The Biotechnological Aspects Of Agar Biology Essay

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The growing seaweed production offers a natural source of applicable substances used in a variety of industries responsible for the production of food, animal feed-stock, fertilizer, biofuel and cosmetics. Seaweed is harvested either from its natural habitat (wild) or from cultivated crops. Because of the rapidly expanding demand of seaweed products which exceed natural available resources, cultivation has increased dramatically over the years. Seaweeds are classified into three main groups of macro-algae namely: Rhodophyceae (red), Phaeophyceae (brown) and Chlorophyceae (green). Both brown and red algae are directly processed into edible products and are used for the extraction of the polysaccharides alginate (brown), carrageenan and agar (red).

Fig. 1 Images of the main group of macro-algae: Chlorophyceae (green), Rhodophyceae (red), Phaeophyceae (brown).

1. Cultivation of red algae in the seaweed industry:

1.1 Methods for cultivation of algae

Two general procedures are used for the growth of seaweed, one is by vegetative method and the other is by separate reproductive cycle involving alternation of generation. The vegetative method is mostly used for the production of hydrocolloids (agar and carrageenan) while the principal seaweeds used as foods must be taken through the alternation of generation for their cultivation. The vegetative method is simply the straightforward cultivation of seaweed placed in an environment that will sustain their growth, depending on salinity, nutrients, light, temperature and movement of the water. To obtain optimal growth, seaweed is attached to ropes or nets that are tied to a floating wooden frame. Cultivation involving a reproductive cycle, with alternation of generations, is necessary for many seaweeds, especially the Phaeophyceae algae. For these, new plants cannot be grown by taking cuttings from mature ones. The sporophyte is what is harvested as seaweed. To grow a new sporophyte it is necessary to go through a sexual phase involving the gametophytes. The mature sporophyte releases spores that develops and grow into microscopic gametophytes.

The gametophytes become fertile, release sperm and eggs that join to form embryonic sporophytes. These slowly develop into the large sporophytes that are harvested(1).

1.2 Agarophytes

Two general species of seaweed, Gelidium and Gracilaria, account for most of the raw material used for the extraction of agar.

Gelidium: Extraction of Gelidium gives the higher quality agar (measured by the gel strength). All Gelidium used for commercial agar extraction comes from natural resources, principally from France, Indonesia, the Republic of Korea, Mexico, Morocco, Portugal and Spain. Gelidium is a small, slow growing plant and while efforts to cultivate it in tanks/ponds have been biologically successful, it has generally proved to be uneconomic.

Gracilaria: Gracilaria species were once considered unsuitable for agar production because the quality of the agar was poor (gel strength too low). In the 1950s, it was found that pre-treatment of the seaweed with alkali before extraction lowered the yield but gave a good quality agar. This allowed expansion of the agar industry and led to the harvesting of a variety of wild species of Gracilaria in countries such as Argentina, Chile, Indonesia and Namibia. Due to overharvesting of the wild crop cultivation methods were developed, both in ponds and in the open waters of protected bays. These methods have spread to other countries, such as China, the Republic of Korea, Indonesia, Namibia, the Philippines and Viet Nam, usually using species of Gracilaria native to each particular country. Gracilaria species can be grown in both cold and warm waters.

1.3 Harvesting methods for wild agarophytes

Industrial harvesting techniques for agarophytes vary, depending on circumstances, but they can be classified as follows:

- gathering of seaweeds washed to the shore;

- gathering seaweeds by cutting or rooting them

out from their beds;

- cultivation.

Gathering of seaweeds washed to the shore. In some countries these seaweeds called "argazos", "arribazon" or "beach wash". These are dead seaweeds that, after completing their biological cycle, are separated by seasonal storms. They are gathered by hand or by mechanical means from the coast or by compressed air ejectors from boats that gather the seaweeds settled in cavities at depths of about 25 metres ("wells"). To avoid fermentation, the seaweed should be gathered shortly after it has separated from its holdfast.

Gathering seaweeds by cutting or rooting them out from their beds. This work is done with rakes or grabs handled from boats or by divers. Gelidium usually occurs on rocky beds, Gracilaria on sandy ones. In general it is feasible to operate with divers in depths between 3 and 20 metres.

Cultivation. Nowadays the need for greater quantities of agarophytes has brought about the introduction of cultivation of Gracilaria crops. However this cultivation has had only limited success and there are some aspects to be solved before it can be generally adopted. At the present time, cultivation for industrial purposes is undertaken in the People's Republic of China, its Taiwan Province and it is now being initiated in Chile.

1.4 Post-harvest treatment

The preservation of seaweeds, between the time of harvesting and their actual use by the agar manufacturer, is very important. To build a seaweed processing factory, which consumes seaweeds at the rate they are harvested, is not practical. Large scale agar manufacture makes it necessary to have available quantities of agarophytes stabilized in such a way that they can be carried long distances, at the least possible cost, and stored for a long time before processing.

The first step is preservation through dehydration, to avoid fermentation that first destroys the agar and then the seaweed. The second step is pressing the weed with a hydraulic press in bales of about 60 kg, to reduce the volume and return transportation and storage costs. Dehydration must be sufficient to guarantee the seaweed's preservation, otherwise an anaerobic fermentation will occur inside the bales causing high temperatures and even carbonization of the seaweeds during storage in warehouses. In general, the moisture content is best reduced to about 20% by natural or artificial drying. In the case of Gracilaria the problem is more difficult to solve. The enzymatic hydrolysis of its agar occurs spontaneously even at relatively low moisture contents, but at variable rates depending on the Gracilaria species and its origin. Gracilaria harvested in India, Sri Lanka,Venezuela, Brazil, and generally in warm waters, has an agar (agarose) less resistant to enzymatic hydrolysis than the Chilean Gracilaria which is the most stable.

Nevertheless, the stability of agar contained in Gracilaria is less than that of Gelidium; Gelidium agar can be preserved in seaweeds indefinitely provided they have been well treated.

Hydrolysis of agar contained in Gracilaria can be due to endogenous enzymes or to the growth of Bacillus cereus(2).

2. Production process of Agar:

2.1 Agar production methods

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. There are some differences in the treatment of the seaweed prior to extraction, depending on the agarophytes 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(3).

2.2 Forms of Agar for industrial uses:

2.2.1 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. Prior soaking makes them easier to dissolve in boiling water.

2.2.2 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 gives agar that gel at 41°C or higher. bacteriological 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.

2.2.3 Agarose

Agar can be divided into two principal components: agarose and agaropectin. Agarose is the gelling component; agaropectin has only a low gelling ability. Agarose is a linear polymer, made up of the monomer agarobiose. Agarobiose is a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose. Agaropectin is a heterogeneous mixture of smaller molecules that occur in lesser amounts. Their structures are similar but slightly branched, sulfated, and may have methyl and pyruvic acid ketal substituents.

Fig. 2 Chemical structure of agarobiose (monomer) and agarose (polymer).

Some agars, especially those extracted from Gracilaria, 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.

Fig 4. The Japanese sweet dish 'mitsumame'.

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.

3.3 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.

Fig 5. Agar used as a growth substrate.

The gel network of agarose contains double helices formed from left-handed threefold helices(4). These double helices are stabilized by the presence of water molecules bound inside the double helical cavity. Exterior hydroxyl groups allow aggregation of up to 10,000 of these helices to form suprafibers.

3. Applications of Agar-based products:

3.1 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(5). In contrast to gelatin gels, that melt around 37°C, agar gels do not melt until heated to 85°C or higher.

Fig. 3 Example of agar-agar used as a gel in food.

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.

3.2 Food

About 90 percent of the agar produced is for food applications, the remaining 10 percent being for bacteriological and other biotechnology uses. 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.

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.

3.4 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.

Fig 6. A petri dish with bacterial colonies on a agar-based growth medium.