Gracilaria And Agar Quality Standards

Gracilaria is the raw material for the agar industry world-wide. Chile is the largest producer of Gracilaria and Japan is the largest producer of agar. World agar production is currently estimated at 10,000 tonnes per annum, about half of which is from Gracilaria. Japan is the largest producer and consumer of agar. Thailand, Malaysia and Indonesia are major importers of agar as shortages of raw materials and technological expertise are a constraint to agar production in these countries. Vietnam can produce a large quantity of seaweeds (Gracilaria) and has an agar industry, but agar quality is not up to standard. As the price of Gracilaria increased year after year, agar also became expensive, both commercial and bacterial grade. In Japan, South Korea and Taiwan, which are top agar producers, labour and land costs were the reason for the increasing the price of agar. These countries switched production to other Asian countries to increase profits.

The annual world production of agar stands at between 7 and 10,000 tonnes per annum, approximately half of which was produced from Gracilaria and the remainder came mainly from Gelidium (Coppen, 1989). The world agar industry basically uses the following seaweeds :

• 1.    Different species of Gelidium harvested mainly in Spain, Portugal, Morocco, Japan, Korea, Mexico, France, USA, People’s Republic of China, Chile and South Africa.

• 2.    Gracilaria of different species harvested in Chile, Argentina, South Africa, Japan, Brazil, Indonesia, Philippines, People’s Republic of China (including Taiwan Province), India and Sri Lanka.

• 3.    Pterocladia capillace from Azores (Portugal) and Pterocladia lucida from New Zealand.

• 4.    Gelidiella from Egypt, Madagascar and India.

Other seaweeds used include: Ahpheltia plicata from North Japan and the Sakhalin Islands; Acanthopheltis japonica, Ceramiun hypnaeordes and Ceranium boydenii (Armisen and Galatas, 1987). A country breakdown of world agar production is given in Table 1.

Japan is the largest producer of agar in the world and, in 1987, Japan exported 3,729 tonnes of agar to the countries shown in Table 2.

Table 1 World production of agar, 1984 (tonnes).

Source: Coppen, 1989

Table 2: Japanese agar exports, 1987.

Source: Coppen, 1989

Japan is highly dependent on imported raw material for its agar production and accounts for most of the world trade in agarophytes (Table 3). The total raw material requirement to produce 7,000 tonnes of agar is around 35,000 tonnes of seaweed, which means that almost a third of the seaweed used enters world trade. The dominant position of Chile as a supplier of Gracilaria means mat, including their own domestic production of agar, they are the world’s largest source of Gracilaria derived agar. Chilean sources estimated the 1985 harvest to be almost 16,000 tonnes (dry weight). The majority of this was collected from the wild but 400 tonnes was cultivated.

The need for greater quantities of agarophytes has encouraged Gracilaria cultivation. Seaweed cultivation has only had limited success, however, and there are still problems to be solved before it can be generally adopted. At present, cultivation is used for industrial purposes in the People’s Republic of China and its Taiwan Province and it is now being initiated in Chile.(Armisen and Galastas, 1987).

The production, utilisation and international trade of commercial seaweeds and seaweed products are important for the countries of Asia-Pacific, especially Gracilaria and agar. In the case of Gracilaria the problem is more difficult to solve. The enzymatic hydrolysis of agar occurs spontaneously even at relatively low moisture contents, but at variable rates depending on the Gracilaria species and its origin.

Table 3 Agarophyte (Gracilaria) imports to Japan (tonnes).

Source: Coppen, 1989

Gracilaria harvested in India, Sri Lanka, Venezuela, Brazil and generally in warm waters, contains an agar less resistant to enzymatic hydrolysis than the Chilean Gracilaria which is the most stable one known. Nevertheless, the stability of agar contained in Gracilaria is less than that of Gelidium.

The world production of red seaweeds was 1,256,918 metric tonnes in 1992 (Table 4).

Table 4: World production of red seaweeds in 1992.

Source: FAO Yearbook of Fishery Statistics, 1992

Marketing of industrial agar is done through trading companies operating from Japan, Europe or the United States, where the most important trading companies are located in the area close to New York. There are, however, different standard specifications as shown in Table 5.
Table 5: Standard specifications of agar for FCC, USP, EEC and FAO
(* negative).

Source: Tengtein Y.,and Wattanaoran P., 1989

FCC – Food Chemicals Codex
USP – The United States Pharmacopoeia
EEC – European Economic Countries
FAO – Food and Agriculture Organisation of the United Nations,

It is difficult to get an idea of the prices of commercial agar because the usual trade statistics list agars with different specifications and applications and therefore with different prices, as shown in Tables 6 and 7.

Table 6: Agar Imported and Exported by Japan in 1986 (January – October).

Exchange rate: 1 US$ = 154.23 Yen Source : Armisen and Galatas, 1987

 Table 7: Japanese export/ import, average price for agar in 1986.

Exchange rate: 1 US$ = 154.23 Yen Source : Armisen and Galatas, 1987

 The major parameters of the Japanese Specifications of Processing agar to show the different of qualities of agar, is shown in Table 8. Table 9 shows the chemical composition of Gracilaria.

Table 8: Major parameters of the Japanese Specifications of Processing Agar.

Table 9: Chemical composition (%) of Gracilaria.

Source: Arasaki, S. and Arasaki, T. 1983

The Asian nations which would participate in the expansion of Gracilaria production and other agar-bearing seaweeds are Thailand, Malaysia and Indonesia. Vietnam can produce a great quantity of Gracilaria and agar, but they are still problems reaching quality standards for agar in the world market. Vietnam will be developing their Gracilaria production and processing in the near future. Japan, South Korea and Taiwan are now looking for joint ventures in other Asian countries to transfer funding and technology for the agar industry because the labour and land costs in their countries were very high. Vietnam would be especially good for investment because of their high production of Gracilaria.

Exporters of seaweed and seaweed products
Marcel Trading Corp., P O Box 241, Manila, Philippines.

REFERENCES

• Arasaki, S. and Arasaki, T. 1983. Vegetables from the sea to help you look and feel better. Japan Publications Tokyo.
• Armisen, R. and Galatas, F. 1987. Production, properties and uses of agar. In: M°Hugh, D. J. Production and utilisation-of production from commercial seaweeds. FAO Fisheries Technical Paper.
• Chandrkrachang, S. and Chinadit, U. 1988. A New Approach to Seaweed Production and Processing. INFOFISH International No.4/88.
• Coppen, J. J. W. 1989. International Trade in Agar for countries in the Bay of Bengal Region . Songkhla Thailand 23–27 October 1989 BOBP/REP/45
• International Trade Centre UNCTAD/GATT 1981. Pilot Survey of the World Seaweed Industry and Trade. Geneva, Switzerland:(l 11 Page)
• FAO, 1992. Yearbook of Fishery Statistic’s, 1992. FAO,Rome.
• Lima Dos Sandos, C. A., Roessink, G. L., Richards-Rajadural, P. L., Taylor, C. T. and Kano, I. 1988. 
• Seaweed processing and marketing in Asia/Pacific. Working paper prepared for the :Seventh Session of the IPFC Working Party of Experts on Aquaculture (Bangkok, Thailand, 1–6 August 1988) UNDP/FAO project RAS/84/027.
• Suo, R. Y. and Qingyin (1992). Laminaria Culture in China. INFOFISH International No./92
• Tangtein, Y. and Wattanaoran P.(1989) “Standard specifications of agar” in Seaweed production and processing Biopolymer Research Unit Srinakarinwirot University Bangkok Thailand (in Thai).

What Is Agar?

Agar, more correctly known as agar-agar, has been used in the East for several hundred years and certainly since the seventeenth century. Agar is traditionally claimed to have been discovered by Trazaemon Minoya in 1658 in Japan.

Agar is typically a strong gelling polysaccharide derived from red seaweeds and is characterised by its chemical repeat units of 3-6,anhydro L-Galactose. Agars also contain sulphate esters in low levels and some methoxy groups.

Agar is eaten extensively in Asia has a flavoured jelly where the brittle texture is appreciated. However for western tases, brought up on gelatin, this is less acceptable. Agar is useful in the food industry as a gelling and thickening agent with particularly good properties in acidic dairy products where it is used as a stabiliser. Another key property of agar is the large hystersis between the melting and setting temperatures which is unusual for a polysaccharide. One of the single biggest users of agar in the west is in the baking industry where the very high melting points of the agar gels make them particularly suitable to the baking process. Agar has good compatibility with sugar and can be used in very high sugar environments that would precipitate most other gums. Agar is often used to provide structure to high sugar systems such as doughnut icing.

Structure
The original structure of agar was was believed to be a simple sulphated poly galactose. However in in 1937 showed that agar consisted of at least two separate polymers that could be fractionated. One was called agarose and the other agaropectin. Essentially agarose is the gelling fraction of agar. Later in 1957 agarose was assigned a linear polymer structure consisting of alternating D-galactose and 3,6 anhydro-L-galactose as shown in figure 1. However agar is actually a very complex polysaccharide and varies considerably depending on the source. In 1991 showed that at least eleven different agarobiose structures could be identified in different agar bearing weeds depending on gender, species environmental conditions and time of the year. In summary agar can be considered to consist mainly of alternating β-(1-3)-D and α-(1-4)-L linked galactose residues. Most of the α-(1-4) residues are modified by the presence of a 3,6 anhydro bridge. The other modification that can be found are mainly substituents of sulphate, pyruvate, uronate or methoxyl groups. Modern alkalie treatment methods tend to increase the level of anhydro bridging in the molecule which subsequently improves the gelstrength. The level of methoxy content appears to be one of the main structural moieties that determines the gel setting temperature with very low methoxy contents giving the lower setting temperatures.

Figure 1. Original structure of agar repeat unit

Agarose is typically high in molecular weight and low in sulphate. Agaropectin is typically a lower molecular weight and also higher in sulphate at about 5-8%. Xylose has been found in some agars

Production
Agar producing seaweeds are available from a wide variety of sources (table 1). Agar weeds are typically wild harvested although commercial farms have been used in Chile and Namibia.

Table 1. Primary agar producing species

Acanthopeltis Japonica           Japan
Gelidiella Acerosa                    Japan, India
Gelidium Amansii                     Japan
Gelidium Cartilagineum           USA, Mexico, South Africa
Gelidium Caulacanthum           New Zealand
Gelidium Corneum                   South Africa, Iberia, Morocco
Gelidium Liatulum                    Japan

Gelidium Ligulatum                  Japan
Gelidium Pacificum                  Japan
Gelidium Pristoides                 South Africa
Gelidium Sesquipedale           Portugal, Morroco
Gracilaria Conferviodes          Egypt, Japan, New Zealand
Pterocladia                               Lucida New Zealand

Agar can be extracted in a variety of ways but the basic methodology revolves around dissolving the agar from the seaweed with hot water, separating the agar from the cell wall residues by filtration and then isolating the agar from the dilute solution. Various methods have been developed to isolate the agar from solution, the traditional method relies on cutting the gel into strips and allowing it to freeze overnight and thaw out the next day in the sun. Due to the high level of syneresis produced in an agar gel the strips loose water on each freezing and cooling cycle until a dry strip is formed. this strip is known in Japanese as Kanten which literally translates as “Frozen sky”.

Industrially Agar can be made in several ways. One metho involves freezing agar solutions in ice tanks in a simply scaled up version of the traditional method. A newer method, which only works for agar types that have significant syneresis such as gracillaria, involves forming a blocks of gel wrapped in clothes and literally pressing the water out of the blocks. The pressing is usually done with large static concrete weights. The pressed agar is then usually pressed again in hydraulic presses to reduce the water content even further prior to drying. A variation of the gel pressing method involves pumping broken agar gel into large filter presses and using the pressure from the feed pumps to force water out of the matrix. This technology was pioneered by Hispanagar in the 1960’s and is now the dominant method of pressing agar. Another methodology involves roller drying the extracted agar.

This method has the advantage in that it can utilise a variety of agar species including gelidium which cannot be pressed easily. Agar gel strength can be improved by removing some of the ester sulphates from the agar chain by alkali treatment. Alkali treatment also increases syneresis and makes pressing the agar easier. Traditional agar is sold in strip or block form. Commercial agar is normally milled into a coarse powder. The agar gel press method is the basis for gel repss methods used in the newer carrageenan processing techniques. As in carrageenan processing not all agar weed types can be pressed and some of the weeds favoured for their low syneresis such as gelidium types are very difficult to press. Very low syneresis agar is favoured in microbial plates.

Properties
Agar gels due to the presence of the agarose fraction in the crude agar at typical concentrations between 0.5% and 2.0%. Unlike carrageenan agar does not require the presence of any particular ions to gel. One of the classic uses of agar is for the preparation of microbial plates where the combined properties of low syneresis, ion independent and a low set temperature make agar ideal. Agar has a uniquely large hysteresis between its melting and setting temperature. Typically agar need to be heated above 90oC to form a good solution and depending on the seaweed source the setting temperate can be as low as 30oC and is typically between 30-45oC for a 1.5% solution. To overcome the very high dissolution temperature of agar several companies manufacture a form of agar that has been specially dried to allow the agar to dissolve at lower temperatures. According to Rees agar forms antisymmetric double helices on cooling that hydrogen bond to form clumps of helices. These clumps can then form larger groupings that from a large porous gel structure. Agar is known to form a very porous gel and the pore size can be roughly measured by assessing the size of particulates that are excluded from the gel in a gel permeation experiment. It has been shown that agar gels can allow molecules up to 30M daltons in size to percolate through it structure. An agar gel as the unusual property of behaving like a sponge. An agar gel of a particular shape can be dried and upon rehydration it will swell to its original size and shape.

Agar synergy’s are not as commercially important as they are for xanthan or carrageenan and tend to be rather small in magnitude. Gelidium agar is known to form a small synergistic interaction with locust bean gum that is not seen in products based on gracilaria. Agar forms a synergistic interaction with sucrose and is used in some confectionery products. Tannic acid on the other hand may actually inhibit gelation. Agar is reasonably acid stable compared to other polysaccharides and does not show any protein reactivity. Agar can be used in acidic dairy products such as yoghurts where carrageenan would cause excessive flocculation due to the protein reactivity of the carrageenan. Recently a synergy has been reported between low gel strength agar and guar gum in patent by Rachid Lebbar of Setexam Agars all have negative optical rotations whereas carrageenans are positive. This can be used to distinguish the two when identification is tricky. Sulphate level is often used and whereas a low sulphate level would indicate an agar you cannot definitively say that a high sulphate level is always a carrageenan.

Agarose

 

The composition of this agar fraction has already been explained in the section dealing with the chemical structures of agar. In the literature we have found that agarose had been prepared according to at least 15 basic principles starting with the acetylation procedure of Araki (1937). A list of these methods follows even though they are interesting mainly for historical reasons.

    1. Acetylation. This method is based on the different solubility in chloroform of the acetates of agarose and agaropectin.

    2. Selective solution. This is based on solubility differences between agarose (less soluble) and agaropectin (more soluble) in aqueous media in well established conditions.

    3. Quaternary ammonium precipitation. This is classical method worked out by Hjerten (1962) and based on the insolubility of products resulting from the reaction of agaropectin with some quaternary ammonium salts.

    4. Polyethylene glycol. The classical method of Polson (Russell, Mead and Polson, 1964; Polson, 1965) based on the reduced solubility of agarose in media that contain polyethylene glycol.

    5. Dimethyl sulfoxide extraction. Tagawa (1966), the method is based on the different solubilities of agarose and agaropectin in this solvent.

    6. Ammonium sulfate precipitation. Azhitskii and Kobozev (1967), the method is based on the precipitation of agaropectin with ammonium sulfate.

    7. Ion exchange. Zabin (1969), the method is based on ion exchange in citrate or acetate forms.

    8. Insoluble support absorption, Barteling (1969), the method is based on the absorption of agaropectin on a non-reactive support such as aluminium hydroxide gel.

    10. Chromatography. Izumi (1970), the method is based on a chromatographic separation of agarose and agaropectin.

    11. Acrinol precipitation. Fuse and Gotto (1971).

    12. Electrophoresis. Hjerten (1971), the method uses electrophoresis over granulated or non-granulated agar gels or over powdered agar.

    13. Rivanol precipitation. Svridov, Berdnikov and Ivanov (1971), the method depends on the precipitation of agaropectin with rivanol.

    14. Chitin and chitosan precipitation. Allan et al. (1971), this method uses the absorbent chitin or chitosan to eliminate agaropectin.

    15. Ethanol or 2-methoxyethanol precipitation of agarose dissolved in a urea buffer. Patil and Kale (1973).

Based largely on these methods, other publications and patents have appeared modifying or maintaining these principles for processes for the preparation of agarose. Sometimes two or even three fractionating methods have been used successively in attempts to improve the agarose quality. At this time we have records of four companies manufacturing agarose and only one of them is an agar manufacturer, very different equipment is needed for the two kinds of production. For agarose, a quality control laboratory with very sophisticated analytical equipment to analyse the finished product is essential. Continuous improvement in technology is essential to adapt to modern applications in biochemistry which have required the introduction of modifications in the chemical structure of agarose, by synthetic organic chemistry in many cases. Thus, an agarose sample obtained from a manufacturer of biochemical reagents does not correspond normally to what we can extract from agar by any of the methods previously mentioned.

Criteria for judging agarose are multiple and they can be grouped in the following way.

    A. Physico-chemical properties. In this case the same basic criteria as for agar are followed: colour, transparency in solution, moisture, ash (in this case much lower due to the absence of polar groups), gel strength, gelling and melting temperatures.

    B. Purity critera. Reduction of electronegative groups to the minimum, the effects of such groups include an electroendosmosis increase and also an increase in the fixation of electrically charged substances, such as an increase in non-selective fixation of proteins. The increased presence of electronegative groups can also be produced by poor separation from the agaropectins. Likewise it is very important to assure the absence of residues of reagents used in the agarose production process.

    C. Specifications are necessary for practical applications, such as protein electrophoresis, DNA residues, non-selective fixation of proteins. For example the absence of inhibitors that could hinder the DNA recombining fragments split by agarose techniques. Controls that will prove agarose to be acceptable for biochemical techniques are included in this group.

Generally, the first two groups appear in specifications even though in some cases the data offered causes confusion, as happens for example in electroendosmosis. Although an accepted criteria for purity is a low electroendosmosis (less electronegative groups present) there are agaroses that have a greater electroendosmosis and yet are better in some specific biochemical separations. Values given to electroendosmosis vary widely for the same agarose when analytical conditions change, such as buffer pH, ionic strength, protein standards and non-charged molecules as well as other conditions dependent on the equipment such as voltage, operating cycle, refrigeration or electrical contact strips. The growing biochemical applications of agarose imply modifications in its structure to expand its range of uses. Thus, it is not realistic to set detailed specifications for a continuously evolving product and none have been set at a national or international level. Some typical specifications for commercial agarose can be found in the Sigma Catalogue (Sigma Chemical Co. 1987) and FMC offer their analytical methods to scientists in their catalogue, “Marine Colloids 1981 Bioproducts Catalog”.

Source: FAO

Agar Manufacturing Process

The production of agar, bacteriological agar and agarose are considered in this section.
Agar manufacturing processes have developed since the early freezing method was used to concentrate the extracts of agarophyte seaweeds. Whichever process is used, the following criteria should be taken into consideration. Firstly, it is necessary to obtain an extract from agarophyte seaweeds that contains the largest possible amount of the existing agar in the agarophytes. Secondly, the agar obtained should have the best possible characteristics to satisfy the standards expected for this product, especially as far as the gel strength is concerned. To achieve this it is necessary to consider the following basic points for the manufacturing process.

    1. The seaweed treatment prior to extraction.
    2. The control of molecular weight distribution during the extraction.
    3. The removal of undesired products.
    4. The need to work with large volumes of dilute extracts.
    5. The economics of dehydrating the dilute extracts.

1. SEAWEED TREATMENT PRIOR TO EXTRACTION
The seaweed treatments prior to extraction are very important as they will condition to a high degree the characteristics of the agar obtained. For example Gracilaria agar was once called an agaroid because at that time Gracilaria was not preteated properly resulting in a product softer than that obtained from Gelidium. Now Gracilaria is given a strong alkaline treatment before extraction. This causes hydrolysis of sulfate groups and transforms important quantities of L-galactose 6-sulfate into 3,6-anhydro-L-galactose, thereby significantly increasing the gel strength of the agar obtained. Tagawa and Kojima (1972) say the industry uses 0.25-0.5M sodium hydroxide solution at 80-90°C for 3-5 hours. Okazaki (1971) gives more detail, showing how the treatment varies depending on the country of origin of the Gracilaria (Okazaki is a useful reference for details of all methods used in the Japanese agar industry). Yang (1982) gives references to the methods used in Taiwan Province. The treatment, also called sulfate alkaline hydrolysis, must be adapted to the class of seaweed used, to obtain as much desulfation as possible while still avoiding the yield losses that this process can cause. These losses can be very important if agar is dissolved in the alkaline solution. The way these treatments are applied is variable and constitutes a part of the manufacturing process that has to be constantly adapted, according to the changing seaweeds, as it becomes a double-edged tool that can substantially reduce the yield if it is wrongly applied.

2. CONTROL OF MOLECULAR WEIGHT DURING EXTRACTION
Agar, as it occurs in seaweed, when extracted is insoluble in cold water and also practically insoluble in hot water. It is therefore necessary to extract it using suitable pH and redox conditions so that some hydrolysis occurs, thereby increasing its solubility. During this fractionation or cracking, it is necessary to avoid the subsequent reduction, by hydrolysis, of the molecular weight of the fragments which have dissolved. As all manufacturing methods are based on agar being soluble in hot water but insoluble in cold, excessive molecular weight reduction of the agar in solution would cause reduction of yields during the process, whenever molecular weights are reached for which cold solutions are possible. On the other hand it is important to avoid molecular units, in the agarophyte residues, that are not soluble either for lack of the necessary solution time or because of an excessive molecular weight that curtails solution under the conditions of extraction.

Figure 10 attempts to clarify a complex process in a simplified way since what we are putting into solution is not only agarose, with a quite uniform chemical structure, but also a mixture of agaropectins carrying electronegative charges, with a minimum solubility temperature that is above the one for agarose. We can see in the figure that all those molecules with molecular weights below PM1 will be easily extracted from the seaweed but will be lost due to their cold water solubility. In contrast, those molecules that remain in the seaweed with molecular weight above PM2 will not be extracted and will remain with the cellulose residues after extraction. The agar manufacturer has to establish working methods that enable the preparation of a molecular weight distribution curve that avoids both losses as much as possible. An ideal result would be that shown by the middle graph of the three shown in Figure 10. It is very difficult to modify the PM1 value but it is possible to increase the PM2 by raising the water temperature in the extraction; this is done by working under pressure whenever the seaweeds permit it. Naturally the differing stabilities of agars to hydrolysis poses limits to such temperature increases.

The industrial objective aims toward narrowing the type of Gaussian curve shown in Figure 10. This reduces losses and increases the molecular weight to the corresponding maximum in the chart which is accompanied by an increase in the agar gel strength. Such considerations will be correct whenever a constant agarose-agaropectin ratio is maintained.

3. REMOVAL OF UNDESIRED PRODUCTS
During the extraction process, a myriad of undesired products will be obtained as well as agar. Such products are soluble salts, seaweed pigments, cellulose, hemicellulose and many extracts coming from impurities and foreign materials contained in the weed, since commercial seaweeds differ greatly from those with which scientists work. Therefore in order to obtain the purest possible extracts in industry, seaweeds are selected and washed carefully and subjected to previous corrective treatments in which generally an alkaline solution eliminates a large quantity of foreign substances, particularly red pigments (phycoestrine), changing the weed to a green colour. This alkaline treatment is with sodium carbonate; it is milder than the alkaline treatment with sodium hydroxide which is used to improve the gel strength of Gracilaria agar.

A careful filtration will purify the extract but this is quite a difficult operation which requires a high temperature (85-100°C) because of the extract’s viscosity and high gelling power. Also cellulose and seaweed “floridean starch” residues, and even clay particles, make the filtration very difficult. Pressure filters are commonly used. Filter presses are the most useful ones, although modern factories use filters specially designed for this purpose.

Differences in the raw material greatly influence the operating methods and this makes further generalizations impossible.

Figure 9 Agarose gelification
Figure 10 Distribution of molecular weights in agar extracts

4. LARGE VOLUMES OF DILUTE EXTRACTS
Due to the high seaweed cost, high yields of agar are essential. However the extract concentrations range from 0,8% to 1.5% as a maximum; it is difficult to work with a more concentrated extract, for filtration as well as in the rest of the process. So the more agar that is extracted, the more water must be added to keep the concentration in the above range. This means that it is necessary to work with large volumes of extracts.

5. THE ECONOMICS OF DEHYDRATING THE DILUTE EXTRACTS
An important aspect to consider is the economics for dehydrating the large volumes of dilute extracts discussed in (4), This is a characteristic problem for this industry and its solution lies in methods based on the insolubility of agar when the extracts are cooled. Sometimes, because of lack of experience with the industry, projects are encountered in which evaporation or precipitation are recommended as the means of removing the large quantities of water from the extracts. We would first like to show why these methods are not feasible and afterwards discuss the methods actually used by the industry.

A. EVAPORATION
Starting from a 1% extract, 99 litres of water have to be eliminated for each kilogram of agar and since the latent heat enthalpy for water at 100°C is 539 kcal/kg, we need 53 361 kcal/kg (539 x 99 = 53 361). In our calculations we shall compare the heat requirements at a theoretical yield (impossible to obtain and far from the obtainable one) and consider only the heat for change of state; any heat requirement derived from specific heat will not be considered because of its small relative importance.

Working in an evaporator with liquids above a 2% concentration is impossible, problems are also posed by the gelling temperature of the extract and its large non-newtonian viscosity at temperatures close to gelling. All this prevents the thermal savings that could be gained by the use of multiple-effect vacuum evaporators.

B. PRECIPITATION
A working method similar to that used for carrageenan, using alcohol precipitation, could be considered. An economical process using this technique has not been achieved so far but the process is feasible chemically. Agar precipitation in alcohol media is more difficult than for carrageenan because the precipitate is more flocculant (has low cohesion) and is difficult to recover quantitatively. A high heat consumption is required because we have to add the heat needed to evaporate the alcohol, to the 53 361 kcal needed to evaporate the water in the mixture. In addition, the alcohol used for precipitation has to be recovered by distillation for reuse.

If we make our calculations using isopropanol, which is used for producing carrageenan for economic reasons, and consider that we start with an azeotropic mixture, previously recovered, of 87% by weight we are forced to work at least 3 litres of azeotropic isopropanol for each litre of extract to be precipitated. Assuming that such a mixture has a density of 0.8234 kg/L, then for each kilo of agar it would be necessary to evaporate:

    99 kg of water extract;
    22.5 kg of azeotropic isopropanol.

This second item is composed of 213 kg of isopropanol and 41.5 kg of water. The latent heat enthalpy for isopropanol is 175.8 kcal/kg. Therefore the theoretical heat energy consumption would be:

    water: 539 x (99 + 41.5) = 75 729 kcal;
    isopropanol: 175.8 x 213 = 37 445 kcal.

This means an energy consumption of 113 174 kcal/kg of agar which is double the heat energy need to evaporate the water contained in the extract, and all of this without taking into consideration the need to concentrate the used alcohol back to the azeotrope plus the recovery of isopropanol vapors that entail a considerable amount of energy. From this it can be seen that the precipitation/dehydration process analogous to that used for carrageenan has a high energy consumption when applied to agar.

For agar, concentration methods are based on its insolubility when cooled and are used in all factories according to two basic principles: freezing or syneresis under pressure.

C. FREEZING
This consists of freezing and thawing the extract, previously gelled, and profiting from the insolubility of agar in the cold to eliminate the greatest part of the water contained in the extract. Freezing should be slow, to allow both the growth of ice crystals and the separation of agar in the highest possible concentration; this is usually followed by draining with a water-extracting centrifuge. Only slow freezing permits large ice crystals to be formed, surrounded by fine sheets of agar. Efforts to speed up freezing produce spongy masses, with high water content and less agar concentration, that dialyse poorly and produce an impure agar, because the impurities which are soluble in cold water do not move so well from the gel to the water.

As far as the economics for this process are concerned, we should consider that if we start with a 1% agar extract, we have to eliminate 99 litres of water per kg of agar; after melting and draining, this agar at best reaches a dry extract content of 15% (1 kg in 6.66 L) but is normally 11-12%. Presuming a 15% agar in the product, the cycle of freezing-defrosting eliminates (99 L – 6.66 L) 92.34 litres water per kg of agar. Furthermore the energy consumption for freezing 1 L or 1 kg of water is 79.67 kcal. To freeze the 99 litres of water contained in the 1% extract would require:

    99 x 79.67 = 7 887 kcal

To remove the water remaining in the melted and drained agar requires a heat consumption of:

    6.66 x 5 390 = 3 590 kcal

We can see the difference between the sum of these figures (11 477) and those for:

    evaporation method = 53 361 kcal,
    precipitation method = 113 174 kcal.

Naturally there is a cost difference between obtaining a difference of a kilocalorie by heating or cooling but the figures leave us in no doubt (even though we have ignored the energy consumption derived from the specific heat of the water that is eliminated) there are enormous energy differences between the working methods considered.

D. SYNERESIS
Syneresis is usually described as the process in which a gel contracts on standing and exudes a liquid. Here the term syneresis is used to describe the process where pressure is used to exude liquid from the gel. The water that soaks the colloidal net of the gel is eliminated by applying, by suitable means, a force that will favour such loss. Energy consumption is very low when working in these conditions but not everybody can benefit from it because the industrial technology is not simple. Pressure has to be applied very carefully to avoid gel losses by extruding the gel through the containing system. The advanced factories that use this process have been obliged to develop a very specific technology, not only producing extracts in the appropriate conditions for good syneresis but also equipment design that will allow the efficient treatment of large quantities of extracts.

Initially long syneresis periods were required, with cycles longer than 24 hours, that would start with a gradual and slow increase in pressure by placing, successively and at a prefixed rate, stone blocks on top of the gel containers; the agar gel was wrapped in canvas cloths and placed in a series of steel boxes fitted between the fixed and movable heads of a vertical hydraulic press. This treatment was followed by hydraulic pressing, once the product was consistent enough to withstand extrusion. Usually a modified platen press is used which is similar to a box press but the cloth bags are not enclosed on the sides during pressing and the press is usually built in horizontal form. Nowadays some agar manufacturers have designed their own modern equipment which permits this syneresis to be carried out automatically in relatively short periods of time and operating with large volumes.

Starting with a 1% agar extract, syneresis increases concentration to a maximum of 25% (1 kg agar per 3 L water). If we consider an average of 20% for the dried extract from industrial runs, the heat energy necessary to remove the rest of the water will be:

    4 x 539 = 2 156 kcal/kg

which is much less than the heat energy needed to dry the agar obtained by freezing where moisture was calculated in ideal conditions, that are difficult to obtain in reality.

Compared to freezing, syneresis results in large electrical energy savings as the electrical energy needed to maintain a pressure on a quite incompressible product is much less than that necessary to freeze 99 litres of water for each kg of agar produced. The cost of electrical energy makes many freezing factories increase extract concentration but this is possible only up to 1.5% before producing a harmful effect from yield losses. Syneresis, when properly applied, will also produce a purer agar, eliminating a larger quantity of soluble matter.

6. GENERAL
Figure 11 is a flow chart showing the steps used in both of the dehydration processes used to produce agar. Treatment and reagents used in each case will be very variable depending on the species of seaweed used, its origin and even the time of the year when it was harvested. All these factors can cause drastic modifications to the treatment.

Figure 11, Agar production diagram

Nevertheless, we should consider some general rules. Seaweeds such as Gelidium, Pterocladia and Gelidiela can be Created by different diffusions, the most usual ones being sodium carbonate solutions, at about 80-95°C. Other reagents such as calcium or aluminium hydroxides or salts can also be used for several purposes. Other treatments with sodium hydroxide solutions of very variable concentration can be used, but the concentration will vary depending on what purposes they are for. As far as Gracilaria is concerned, 0.1 M sodium hydroxide solutions are commonly used; higher concentrations can also be used. The reagents named “Reagents I” in Figure 11 are basically the ones mentioned above. The ones shown as “Reagents II” are Chose used to adjust the extracting conditions and, in general, are organic or inorganic acids or salts with which pH and other extraction parameters are fixed.

The variables in the manufacturing process make it hard for a factory to change the seaweeds it uses as raw materials. Agar manufacturing history is full of fiascos caused by industries trying to change their seaweeds without having adequate technology to adapt to the change.

Water consumption in an agar factory varies widely depending on the seaweed used but it is always very high. Normally factories working Gracilaria seaweeds have a higher water consumption than others. Consumption also increases when an agar of better quality is required, although, in general, it can be reduced by a suitable design of the factory; however this can lead to an increase in investment and therefore to a more difficult project profitability. Factories using the freezing process have very high water consumption as cooling water is needed for the freezing equipment.

Using recycled water, after appropriate treatment, would reduce its consumption but, in general, would increase the plant operating cost. If poor quality water is going to be used, a prior treatment will be required but it is very important to know its cost before the location is decided since a mistake in this point could make the operation of the factory economically impossible.

The above-mentioned problems about water, and those originating from changing to seaweeds of a different origin, are the ones which have led many factories to bankruptcy.

A manufacturer of good quality agar must be ready to monitor his process and so be able to spot readily any variations that seaweeds cause in the yield or in the quality of the final product. For this purpose a well equipped control laboratory is required together with a pilot plant that will enable any modifications needed in the process to be studied prior to the industrial treatment of each batch of raw material. An adequate pilot plant can process from 1-10 kg of seaweeds, depending on the size and importance of the factory. In general small factories with elementary technology do not achieve international quality standards and their products have to be sold at lower prices in local markets. Bacteriological contamination particularly is usually too high and sometimes dangerous in such plants, closing them to many markets.

A food grade agar should have a moisture content of less than 18%, ash below 5%, gel strength above 750 g/cm2 (Nikan-Sui method) and a bacterial count below 10 000 bacteria per gram. Escherichia coli and Salmonella must be absent (other pathogenic bacteria may also be specified). Usually the lead content is specified as less than 5 ppm and arsenic less than 3 ppm. These specifications are for agar produced on an industrial scale. In the Orient, large quantities of “natural agar” are sold by very small producers and consumed in the form of threads (“strip”) or bars (“square”) that are usually produced from Gelidium and do not have to meet the above-mentioned specifications. Generally its gel strength is 450 g/cm2 by the Nikan-Sui method.

Figure 4b shows as closely as possible what we consider the present situation for the world production of agar. This table has been prepared taking into account the results obtained from an enquiry made among the most important agar manufacturers in countries such as Spain, Chile, Morocco, Portugal, Argentina, Mexico, France, New Zealand, Brazil, etc., and the available Japanese statistics. All these data along with others from Korea, People’s Republic of China, its Taiwan Province and Indonesia have been updated during the XIIth International Seaweed Symposium held in Brazil, August 1986.

BACTERIOLOGICAL AGAR
The use of agar in bacteriology is one of the most important uses and requires strict physical-chemical control as well as the absence of hemolytic substances and what is more important and difficult, the absence of any bacterial inhibitors. Robert Koch started using agar in 1881 to gel culture broths when preparing solid culture media and this was the first introduction of this oriental product to Europe.

Its uses in microbiology are based on the special properties: a gelling temperature of 32-36°C, a melting temperature of 85-86°C, a lack of hydrolysis by bacterial exoenzymes and its ability to be prepared without bacterial inhibitors. The above temperatures refer to culture media gelled with agar and which contain 10-11 g agar per litre of culture media.

Bacteriological agar is prepared from Gelidium and Pterocladia because Gracilaria and Gelidiella give agars with gelling temperatures above 41°C. It is manufactured in a limited number of highly specialized factories and under rigid physical-chemical and bacteriological controls.

There are no specifications for a universal application for bacteriological agar as the different microbiological schools evaluate the parameters in various ways. There are neither international nor national specifications. There are many differences between food grade and bacteriological grade, in physico-chemical and bacteriological controls, but this information is confidential and is shared only by the bacteriological agar and culture media manufacturers.

As agar is used only as a gelling agent in solid media, it is essential to avoid interactions with the rest of the media components such as meat extract, peptones, proteins, amino acids, sugars and other carbohydrates, as well as pigments, indicators, inhibitors, mineral salts, etc., used in their formulation. It has to mix with these components without producing problems such as colour changes, precipitate formation or gel strength losses, even after autoclave sterilization. Therefore actual specifications are different depending on each user and each culture media manufacturer. In general, bacteriological agars are very transparent agars in solution as well as in gel form and they represent the purest qualities in the world market. The rest of the parameters vary as the agars are adapted to the individual requirements of the manufacturer and end user.

In much smaller quantities, and at a much higher price, another type of agar called “Purified Agar” is also available. These are bacteriological agars that could also be used in biochemistry for electrophoresis or immunodiffusion; they can be considered as agarose forerunners, being still used for economic reasons.

Source: FAO

Chemical Structure Of Agar

Early studies of agar showed that it contained galactose, 3,6-anhydro-galactose (Hands and Peats, 1938; Percival, Somerville and Forbes, 1938) and inorganic sulfate bonded to the carbohydrate (Samec and Isajevic, 1922).

Structural studies have been based on the fractionation of agar by several methods, followed by chemical and enzymatic hydrolysis. The enzymatic hydrolysis studies of W. Yaphe have been of great importance. Subsequently the spectrochemical studies using infrared spectroscopy and nuclear magnetic resonance spectroscopy, particularly 13C n.m.r., have explained many important points in the structure of these intricate polysaccharides.

Infrared spectroscopy is the most accessible method for many laboratories. Figure 8a shows different absorption bands that have been characterized for the agar spectrum. The typical bands of a carrageenan spectrum are also shown (Figure 8b) because many of its important uses are similar to those of agar and the spectra are useful for distinguishing the two. The bands at 1 540 and at 1 640 cm-1 are especially noteworthy. They come from the proteins existing in agar and about which only a few comments have been made before. The peak at 890 cm-1 has not been identified up to the present time.

N.M.R. is of great importance when studying these structures. However the technique is difficult and it requires 13C n.m.r. equipment which only a few laboratories can afford. For this kind of work it is best to consult W. Yaphe’s papers, published from 1977 – for example, Bhattacharjee, Hamer and Yaphe, (1979); Yaphe (1984); Lahaye, Rochas and Yaphe (1986).

Agar is now considered to consist of two fractions, agarose and agaropectin. These were first separated by Araki (1937) and the results were published in Japanese so they were not readily available to some research workers. For example Jones and Peats (1942) assigned a single structure to agar defining it as a long D-galactose chain residue, joined by 1,3-glycosidic links; in the proposed structure, this chain was ended by a residue of L-galactose joined to the chain at C-4 and with C-6 semi-esterified by sulfuric acid. This false structure is still mentioned in some books on natural polymers and even in recently published encyclopedias.

AGAROSE

Interest in agarose was lost until Hjerten, working under Tiselius at the University of Uppsala, began to look for an electrically neutral polysaccharide suitable for electrophoresis and chromatography. He published an improved method of separation based on the use of quaternary ammonium salts (Hjerten, 1962). A technique for agarose preparation using polyethylene glycol was reported by Russell, Mead and Polson (1964) and later this was patented with Polson (1965) named as the inventor. Both methods gave agarose of sufficient purity to allow the study of its structure.

Figure 5 shows the type, and approximate relative quantities, of the residues that can be separated from the total hydrolysis of agarose.

Figure 6 shows agarose to be a neutral, long-chain molecule formed by b -D-galactopyranose residues connected through C-1 and C-3 with 3,6-anhydro-L-galactose residues connected through C-2 and C-4. Both residues are repeated alternately. The links between the monomers have different resistance to chemical and enzymatic hydrolysis. 1,3-a links are more easily hydrolysed by enzymes (Pseudomonas atlantica) and neoagarobiose results. 1,4-b links are more easily hydrolysed by acid catalysts and yield agarobiose units. Nevertheless 1,4-b links make the polysaccharide chain particularly compact and resistant to breakage, as is found in the peptidoglycan of bacteria. The molecular weight assigned to non-degraded agarose is approximately 120 000. This weight has been determined by sedimentation measurements and it represents 400 agarbiose (or 800 hexose) units linked together.

Figure 5 Agarose hydrolysis products
Figure 6 Agarose structure
Figure 7 Agaropectins hydrolysis products

 Figure 8a Infrared spectrocopy on agar films

 NOTES:

    (1) Peaks at 1060, 1180, 1250 and 1370 are produced by sulfates but the position occupied in the chain by the sulfates is not clearly seen in Agar due its low content of sulfates (< 2%).

    (2) Peak at 1750 not attributed up to this moment could be caused by methyl groups as Agar with 6-methyl forms a peak at 1780 cm-1.

Figure 8b Infrared spectrocopy on carrageenan films

NOTES. –

    1.- A peak at 831 CM-1 wide is mentioned in the Bibliography to correspond to a 3-Sulfates mixture.

        -O-SO equatorial vibration on C-2 of a galactose linked in (1 ® 3) ring.
        -O-SO vibration on C-2 of a galactose linked in (1 ® 4) ring.
        -O-SO vibration on C-6 of a galactose ring.

    2.- Carrageenans have wide and strong absorption bands in 1,000-1,100 CM-1 region which are typical in all polysacharides.

    3.- Maximum absorption is given by 1,065-1.020 CM-1 for all carrageenan types (Kappa, Iota, Lambda, etc.)

This clarifies the information in Figure 6. However it should be noted that, depending on the origin of the raw material, some units of 3,6-anhydro-L-galactose are replaced by L-galactose. Also some D-galactose and L-galactose units can be methylated and it is said they can be in fact 6-0-methyl-D-galactose and 2-0-methyl-Lgalactose. This methylation, arising from the seaweed used in the process, determines the agarose gel point and therefore that of the agar it comes from. D-xylose has been found in very small quantities from hydrolysed agarose but it has not been possible to assign it a position in the structure.

Polar residues such as pyruvic and sulfuric acids are also found in small quantities. They may come from the small amounts of agaropectin lef in the agarose after its preparation but in our opinion sulfate and pyruvate groups remain linked in small quantities to the agarose structure, depending on the seaweed used in agar production. We follow the traditional definition of agaroses as those products obtained as the non-charged fraction after using a classical separation technique such as the precipitation with quaternary ammonium salts by Hjerten. On the other hand, in spite of the copious bibliography on this matter (we have seen 14 different basic methods to prepare agarose), none of the methods permits an agarose preparation free of electronegative charges. Many researchers have used two or three fractionating methods successively, in order to improve the separation and reduce the amount of electronegative groups present. In spite of all these efforts, these groups could not be eliminated. To cancel the electroendosmotic flow, which might be induced by these electronegative groups, it has been necessary to fix electropositive groups or use some other means so as to reduce the migration of cations (and their solvation water molecules) fixed to electonegative groups. Consequently we consider the agarose theoretical structure a chimerical dream to which we get closer each time by using more refined fractionation methods although perhaps, in practice, it may not exist at all in agar and the agarophyte seaweeds.

Nowadays commercial agaroses for use in biochemical separation techniques have to be chemically modified, so that their structure is different from the agarose as it is extracted from the seaweed, Phycologists should be aware that this is so, unless the manufacturer states that the original chemical structure has not been modified.

Agarose is responsible for the gelling power as we know it in agar. This is a gelation in aqueous media with a very small reactivity with cations and proteins and this differentiates agar from carrageenan.

AGAROPECTIN

Agaropectin (or better, the agaropectins) have a low gelling power in water. At the present time, a specific structure has not been assigned to the agaropectins. It is customary to say that they are formed by alternating units of D-galactose and L-galactose, and that they contain all the polar groups existing in agar.

Figure 7 shows the residues obtained by hydrolysis; among them, sulfated and pyruvate residues are evident. It has been verified that L-galactose 6-sulfate and D-galactose 4-sulfate are the major sulfate residues in agar. From small to moderate quantities of 3,6-anhydro-L-galactose have also been detected. These small quantities vary depending on the origin of the seaweed, on the harvesting season, on the treatment applied during the agar manufacturing process and on the treatment used during the agarose separation process.

The presence of 4,6-0-(1-carboxyethylidene)-D-galactose has also been verified, making the position of pyruvic acid in the structure perfectly clear. This unit is relatively important in agaropectin but in agarose it appears in much lower levels, as mentioned previously, probably because agarose has terminal units of 4,6-0-(1-carboxyethylidene)-D-galactose. The quantity of pyruvic acid in agar and agarose varies widely depending on the seaweeds used as raw material; we have verified quantities between 0.2-2.50% in agar and 0.02-1.30% in agarose. In this regard the work of Hirase (1957) is very interesting and explanatory.

These variations, that sometimes can be very important, appear even in seaweeds of the same class harvested a short distance from each other and seem to be permanent and depend on the growing locations. Over a period of several years (more than 10 in some cases) we have studied different Gelidium or Gracilaria harvesting areas in Europe, Asia and America, verifying the persistence of this phenomenon that can be caused by microclimatic differences. In our opinion the differences in cations existing in certain habitats also can be a cause. Naturally the different types and species cause differences that are very important sometimes in the agarose and agaropectin structures.

In Figure 7, D-galactose 2,6-disulfate has been included because we think we have identified it in small quantities in the agaropectins of some seaweeds grown in difficult conditions (“El Niño” phenomenon). These agaropectins had high viscosity, that was also apparent in the agar from which they came, along with a lower gelling power. In cases where this sulfated residue is found, the agaropectin and the agar have undesirable properties. Also shown in Figure 7 are D-galactose and L-galactose which appear to be modular units of agaropectin. Glucuronic acid is present only in traces (like the D-xylose found in agarose).

So while the basic structure of agaropectin consists of alternating D-galactose and L-galactose, D-galactose can be substituted by D-galactose 4-sulfate, by 4,6-0-(1-carboxyethylidene)-D-galactose in certain terminal chain positions or even possibly by D-galactose 2,6-disulfate, while part of L-galactose can be replaced by 3,6-anhydro-L-galactose. These different substitutions of the basic monosaccharide give an enormous number of possible structures.

McCandless used an immunochemical method to detect different carrageenan fractions with great sensitivity (Di Ninno and McCandless, 1978 and 1978a). A similar method might be applied to studying the different kinds of agaropectins in regard to their different seaweed origins, as well as the posible evolution of the structure of agaropectins during the life of the seaweed. To do this it is necessary to take into consideration the different fractions preceding the series of biochemical transformations produced by the algal enzymatic mechanisms which result in certain terminal fractions (one of which may be agarose). The current possibilities through monoclonal antibodies would allow an improvement of the sensitivity and selectivity of the method used by McCandless.

Source: FAO

Agar-Agar

Gracilaria

Another name that is used for agar is agar-agar. Agar is a natural polysaccharide, present in the different seaweeds of the family Rhodophyceae (Gelidium spp. and Gracilaria spp.).
These seaweeds are harvested mainly in the USA and Japan. After harvesting, the agar is extracted from the seaweed and purified.

Production Process of Agar
The basic extraction of agar from seaweeds, such as Gelidium and Gracilaria, starts with washing the seaweed to remove foreign matter. There are some differences in the processing of the various seaweeds prior to extraction, depending on the type of seaweed used.

Gelidium is simply washed to remove sand, salts, shells and other foreign matter and is then placed in tanks for extraction with hot water.

Gracilaria is also washed, but it must be treated with alkali before extraction; this alkaline pre-treatment causes a chemical change in the agar from Gracilaria, resulting in a product with an increased gel strength. Without this alkaline pre-treatment, most Gracilaria species yield an agar with a gel strength that is too low for commercial use.

The alkali treatment is heating the seaweed in a 2-5 percent sodium hydroxide at 85-90°C for 1 hour; the strength of the alkali varies with the seaweed species and is determined by testing on a small scale. After removal of the alkali, the seaweed is washed with water, and sometimes with a very weak acid to neutralize any residual alkali.

In the next processing step, the seaweed is heated with water for several hours during which the agar dissolves in the water. Gelidium seaweed is more resistant and extraction under pressure (105-110°C for 2-4 hours) is faster and gives higher yields. Gracilaria seaweed is usually treated with water at 95-100°C for 2-4 hours. The remainder of the process is the same for both types of raw material. The hot extract is given a coarse filtration to remove the seaweed residue, filter aid is added and the extract is pumped through a filter press. The extract is thick and will gel if allowed to cool, so it must be kept hot during the filtration processes. The hot filtrate is cooled and forms a gel which contains about 1 percent agar. The remaining 99 percent is water containing salts, proteins, carbohydrates and color molecules. The gel is broken into pieces, and, if necessary, it is bleached to reduce the color. Then it might be washed again to remove the bleach and remaining salts.

The next step is removing the remainder of the water from the gel. There are two methods commonly used: either a freeze-thaw process or by using pressure. In the freeze-thaw process, the gel is slowly frozen so that large ice crystals form. The structure of the gel is broken by the ice crystals and when it thaws again a lot of the water drains away, leaving a concentrated gel which contains about 10-12 percent agar. Sometimes, this gel is placed between porous filter cloths and squeezed in a hydraulic press to remove more water. However, this is a slow process, and usually the thawed material is simply drained and placed in a hot-air dryer. After drying, it is milled to the required particle size. Because of the refrigeration costs, this freeze-thaw process is relatively expensive, compared to the alternative described next.

The alternative process relies on syneresis. To seperate the water from the agar gel, pressure is used. The 1 percent agar gel is placed in a press. Pressure is applied and very slowly increased over about 24 hours, forcing liquid out of the gel. The remaining gel, now containing about 20 percent agar, is removed from the press, shredded and dried in a hot-air oven before being milled to the required particle size. With no refrigeration required, the energy consumption is much lower than for the freeze-thaw method, and, since more water has been removed, less soluble matter remains, so the agar is more pure. Less energy is also needed in the drying process since less water needs to be removed.

This process based on syneresis has been widely adopted by large agar producers who can afford the higher capital costs for this equipment.

Chemical Structure and Properties
Agar is a combination of neutral (agarose) and electrically charged (agaropectine) molecules. Agarose is a linear polymer,made up of the repeating monomeric unit of agarobiose. Agarobiose is a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose.

Agar is soluble in hot water. It forms a reversible gel when it cools down. The temperature at which agar gels is between 32 and 43°C. The gel can be melted again when heated to 85ºC. Due to its high melting point, it does not melt in the mouth. It also does not need refrigeration, making it very suitable for warm climates.

Agar has a high gel strength. It can be used in an broad pH range. It can used alone or mixed with other hydrocolloids, carbohydrates and proteins.

Some agars are sugar reactive, combining the agar with high sucrose solutions increases the gel strength.

Agar does not have a flavor or odor.
Agar has a slight yellow – off white color. It is available in a powder or strip form.

Functionality in Food
Agar is used as a thickening agent and stabilizer. It can also be used as a non-caloric fiber as it is not digested by the human body.

Applications
About 90 percent of the agar produced is used in foods. In the baked goods industry, the ability of agar gels to withstand high temperatures makes it very suitable as a stabilizer and thickener in pie fillings and meringues. Adding agar to icing creates a more stable, non stick and smooth icing.

Some agars, especially those extracted from Gracilaria chilensis, can be used in confectionery with a very high sugar content, such as fruit candies.

Agar is traditionally used in Asian countries, as the gelling component of jellies. A popular Japanese sweet dish is mitsumame: cubes of agar gel containing fruit and added colors. It can be canned and sterilized without the cubes melting.

Agar is also used in gelled meat and fish products, and is often preferred to gelatin because of its higher melting temperature and gel strength.

In combination with other gums, agar has been used to stabilize sherbets and icecreams. It can improves the texture of dairy products like cream cheese and yoghurt. It has been used to clarify wines, especially plum wine, which is difficult to clarify by traditional methods.

Agar is used in vegetarian foods such as meat substitutes.

Market, Marketing And Future Prospect Of Agar

A summary of the agar markets is shown in Table 3. It does not include production from Gelidiella acerosa and Gracilaria species in India, where 800-1 300 dry tonnes of seaweed are used to produce 100-160 tonnes/year of agar.

All the companies previously listed as agar producers sell directly to agar users. However, there are other companies that buy from producers and re-sell the agar, either alone or in admixture with other hydrocolloids, to users. These companies specialize in supplying food ingredients, usually defined as food additives that improve the quality, texture, stability or presentation of a food product. Because they are more active in the carrageenan and alginate industries, further discussion about them can be found later, in the relevant sections.

Future prospects
The market for food grade agar is stable and not likely to expand very much in the near future, unless new uses are developed, and this does not seem likely at present. During the last 30-40 years agar has gradually been replaced in some of its traditional uses by other hydrocolloids that either gave a better result in particular applications or are cheaper. Uses now are restricted to those that depend on the unique gelling properties of agar. There are many producers, some endeavouring to capture market share with low price or low quality material, so it is becoming a very tight market. The bacteriological agar market is also stable, but present prospects are that it is unlikely to show much expansion in the next five years. The market for agarose will expand during the next five years as its uses in biotechnology increase and probably diversify as new techniques are developed. However, it is a specialized and relatively small market; users often purchase in lots of 100 g, with a total worldwide consumption of about 50 tonne/year.

Agar Uses

The uses of agar centre around its ability to form gels, and the unique properties of these gels. Agar dissolves in boiling water and when cooled it forms a gel between 32° and 43°C, depending on the seaweed source of the agar. In contrast to gelatin gels, that melt around 37°C, agar gels do not melt until heated to 85°C or higher. In food applications, this means there is no requirement to keep them refrigerated in hot climates. At the same time, they have a mouth feel different from gelatin since they do not melt or dissolve in the mouth, as gelatin does. This large difference between the temperature at which a gel is formed and the temperature at which it melts is unusual, and unique to agar. Many of its applications take advantage of this difference.

 Food
About 90 percent of the agar produced is for food applications, the remaining 10 percent being for bacteriological and other biotechnology uses. Agar has been classified as GRAS (Generally Recognized As Safe) by the United States of America Food and Drug Administration, which has set maximum usage levels depending on the application. In the baked goods industry, the ability of agar gels to withstand high temperatures means agar can be used as a stabilizer and thickener in pie fillings, icings and meringues. Cakes, buns, etc., are often pre-packed in various kinds of modern wrapping materials and often stick to them, especially in hot weather; by reducing the quantity of water and adding some agar, a more stable, smoother, non-stick icing is obtained.

Some agars, especially those extracted from Gracilaria chilensis, can be used in confectionery with a very high sugar content, such as fruit candies. These agars are said to be “sugar reactive” because the sugar (sucrose) increases the strength of the gel. Because agar is tasteless, it does not interfere with the flavours of foodstuffs; this is in contrast to some of its competitive gums that require the addition of calcium or potassium salts to form gels. In Asian countries, it is a popular component of jellies; this has its origin in the early practice of boiling seaweed, straining it and adding flavours to the liquid before it cooled and formed a jelly. A popular Japanese sweet dish is mitsumame; this consists of cubes of agar gel containing fruit and added colours. It can be canned and sterilized without the cubes melting. Agar is also used in gelled meat and fish products, and is preferred to gelatin because of its higher melting temperature and gel strength.

In combination with other gums, agar has been used to stabilize sherbets and ices. It improves the texture of dairy products like cream cheese and yoghurt. It has been used to clarify wines, especially plum wine, which is difficult to clarify by traditional methods. Unlike starch, agar is not readily digested and so adds little calorific value to food. It is used in vegetarian foods such as meat substitutes.

Other uses
In the pharmaceutical industry agar has been used for many years as a smooth laxative.
In orchid nurseries, agar gels containing appropriate nutrients are used as the growth substrate to obtain clones or copies of particular plants. Meristems – the part of the plant with actively dividing cells, usually the stem tips – are grown in the gel until there has been sufficient root development and growth for them to be transplanted. An advantage of this system is that the plants can be cultured in a sterile environment.

Microbiological agar
Bacteriological agar is used in testing for the presence of bacteria. It is specially purified to ensure that it does not contain anything that might modify bacterial growth. It is therefore more expensive, frequently at least twice the price of food grade agar. A hot agar solution (1-1.5 percent) is prepared and as it cools, nutrients or other chemicals specific for the type of bacteria being tested are added. When the solution has cooled below its gel point, the sample suspected of containing bacteria is spread on the surface of the gel, which is then covered and stored at a temperature suitable for bacterial growth. The agar gel should be as clear as possible so that any bacterial growth can be easily seen.

For further details
Further information about the uses of agar can be found in Glicksman (1983) and Armisen and Galatas (1987, 2000). Armisen and Galatas (2000) also contains some interesting recipes for yokan (traditional Japanese), sweet potato dessert (traditional Argentinian) and sugar icings, all of which illustrate typical methods for using agar in foods. Armisen (1997) lists eleven important advantages enjoyed by agar in food applications. Armisen (1995) is a paper about the use and importance of Gracilaria, but it also has useful discussions about natural and industrial agars, compares the characteristics of agars from Gelidium and from Gracilaria, and is useful background reading for those wishing to learn more about the agarophyte and agar industries. Source FAO

Inasea Seaweed Gallery

http://apps.bravenet.com/go.js?service=photocenter;id=1;usernum=1040606654

Agar Production Methods

Food grade agar    A short and simplified description of the extraction of agar from seaweeds is that the seaweed is washed to remove foreign matter and then heated with water for several hours. The agar dissolves in the water and the mixture is filtered to remove the residual seaweed. The hot filtrate is cooled and forms a gel (jelly) which contains about 1 percent agar. The gel is broken into pieces, and sometimes washed to remove soluble salts, and, if necessary, it can be treated with bleach to reduce the colour. Then the water is removed from the gel, either by a freeze-thaw process or by squeezing it out using pressure. After this treatment, the remaining water is removed by drying in a hot-air oven. The product is then milled to a suitable and uniform particle size.

However, for a better understanding of the process, some of the details and difficulties need to be described.
There are some differences in the treatment of the seaweed prior to extraction, depending on the genus used. Gelidium is simply washed to remove sand, salts, shells and other foreign matter and is then placed in tanks for extraction with hot water. Gracilaria is also washed, but it must be treated with alkali before extraction; this alkaline pre-treatment causes a chemical change in the agar from Gracilaria, resulting in an agar with an increased gel strength. Without this alkaline pre-treatment, most Gracilaria species yield an agar with a gel strength that is too low for commercial use. For the alkali treatment, the seaweed is heated in 2-5 percent sodium hydroxide at 85-90°C for 1 hour; the strength of the alkali varies with the species and is determined by testing on a small scale. After removal of the alkali, the seaweed is washed with water, and sometimes with very weak acid to neutralize any residual alkali.

For the hot-water extraction, Gelidium is more resistant and extraction under pressure (105-110°C for 2-4 hours) is faster and gives higher yields. Gracilaria is usually treated with water at 95-100°C for 2-4 hours. The remainder of the process is the same for both types of raw material. The hot extract is given a coarse filtration to remove the seaweed residue, filter aid is added and the extract is pumped through a filter press equipped with a fine filter cloth. The extract is thick and will gel if allowed to cool, so it must be kept hot during the filtration processes.

The filtrate is now cooled to form a gel, which is broken into pieces (Figures 7 and 8). This gel contains about 1 percent agar. The remaining 99 percent is water that may contain salts, colouring matter and soluble carbohydrates. The gel may be treated with bleach to reduce any colour, washed to remove the bleach, and allowed to soak in water so that most of the salts can be removed by osmosis. The wash waters are drained and the remainder of the process is concerned with the removal of the 99 percent water in the gel. Either of two methods can be used for this.

The original method of water removal is the freeze-thaw process. The gel is slowly frozen so that large ice crystals form. The structure of the gel is broken down by the freezing so that when the material is thawed most of the water drains away, leaving a concentrated gel that now contains about 10-12 percent agar (this means about 90 percent of the original water content has been removed, and with it went a high proportion of any salts, soluble carbohydrates and soluble proteins that may have been present in the gel). Sometimes this gel is placed between porous filter cloths and squeezed in a hydraulic press to remove more water. However, this is a slow process, and usually the thawed material is simply drained and placed in a hot-air dryer. After drying it is milled to the required particle size, usually about 80-100 mesh size. Because of the refrigeration costs, this freeze-thaw process is relatively expensive, compared to the alternative described next.

Figure:7

Hot agar solution is fed, from the T-shaped PVC pipe,

 as a thin layer onto a stainless steel belt where it is cooled and forms a gel.

Figure 8

Pieces of gel breaking up as they fall off the end of the stainless steel cooling belt.

 A cutting device, consisting of a stainless steel screw and thin wire, is at the bottom of the ramp.

Sometimes the thawing is accelerated by washing the frozen blocks of gel with large quantities of water (Figure 9), but this adds to the already large water consumption of the process.

The alternative process relies on synaeresis. This is the term used to describe the separation of liquid from a gel. A common example is that of a partly used jar of jam or preserves that is left standing for several days: pools of liquid can often be seen at the surface. However, for the agar gel, pressure is used to force the separation of the liquid. The equipment used is based on the following. Two grooved metal plates are covered with porous cloth and the 1 percent agar gel is placed between the cloths, like a sandwich with metal plates on the outside, then the layers of cloth, with the gel in the middle. Pressure is applied to the metal plates and very slowly increased over about 24 hours, forcing liquid out of the gel, through the cloths, down the grooves of the metal plate and away to a drain. The piece of equipment contains about fifty of these sandwich-type units, all in a vertical plane, all being placed under pressure by one hydraulic ram (Figure 10). At the end of the time, the pressure is released, the metal plates are separated and the remaining gel, now containing about 20 percent agar, is peeled off the porous cloth (Figure 11). It is shredded and dried in a hot-air oven before being milled to the required particle size, usually about 80-100 mesh size. With no refrigeration required, the energy consumption is obviously much lower than for the freeze-thaw method, and, since more water has been removed, less soluble matter remains, so the agar is more pure. Less energy is also needed in the drying process since less water is being removed. This process based on synaeresis has been widely adopted by large agar producers who can afford the higher capital costs for this equipment.

Figure 9

Thawing frozen slabs of agar by hosing with water.

Figure 10

Dewatering machine used to squeeze water from agar gel.

Figure 11

A sheet of agar gel after squeezing in the dewatering machine.

Figure 12

Agar blocks (left) and agar strips (right).

Figure 13

Flow chart for the production of agar (after Armisen and Galatas, 1987).

A large and reliable freshwater supply is a requirement for an agar factory. Water consumption is high and the processing of Gracilaria requires more than for Gelidium. Higher water consumption also means larger quantities for waste disposal, so recycling of water is becoming more necessary, depending on the location of the factory.

For further details
Detailed information on the commercial extraction process is not easily available. There are several short publications on the results from laboratory-scale extractions, but commercial agar producers are generally secretive about the details of their processes. Armisen and Galatas (1987) is one of the few publications that gives some details, but there are still many gaps, particularly in the conditions of the alkali treatment and the subsequent hot water extraction; nevertheless, it is the best starting point. The original print version may not be readily available but it can be read and downloaded from the FAO Web site (see References 2 – Internet sources). A later book chapter by the same authors, Armisen and Galatas (2000) gives a useful comparison of the freeze-thaw and synaeresis methods for removing water from the agar gel. Nussinovitch (1997: 4-5) also has a few useful details about extraction.

 Agar strips
Agar for use in food is sold in two forms: strip agar and agar powder. The powder is produced by the method previously described. Agar strip, sometimes called natural agar, is produced on a small scale in China, Japan and the Republic of Korea by the old, traditional method. Gelidium must be used; it was the only raw material used before the Second World War. It is boiled for several hours in water, acidified by the addition of either vinegar or dilute mineral acid. The hot extract is filtered through cotton cloth, then poured into wooden trays to cool and form a gel. The gel is extruded to produce spaghetti-type strips about 30 cm long. The strips are placed outside at night to freeze and allowed to thaw in the day, so water is released and runs off, leaving a more concentrated gel. This process can be repeated, or modern refrigeration can be substituted. The strips are dried in the sun, which also bleaches the strips. Strips are assembled into bundles and sold for domestic use (Figure 12). Prior soaking makes them easier to dissolve in boiling water.

Bacteriological agar
This can only be made from species of Gelidium because the resulting agar has a low gelling temperature (34-36°C) that allows the addition of other materials to the agar with a minimum risk of heat damage. Gracilaria and Gelidiella give agars that gel at 41°C or higher. “Bacto” agars must not contain anything that might inhibit the growth of bacteria, such as trace metals, soluble carbohydrates or proteins, nor should they contain any bacterial spores. They must not interact with any materials that must be added as nutrients for the bacteria under study. The gels must be strong and have good clarity. Manufacturers of bacteriological agar keep all processing details confidential. However, recently Kim et al. (2000) published details [in Korean] of a pilot-scale preparation that they claim gave a product that is superior to commercial bacteriological agar. Armisen and Galatas (1987) and Armisen (1997) discuss the necessary specifications for bacteriological agar.

Agarose
Agar can be divided into two principal components: agarose and agaropectin. Agarose is the gelling component; agaropectin has only a low gelling ability. There are several methods of producing agarose; many rely on removing the agaropectin from the agar. There are only a small number of processors who produce purified, high quality agarose for a small but growing market, mainly in biotechnology applications. These processors use good quality agar as their starting material rather than seaweed, and are often not in the seaweed processing business. Armisen and Galatas (1987) summarize the methods that have been used to isolate agarose from agar, and discuss the specifications expected for a high quality agarose. (Source FAO)