies are dried in the drying chamber and then ground with a milling machine to form agar powder.

B. Gracilaria agar

The manufacture of Gracilaria agar in general is similar to that of Gelidium. However, since Gracilaria contains a considerable amount of sulfated galactan (the precursor of agarose) varying with species, growing season and location, alkali treatment for Gracilaria is extremely important to improve the quality of the agar product. Fig. 9 shows the plant flow sheet diagram of Gracilaria agar production.

Fig. 9 Plant flow sheet diagram of Gracilaria agar production

Alkali treatment: In order to confirm an adequate condition of alkali treatment condition for obtaining quality of agar from Gracilaria, a preliminary test should be done in advance in the laboratory prior to industrial extraction. Table 8 shows the Japanese results for treating Gracilaria samples collected from different countries and regions. It is apparent that the treating conditions with alkali depend on the quality of the algae collected. Generally, the Gracilaria seaweeds are treated with 6–7% NaOH solution for 1–2 hrs at 70–90°C. For some species that contain more highly sulfated galactans, a higher concentration of alkali (10–20%) would be needed. The advantages of this method are low quantity of NaOH used, short production cycle and low pollution. The disadvantage is low yield of agar since a considerable amount of agar will be lost during heating.

Washing: The alkali treated weeds are washed thoroughly with fresh water to remove alkali. A suitable amount of acid may be added to hasten neutralization.

Bleaching: The weeds are put into a metallic basket and suspended in a water tank in which sodium hypochlorite solution has been added with the effective chlorine ca. 0.05% for 15 min. at pH 5–6. After this, about 2% (by wt of dry seaweeds) of sodium thiosulfate is added to reduce excess hypochlorite. The seaweeds are then hoisted and washed with water.

Hot extraction: The weeds are boiled in water (15–20 times by the wt of dry weeds) heated for 1–1.5 hrs. The extract concentrations range from 0.8 to 1.5% as a maximum.

Filtration: The hot liquor is sent to rough filtration with 20 mesh nylon cloth and to fine filtration with filter press or vacuum filter.

Gelling: The filtrate is cooled in boxes at room temperature and the jelly is cut into sticks in the same manner as the Gelidium agar gel.

Freezing: The jelly sticks are placed in the freezing chamber at -15°C to -18°C for 24 hrs.

Thawing and Sun drying: The frozen jellies are thawed with fresh water and centrifuge to dehydrate and sun dried to strips.

Hydraulic press: The cut jelly stick or jelly films are packed into nylon bags, and subjected to the hydraulic press. About 10–12 hrs for dehydration are needed at a pressure ranging from 0.1 to 6–10 kg/cm².

Drying and milling: THe dehydrated jellies are sent to the drying chamber at 70°C and then ground to powder agar (80–100 mesh).

C. Manufacture of agarose

Agar is an anionic polymer containing a small amount of sulfate (for Gelidium agar 1–3% and for Gracilaria agar 4–10%) and composed of agarose backbone. The agarose is almost free from sulfate (ca. 0.1–0.5%) and gives a high gel strength. Nowadays agarose is widely used in the biochemical field since its physical and chemical properties approach those of an ideal gel matrix for diffusion and electrokinetic movement of biopolymers. There are several methods for isolating agarose from agar.

Fractionation: This method was first achieved in 1937 by Araki, who acetylated the agar with acetic anhydride in pyridine and separated the acetylated agarose by chloroform extraction, then deacetylated this fraction by saponification to obtain the agarose. The following three methods are of commercial significance.

Cetyl pyridinium chloride (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 λ-carrageenan, a highly sulfated anionic polymer, may increase the bulk of the quaternary ammonium salt precipitate thus facilitate the removal of sulfated galactan by co-precipitation (Fig. 10).

Fig. 10 Preparation of agarose by CPC method

DEAE-cellulose method: An adequate amount of anionic exchange resin DEAE-cellulose powder is added into the dissolved agar sol and stirred at 80°C. The sulfate galactan in agar is adsorbed by the anionic resin and removed by filtration. The filtrate is sent to gelation, freezing, thawing (or alcohol precipitation) drying and milling. The resultant product is agarose powder.

Polyethylene glycol (PEG) method: 7–20% PEG

(mol. wt. 6,000) and 1% NaCl are added into the boiled agar solution. The agarose is precipitated and isolated by centrifuging. After dehydration by acetone and the dried agarose with sulfate content ca. 0.23% is obtained.

6. Application of agar

Agar has a wide variety of uses. It is employed in the bakery, confectionery, dairy industries and meat packing; in pharmaceuticals; in biomedical and other fields.

A. In food industry

The application of agar in the preparation or manufacture of human food is guaranteed in its safety by more than three hundred years of continuous use by some countries. The World Health Organization permits agar for use in the human food industry and it has also been accepted and authorized by the regulations of various countries including the U.K., Federal Republic of Germany, U.S.S.R., France and Poland. The FDA of the United States assigns the agar a grading of GRAS (Generally Recognized As Safe).

Agar is used in food predominantly by its stabilizing and gelling characteristics as shown in Table 9.

B. Pharmaceutical uses

Microbiological culture medium: Agar is most valuable in microbiology being used as the culture medium for practically all pathogenic and non-pathogenic bacteria. Agar is not easy to metabolize and has good gel firmness, elasticity, clarity and stability. None of the pathogenic bacteria is known to digest agar. Usually 1.2–1.5% agar is used for this purpose. About one-sixth of all the agar in the U.S.A. is normally used for culture medium.

Dental impression materials: In prosthetic dentistry it is necessary to make accurate casts of intricate undercut objects. Agar, mixed with other substances, serves as the ideal dental impression material since it makes it to make better and more precise reproductions. The concentration of agar in the impression material is up to 13–17%. Nowaday agar encounters competition from alginate in the use of dental impression material.

Laxative preparation: The value of agar as a laxative is in the prevention of constipation. Agar is a hygroscopic substance and can absorb water and expand considerably, thus increasing the bulk and stimulating peristalsis of intestine, facilitating waste elimination.

C. Biomedical applications of agarose

As mentioned above, agarose is an ideal gel matrix for diffusion and electrokinetic movement of biopolymers, and its gel is an anti-convection medium, which is biologically inert and with controlled ionic properties. Thus, agarose is widely used in biomedicine and biotechnology.

Electrophoresis: Agar gel electrophoretic media have been used for many years to separate and identify serum and spinal fluid proteins and other biological mixtures, facilitating the diagnosis of illnesses of patients. The electrophoretic separation applications using agarose gels includes nucleic acids, lipoproteins, lactic dehydrogenase isoenzymes, serum proteins, glycoproteins, heparin, acid mucopoplysaccharides, bacterial proteins, plant viruses, etc.

Chromatography: Columns of agarose gel particles sold under such tradenames as Sepharose (Pharmacia) and Bio-Gel A (Bio-Rad) are extensively used as media for molecular weight separations, e.g. for the molecules greater than 250,000 daltons, and for separation of artificial mixtures of proteins and viruses, and of ribosomes.

Immunology: The application of agarose in immunology is extensive. Briefly, the uses in this field include the techniques as agarose gel diffusion, radial immunodiffusion, immunoelectrophoresis, electroimmunoassay, counterelectrophoresis, and many others.

Immobilized enzymes and cells: Agarose plays an important role as a biologically inert carrier to which enzymes or cells are bound or introduced during gel formation or by subsequent diffusion and then insolubilization. Such agarose beads are used as bioconverters to transform one chemical to another.

Meanwhile, new applications in biotechnology are continually being discovered for agarose and its derivatives.

2. CARRAGEENAN -- A RED SEAWEED POLYSACCHARIDE

I. Introduction

Carrageenans are the polysaccharides extracted with hot water from certain genera of red seaweeds such as Chondrus, Gigartina, Eucheuma, Furcellaria, Phyllophora, etc.

About 600 years ago, people living along the coast of Carragheen County on the southern coast of Ireland started using the plant, known as Irish moss, a common name for Chondrus, in foods, medicines, and as fertilizer. They subsequently noted its milk reactivity. Irish moss has also been known as carrageen from the same Irish word which means “rock moss”. Irish settlers to America in the 1700's brought with them a taste for Irish moss, and it was soon recognized as a component of the natural flora off the coast of Massachusetts.

Isolation of the polysaccharide extract of Irish moss was reported in 1837, and its purification by alcohol precipitation was done as early as 1871. Stanford (1862) used the name “carrageenin” for the extract from Chondrus crispus. The present spelling, “carrageenan” was recommended and adopted by the Committee on Carbohydrate Nomenclature of the American Chemical Society, to be consistent with the use of the-an suffix for the name of polysaccharides. Since the extract possesses high viscosity and gelling properties and was found to have some uses, several firms on the Eastern coast of the United States began to produce the extract in the 1930s.

2) Carrageenophytes and carrageenan production

Chemical investigations on the structure of the polysaccharides from different species of the red seaweeds revealed that the polysaccharides extracted from Chondrus, Gigartina, Eucheuma, Hypnea and Furcellaria are all the carrageenan-type (see below).

Irish moss, Chondrus crispus was first used as the raw material for carrageenan extraction. Later Gigartina sp. was found to be another important source of carrageenan. These two genera have been the chief raw material for carrageenan production in the United States for long time.

Eucheuma growing in the Far East has a long history of use as articles of food. It distributes chiefly in the tropical far western Pacific including wild and cultivated sources. But it was not until the Eucheuma were recognized as valuable carrageenophytes by the western carrageenan industry that the large-scale export of these species became established. Table 10 shows the annual world production of carrageenophytes in 1984. The Philippines was the main producer of carrageenophytes due to heavily increased farming of Eucheuma.

Table 10. Annual world production of carrageenophytes in 1984 (Tons)

More recent estimates show that the annual production of Eucheuma has already approached ca. 40,000 tons, 95% of which comes from the Philippines.

Various species of Eucheuma such as E. cottonii, E. striatum, E. spinosum, E. gelatinae, E. alvarezii and E. denticulatum are being cultivated in the Philippines. Of these only E. gelatinae is predominantly from wild crops, most others being farmed. In Indonesia the cultivation of Eucheuma is also in progress. Most of the Eucheuma crop in these regions goes to United States, Denmark, Japan, France, Australia, Spain and China for the carrageenan production.

In China, along the coasts of Guangdong, Guangxi and Hainan Provinces some amount of carrageenophytes such as Eucheuma and Hypnea grown in wild are used for carrageenan production.

The Furcellaria fastigiata growing along many coasts of the northern part of the Atlantic Ocean such as the coasts of Norway, Sweden, Denmark, Baltic Sea and Canada was the main source for carrageenan production in Denmark. Formerly the local producer in Denmark gave the Furcellaria extract the trade name, “Danish agar”, which has a strong gelling ability and is akin to the properties of agar. Later, chemical studies revealed that the structure of Furcellaran is also the carrageenan-type (see below).

Phyllophora nervosa is collected in the Black Sea by U.S.S.R. and Turkey. Its extract, erroneously called “agar,” gives the gelling property similar to agar, but subsequent chemical studies confirmed that the structure of the hot water extract is carrageenan-type, the main component being rich in K-and β-carrageenans (see below).

The distribution of carrageenophytes over the world is shown in Fig. 1.

Table 11 indicates the world production of carrageenan estimated by 1980's information.

Table 11. The annual production of carrageenan from main producing countries in 1980

The annual production in Denmark, France and the United States amounts for almost 90% of world carrageenan production. Recent estimates have placed the annual production of carrageenan at about 15,000 tons.

3. Chemistry of carrageenans

Carrageenans have long been known to be the highly sulfated galactans. The common structural feature in all carrageenans was verified to be a linear polymer alternating with the repeating disaccharide, 1, 3-linked-β-D-galactopyranosyl and 1, 4-linked-α-D-galactosyl sugar residues (Fig. 11). The presence of substitute groups, replacing hydroxyl groups, or other modifications of this disaccharide unit, such as anhydride ring formation, gives rise to the structural variation appeared in carrageenans. Therefore, carrageenans are a spectrum of structurally related polysaccharides differing primarily in the proportions of galactose, ester sulfate (also in the position and content) and 3, 6-anhydro galactose depending upon the species of carrageenophytes. The earliest investigations classified the carrageenans based on their solubity in KC1 solution as K-carrageenan (insoluble) and λ-carrageenan (soluble). Later through a vast amount of studies using various chemical and instrumental techniques such as alkali-treatment, methylation, partial acid hydrolysis, enzymic degradation and ¹³C-NMR and IR spectroscopy, this has been replaced, for the most part, by classification based on chemical structure. As a result, the carrageenans are divided into three families according to the position of sulfate groups in the 1, 3- and 1, 4-linked galactose residues. Fig. 11 shows the idealized repeating disaccharide structures of the members of these three carrageenan families.

(a) In the beta (β) family the 1, 3-linked galactopyranosyl residues are not sulfated such as β-and γ -carrageenans;

(b) in the kappa (K) family the 1, 3-linked units are sulfated at C-4 such as K -,ι-, μ-, and -carrageenans;

(c) and in the lambda () family the sulfation occurs at C-2 on the 1, 3-linked units such as λ-, ξ- and π-carrageenans.

The repeating disaccharide types that have been identified in carrageenans from different red algae are summarized in Table 12.

The biogenetic precursors, δ-, γ-, μ-, κ - carrageenans after alkali modification will be transformed to β-, α-, K -, ι -carrageenans, respectively (Fig. 11), β- and α-Carrageenans in β-family give the strong gel strength on addition of K⁺. K-and κ-carageenans in K-family give the strong gel strength in the presence of K⁺ and Ca²⁺, respectively. All carrageenans in λ-family do not gel regardless the alkali modification due to the presence of 2-sulfate group in 1, 3-linked D-galactose residue, which are resistant to alkali action.

More recently a new type of carrageenan, ω-type, not belonging to above three families, was found in the hot water extract of Ressoella verruculosa (Gigartinales), its structure being confirmed by using ¹³C-NMR spectroscopy as shown in Fig. 11 and Table 12.

As shown in Table 12 the polysaccharides from Chondrus, Eucheuma cottonii, E. striatum, Hypnea sp., Furcellaria sp., etc are mainly composed of K-carrageenan, and those from E. spinosum, and Gigartina spp. are composed of ι- and λ-carrageenan, respectively.

Up to date 12 types of carrageenans were identified from the polysaccharides of various carrageenophytes, indicating the complexity of the carrageenans. Among them only K-, ι- and λ-carrageenans are of commercial significance.

These idealized carrageenans are not present in the nature, but some are present predominantly in some molecules, which are mixed together, and some are connected together in the same molecular chains, i.e. in molecular hybrid.

Fig. 11 The repeating (idealized) disaccharide structures of the different carrageenans

Table 12 The repeating disaccharide residues in different carrageenans from various carrageenophytes

3Alβ — 4Blα — 3Alβ — aBlα —

4. Properties of carrageenans

The charged nature of the sugar units in carrageenan and their structural arrangement within the macromolecule render the carrageenans highly reactive chemically and account for their physical properties.

A. Solubility

The solubilities of carrageenans in various media are summarized in Table 13.

Hot water: The carrageenan solution can be prepared with concentration up to 10% in hot water.

Cold water: Sodium salts of K- and ι -carrageenans are soluble in cold water, while K⁺ and Ca²⁺ of Kappa and iota are not soluble. Ca²⁺ salt gives thixotropic dispersion in ι-carrageenan solution system.

Cold milk: λ-Carrageenan has the greatest ability to disperse in 5–10°C milk and thicken it without adding solubilizing salts. λ-carrageenan is insensitive to K⁺ and Ca²⁺, for K- and ι-carrageenans, due to the higher content of 3, 6-AG, and the lower content of ester sulfate, they are more sensitive to K⁺ and Ca²⁺, which are constituents of the milk. Even though K-and ι-carrageenans are practically insoluble in cold milk, they may be used effectively for thickening and gelling if tetrasodium pyrophosphate (TSPP) is added.

Concentrated sugar and salt solution: K- and λ-carrageenans are soluble in hot sucrose solutions with concentrations up to 65% after heating to 70°C, while ι-carrageenan is not easily soluble in hot sucrose solution at any temperature. ι-carrageenan solution alone will tolerate high concentrations of electrolytes such as NaCl up to 20–25%, while K-carrageenan will be salted out. In theory, λ-carrageenan is also soluble in concentrated salt solutions, but in practice, λ-carrageenan always contains some K-compound which makes them less salt compatible.

Water-miscible solvents: Alcohol, propylene glycol, glycerin, and similar solvents may be mixed with carrageenan solutions. The amount of solvent tolerated depends upon the molecular weight and type of carrageenan present. These water miscible solvents make excellent dispersants to carrageenan. In general, these factors which tend to make the carrageenan more hydrophilic will increase its solvent tolerance. For example, the higher the sulfate content and the lower the molecular weight of the carrageenan, the greater the solvent tolerance.

Viscosity and molecular weight: The viscosity of carrageenan depends on concentration, temperature, the presence of other solutes and the type of carrageenan and its molecular weight. The viscosity increases almost exponentially with concentration. For the gelling type of carrageenan, the viscosity measurement is carried out at high temperature (e.g. 75°C) to avoid the effects of gelation, usually 1.5% of concentration being used, while for the cold water soluble (non-gelling) carrageenans, their viscosities are measured at 25°C with 1.0% concentration of solution. Viscosity is usually measured with the easily operated rotational viscometers such as Brookfield's. Commercial carrageenans are generally available in viscosities ranging from 5 to 800 cps (1.5% at 75°C). The salts lower the viscosity of carrageenan solutions by reducing the electrostatic repulsion among the sulfate groups. The solutions of carrageenans having viscosities <100 cp. display the Newtonian flow, varying degrees of pseudoplasticity for sodium and lambda carrageenan, and thixotropic characteristic in the case of calcium iota carrageenan. The latter is typified by a decrease in viscosity with increasing shear or agitation and return to normal viscosity with stopping of agitation.

The viscosity of carrageenan sol increases with molecular weight (Fig. 12) in accordance with Mark-Houwink equation:

[η] = KM^α

where [η], the intrinsic viscosity, is defined as the limit of the ratio of specific viscosity to concentration (ηsp/C) on extrapolation to zero concentration. Intrinsic viscosity correlate closely with normal viscosity measured at 1.5% concentration and 75°C. M is an average value, the viscosity-average molecular weight. K and α are constants. The average molecular weight by viscosity and other measuring methods such as ultracentrifuge, gel electrophoresis etc. ranges from 74,500 to 900,000. The optimum molecular weight of carrageenans for food uses should range between 100,000 to 500,000. The majority of carrageenan products have a molecular weight of about 250,000.

Average molecular wt.

1. Predominantly sodium salt of carrageenan
2. Predominantly calcium salt of carrageenan

Fig. 12 Carrageenan viscosity as a function of molecular wt.

Gelling and melting temperatures: Carrageenan gels like agar are thermally reversible in that they remelt upon heating and gel again on cooling. The gelling temperature of K-carrageenan ranges between 35°C and 65°C, and melting temperature about 55–85°C. The extent of hysteresis is dependent on the type of carrageenan: for Kapa it is 10°C to 15°C, for iota about 5°C. Potassium and calcium ions may distinctly raise the gelling temperature of the K- and ι-carageenans, respectively, with the concentration added (Fig.13).

K-Carrageenan gel in the presence of K⁺ is rigid and brittle and exhibits marked syneresis due to the higher content of 3, 6-AG and low content of sulfate and the hydrophobic nature, while ι-carrageenan gel in the presence of Ca²⁺ is elastic and gives low syneresis due to more hydrophilic.

Fig.13 Cation effect on gelling temperature of carrageenans

Gel strength: Carrageenans at concentration as low as 0.3% may form thermally reversible water gel in the presence of cation. Gel strength of carrageenans is determined with a gel tester (same as for agar) using 1.5% concentration of gel at 75°C. Fig. 14 illustrates that the gel strength increases with the concentrations of carrageenan and gelling cation added. The consistency of carrageenan gels varies from hard and brittle to soft and elastic. Gels of K-carrageenan tend to be more brittle than those of ι-carrageenan, the increased

Fig. 14 Cation effect on gel strength of carrageenan elasticity of ι-type gel being due to increased amounts of 2-sulfated residues in the polymer.

The gel strength of carrageenans from Eucheuma sp. varies from very weak (ca.20 g/cm²) to 500g/cm², depending upon the species of carrageenophyte and the condition of the alkali treatment (Table 14).

Table 14. The yield and properties of carrageenan extracted from Eucheuma sp. treated with different concentration of alkali (Shi et al, 1986)

The gel strength and the yield of carrageenan in carrageenophyte exhibit seasonal variation. The investigation on the Indian carrageenophytes, Hypnea musciformis and H. valentiae shows that the yield varies parallel to the gel strength, being low in October and thereafter gradually increase to high value in March for the former, and low in November and thereafter increase till April for the latter.

Gelation: The mechanism of gelation of K- and ι-carrageenans is virtually similar to that of agar (see above). According to the investigation by X-ray diffraction and optical rotatory dispersion techniques it was demonstrated that the gel formation is attributed to the formation of double helices bundles. At temperatures higher than the melting point of the gel, thermal agitation overcomes the tendency to form helices and the polymer exists in solution as a random coil (Fig. 15). Upon cooling a three-dimensional network is crosslinked through coaxial double helices, forming small soluble clusters or “domains” consisting typically of about 10 chains without causing gelation (Gel I). Further crosslinking of these “domains” into a cohesive gel structure forming the junction zones in the gel (Gel II) involve side-by-side association of double helices from different domains. Helix-helix aggregation occurs only in the presence of cations (typically K⁺ for K- and Ca²⁺ for ι-carrageenans) which can suppress electrostatic repulsion between the highly charged participating chains by packing within the aggregate structure (Gel II).

Fig. 15 “Domain model” of carrageenan gelation (Morris, 1986)

Fig. 16 The change from Cl to lC conformation as 3, 6-AG ring is formed

The effect of sulfation on gelling properties correlate with the formation of double helices. Gel strength is more sensitive to the presence of 6-sulfate groups than to 2-sulfate or 4-sulfate groups, as the former exert a much greater effect on the conformational regularity of the polysaccharide. For example, sulfate at C-2 of the 1, 3-linked units, such as in λ-family, acts as a wedging group to prevent the double helix from forming. Sulfate at C-2 on the 3, 6-AG units, as in ι-carrageenan, projects outward from the double helix, and so does not sterically interfere with its formation. Sulfate at C-4 on the 1, 3-linked residues, as in K- and ι-, similarly projects outward and does not interfere with double helix formation. Sulfate at C-6 on the 1, 4-linked residues forms kinks in the chains inhibiting double helix formation. As 1, 4-linked unit is sulfated at C-6 as in μ-carrageenan it exists in Cl chair conformation, as do all the 1,3-linked units. This introduces a kink into the polymer chain. If it is treated with alkali (OH^-) or in vivo acted by an enzyme ‘dekinkase’ the ring closure forming 3, 6-anhydride (K-carrageenan) will occur, so 1, 4-linked unit transforms to lC form resulting in the removal of the kink (Fig. 16) increasing the degree of conformation regularity and exhibiting high gelling ability. The presence of even one kink in 200 residues has a distinct effect in lowering gel strength.

Compatibility: Locust bean gum, being non-gelling polysaccharide and belonging to the galactomannan family, exhibits an extremely useful synergism with K-carrageenan. The addition of locust bean gum to K-carrageenan solution results in marked enhancement of the gel strength and the transformation of the gel from brittle to elastic accompanied by a reduction in syneresis (Fig. 17). - and -carrageenans exhibit no synergism with this gum. Guar gum does not give such effect.

Fig. 17 K-Carrageenan-locust bean gum synergism

Carrageenans in different generation of carrageenophytes: The life cycle of carrageenophyte has different stages or generations. It has been known that in the diploid gametophytic plants the K-carrageenan is predominant and in the haploid tetrasporophytic plants the λ-carrageenan predominant. They occur in individual plants and are usually harvested together in mixture. Hence the commercial carrageenan products always contain both K-and λ-carrageenans, which are much valued for some applications such as chocolate milk stabilization. Table 15 shows the properties of carrageenans extracted from two generations of Chondrus ocellatus.

Table 15. Chemical and Physical properties of carrageenans extracted from two generations of Chondrus ocellatus collected from Oingdao Bay (Shi et al, 1986)

♀: Female gametophyte,
+: Tetrasporophyte.

The carrageenan extracted from gametophyte is K-type, exhibiting the gel ability and lower viscosity and sulfate content and higher 3, 6-Ag content, while that from tetrasporophyte is λ-type, exhibiting no gel ability and higher viscosity and sulfate content and lower 3, 6-AG content.

So far we know this phenomenon is only true in all members of the Gigartinaceae (Chondrus spp., Gigartina spp.) and Phyllophoraceae (Phyllophora spp.), but it does not extend to Eucheuma, Hypnea and Furcellaria spp.

Reactivity: Since carrageenans possess strong negative charges, they may react with other polyelectrolytes possessing positive charges, e.g. the milk proteins. Reaction depends on protein/carrageenan net charge ratio, the pH of the system, and the weight ratio of carrageenan to protein. At pH level below the isoelectric point the protein has a net positive charge, then direct electrostatic interaction between the negatively charged carrageenan and the protein occurs and the precipitate will appear (Fig. 18c). Above the isoelectric point of the protein the ca²⁺ or other polyvalent cations in solution act as the bridge between the negatively charged carboxyl groups on the protein and the negatively charged ester sulfates of carrageenans (Fig. 18A). At the isoelectric point of the protein, as shown in Fig. 18B, an intermediate or transitional degree of association occurs.

Figure 8 Protein reactivity of carrageenan

5. Manufacture of carrageenans

A. K-Carrageenan

The manufacturing process and equipment used for K-carrageenan are practically similar to those for Gracilaria agar, and are shown diagrammatically in Fig. 19. Three purification processes (A, B, C) may be used for the production of K-carrageenan.

Fig. 19 Plant flow sheet diagram of Eucheuma carrageenan production

1. KCl precipitation process,
2. Press dehydration process,
3. Freeze - thaw process.

As shown in Fig. 19 some producers apply KCl precipitation process (A) for the K-carrageenan production from Furcellaria or Eucheuma. In this case, the filtrate after hot extraction should be evaporated with the double-effect evaporator to reduce the filtrate volume, and then the filtrate is extruded through spinnerets into a cold 1–1.5% solution of potassium chloride. The resulting gelled threads are washed with KCl solution and followed by pressing, drying and milling to k-carrageenan powder.

B. λ-Carrageenan

λ-Carrageenan is usually manufactured by alcohol precipitation method (Fig. 20).

C. Semi-processed carrageenan

The semi-processed carrageenan (or spoken of a AMF, alka

	sun-bleached and dried. to remove adhering salts, sand, and marine organisms. In open kettles extracted at ca.80°C with 100 times of water for 1 hr. or digest with hot alkaline solution. adjust the pH of the liquor, coarse and fine filtration as for K-carrageenan. with double effect evaporator to reduce the water valume. with iso-propyl alcohol or other alcohols.
	separate the coagulum with basket centrifuge or vibrating screens from the alcohol and wash with alcohol. smash with hammer mill. to 80–270 mesh.

Fig. 20 Plant flow sheet diagram of -carrageenan production

li-modified flour or AMC, alkali-modified carrageenan) is produced simply by alkali treatment of Eucheuma cottonii (K-carrageenan) and drying. By this process the ratio of process water to product is minimized, thereby reducing the cost of isolating the dry product. It is far less expensive than extracted carrageenan. It can usually substitute for extracted carrageenan where a little cloudiness due to the small percentage of cellulose present does not interfere. The process is shown in Fig. 21.

wet or dried weeds, taken from Eucheuma farms chopped wet or dried weeds in baskets handled by a overhead traveling crane. hot alkali solution with about 8.5% concentration of KOH. rinse with water for several times in successive tanks. blended and packaged.

Fig. 21 Process of semi-processed carrageenan

6. Application of carrageenans

A. In food

Carrageenan has been used as a natural food additive for over 600 years, and is today recognized as a harmless food additive without nutritional value. Nowadays, it is used in food primarily as gelling, thickening or stabilizing agents. A vast amount of carrageenans are applied for the dairy products and water products.

Milkshake and instant breakfast powder: -carrageenan is used to suspend the ingredients and to impart a richness and body to these drinks. The use level is 0.1–0.2%.

Cooked flans and custards: Light-bodied custard desserts are prepared by incorporating K-carrageenan with other ingredients. The use level of K-carrageenan is from 1.05 to 2.1 g/L of milk.

Cooked pudding and pie fillings: K-Carrageenan (0.42– 1.05 g/L of milk) provides a more uniform set to these products.

Cold prepared flans and custards: λ-Carrageenan (0.2– 1.0%) produces instant gelling in cold-milk systems. The mixture of λ- or ι-carrageenan in combination with TSPP provides syneresis control and texture modification.

Chocolate milk: A typical chocolate milk containing 1% cocoa, 6% sugar and 0.025–0.035% carrageenan. The latter keeps the cocoa in suspension and gives the drink a rich mouthfeel.

Chocolate syrup: Chocolate milk is sometimes prepared by mixing with a syrup concentrate. One part of syrup is added to 10–12 parts of milk, λ-carrageenan (0.04–0.05%) keeps the cocoa in suspension.

Ice cream and sherbet: λ-Carrageenan used (0.01–0.05%) in combination with a primary stabilizer such as locust bean gum, guar or CMC controls ice crystal formation, and prevents syneresis under freeze-thaw conditions, and also prevents they separation in the unfrozen ice cream mix.

Filled and skim milk: ι- and K-carrageenans are effective (0.02–0.04%) in stabilizing the fat emulsion and in improving the appearance and mouth-feel of these products.

Cottage and cream cheese products: K-carrageenan (0.02–0.03%) in combination with locust bean gum (0.1–0.2%) stabilizes the creaming mixture, induces curd formation, imparts shape retention, and prevents syneresis.

Evaporated milk (canned): K-Carrageenan (0.005% or 50 PPM) is used to prevent fat separation in evaporated milk.

Infant formulations: K-Carrageenan is required (0.02– 0.04%) for fat and protein stabilization in food formulations for infants in both milk and soy milk products.

Ready-to-eat milk puddings (canned): ι-Carrageenan (0.1–0.2%) is used to replace part of the starch in these products, giving advantages during processing and in finished product.

Whipping cream: λ-Carrageenan is added (0.05–0.15%) to the natural cold cream to improve stabilization of the whip in whipping cream. If K-carrageenan is added during pasteurization, 0.02–0.05% is adequate.

Aerosol spray cream topping: K-Carrageenan stabilizes both natural and artificial aerosol-propelled cream toppings in the can at about 0.03% and 0.05–0.10% of K-carrageenan, respectively. Usually, locust bean gum (0.10%) is used in combination with carrageenan.

Yogurt: K-Carrageenan is used to stabilize yogurt to which fruit is added. A typical system includes about 0.25% carrageenan and 0.75% locust bean gum based on the yogurt.

Frozen whipped toppings: A combination of K- and λ-carrageenans at 0.03–0.05% improve the body of frozen whipped toppings and reduce syneresis under freeze-thaw conditions.

Imitation milk: In imitation milk products, sodium caseinate and/or soy protein are used in place of milk solids, and vegetable fat replaces the butterfat. K- and ι-carrageenans are used at about 0.05% in stabilizing the fat emulsion and providing body to the product.

Dessert gels: A mixture of K- and ι-carrageenans at 0.1–1.0%, either alone or in combination with locust bean gum, are suitable for gelled desserts.

Fruit drinks: λ- or the sodium salt of K-carrageenan (0.1–0.2%) can be used with suitable amount of sugar, acid and flavor, forming a fruit-drink mix.

Low-calorie jellies: A jelly containing K- and ι- or K-carrageenans (0.5–1.0%) and locust bean gum in combination with artificial sweetness.

Pet foods: K-Carrageenan (0.2–0.5%) in combination with a similar amount of locust bean gum prevents fat separation during processing and imparts a richness to the gravy which accompanies the canned pet foods.

Fish gels: Combinations of K- and ι-carrageenans (0.5– 1.0%) gel the broth and preserve the flavor of fish packed in cans or jars.

Frozen fish coating: A solution of about 0.4% of the mixture containing K-carrageenan, locust bean gum and potassium chloride forms a gelled film that coats frozen fish, protecting it from freezer burn and mechanical disintegration during processing.

Relishes, Pizza, Barbecue sauces: K- or ι-carrageenan (0.5%) is used to provide texture, sheen, and improved adhesion properties in relishes, pizza and barbecue sauces.

As regards the safety of carrageenans used in food, it has been shown that carrageenans are harmless for food. Investigations have demonstrated that carrageenan is not a carcinogen, although in the former reports, highly degraded carrageenan can induce ulceration in guinea pigs and rabbits. In the late 1970's, carrageenan was listed by FDA (Food and Drug Administration, U.S.A.) as Generally Recognized as Safe (GRAS), and the food grade carrageenan was defined as having a water viscosity of no less than 5 Cps at 1.5% concentration at 75°C, which corresponds to a molecular weight of 100,000.

B. Pharmaceutical uses

Mineral oil and insoluble drug preparations: ι-Carrageenan (0.1–0.5%) gives stable emulsions and suspensions for mineral oil and insoluble drug preparations.

Antacid gels: The chalkiness of antacids can be masked by incorporating them into a glycerin-water mixture, gelled with K-carrageenan (0.7–2.0%) or a mixture of K-carrageenan and locust bean gum.

Drugs for peptic and duodenal ulcers: Carrageenan has been proved to be effective for the symptomatic relief and cure of peptic and duodenal ulcers.

Barium sulfate suspensions: Carrageenan is used as a dispersant for Barium sulfate.

C. Other industrial uses

Toothpastes: Carrageenan (0.8–1.2%) is used to prevent separation of the liquid portion and abrasive, and to impart short texture and good rinseability characteristics to tooth paste.

Lotions and creams: λ-Carrageenan (0.1–1.0%) is used in hand lotions and creams to provide slip and improved rub-out.

Water-based paints: ι-Carrageenan or a mixture of K-carrageenan and locust bean gum is used (0.15–0.25%) to thicken latex emulsion paints.

Air-freshener gels: Gel mixture of K- and ι-carrageenan at a concentration of 1.5–2.5% in combination with locust bean gum and fragrant oils is used as an air-treating gel.

Immobilization of enzymes and cells: Carrageenan gel beads formed with cations are the excellent media for entrapping enzymes or cells used for catalyzing the chemical syntheses and conversions.