were found in the agar quality, obtained from various developmental stages of agarophyte Gracilaria bursa-postoris, Gracilaria coronopifolia, G. verrucosa and Onikusa pristoides. However in carrageenophyte, the quality of the agar gel and its structure doesn’t get affected by the different stages of algal life.
As a consequence, it will be good to use ‘agar’ as a standard term for the repetitive interrelated molecules based on repeated units of agarobiose. These types of polysaccharides differ in their physico-chemical and chemical properties all through their algal genotypes and even within the algal species. Since this polysaccharide family is so diverse in such a way that the nature of agar gel gets affected by physiological, cellular as well as by environmental factors. Thus not only physico-chemical but also chemical alterations due to these factors, should be explicated.
5.8.1 Techniques to Analyze the Fine Chemical Structure of Agar
With the development of various physico-chemical analysis techniques so far, the basic knowledge about the structure of agar and agarose molecules has been inproved. Specific enzymatic activities like; hydrolysis of agar and agarose, the physico-chemical fractionation of these molecules, and specially the molecular structure defining techniques such as nuclear magnetic resonance spectroscopy have enhanced the basic chemical understanding about these substances.
In 1956, Araki and Arai used agarases, also known as agarose 4-glycanohydrolase, to determine the structure of agarobiose. Since then several agarases have been purified and used to study the agar structure of different origins. Therefore agarases helped a lot in predicting the accurate chemical structure of agar.
In 1983, Christiaen and Bodard used infrared spectroscopy to determine the sulfate content in G. verrucosa agar. He also used infrared spectroscopy to track the sulfate and 3,6-anhydrogalactose content variations in different agar fractions. Conversely infrared spectroscopy wasn’t precise enough to determine the definitive polysaccharide structures. Later in 1985 Whyte and his team studied a combination of infrared spectroscopy and high performance liquid chromatography (HPLC) of methanolysates of algal galactans and used it to classify them into agar and/or carrageenan.
The most important discoveries in the field of structural and chemical analysis of agar were made using nuclear magnetic resonance during the period of 1980 to 1991. Izumi in 1973 [30] and Welti in 1977 initially proposed the 1H NMR spectroscopy of agar to study its quantitative data and to detect the minor concentration of particular repeating units of agarobiose. Although it is not generally used due to the complex spectra it provides, still its data quantification is possible. Nevertheless, identification of minor traces of desired repeating units is also possible. 13C NMR spectroscopy provides less complicated and easy to understand spectra by using one nicely resolved signal per carbon molecule. Even though 1H NMR is more sensitive than the 13C NMR spectroscopy, but it delivers complicated spectra of molecules than the 13C NMR spectroscopy.
Therefore a combination of techniques can be used to precisely study the chemical, physico-chemical as well as the molecular structure of agar gel. Specifically extracting, fractioning, enzymatic hydrolyzing the agar, as well as 1H and 13C Nuclear Magnetic Resonance spectroscopy techniques are club together to accurately study the critical chemical structures and distribution of the consecutive units of agarobiose and other similar molecules in different agars of various algae.
Since, 3,6-anhydrogalactose deposit of agar gets hydrolyzed due to acidic pH, thus lost during the procedure. Therefore gas chromatography was not suitable for studying agar components, before 1991 [12]. In 1991, a modified procedure was developed, using hydrolysis and reduction in a two-step process thereby not damaging 3,6-anhydrogalactose. This would retain the anhydro-sugar, which can be quantified conventional capillary gas chromatography.
Thus factors associated with the extensive and monotonous extraction and purification process are avoided during the direct quantification of polysaccharides, extracted from the cell wall of the algae. Helleur and his team in 1985, and Bird and his team in 1987, used the pyrolysis–gas chromatography, with or without mass spectrometry, to verify the critical chemical structure of algal cell-wall polysaccharides, either after extraction or in situ. Solid state 13C NMR spectroscopy chemical study of agarose, verified the spectrum of longer line-width although with the identical peak chemical change detected as from high resolution 13C NMR spectroscopy. These spectra also help in the analysis of polysaccharides of algal cell wall, which also helps in characterization of substituent like methoxyl and pyruvate groups.
5.8.2 Synergies and Antagonisms of Agar Gels
Products that affect the gel strength, gelling property, modify the gel texture and elasticity of agar are called synergies. However, antagonists are the products that decrease the gelling ability of agar or obstruct the gelling process completely.
Traditionally, Nikan values are used to determine the agar gel strength. The name Nikan comes from the traditional Japanese Nikan Sui method. According to this method force applied by a plunger for 20 s, to shatter a gel with the use of a cylindrical piston of 1 cm3 surface value is equal to one Nikan value. This method is still being used over the globe by most industry, even though there are more precise and accurate methods have been developed.
Acid and Alkali Hydrolysis
Like the other polysaccharides, agar also loses its molecular weight and gelling strength due to hydrolysis. If agar stays in lower pH (below 5.5) and higher temperature for longer time, it loses its gelling strength due to acid hydrolysis. It occurs more readily in agars. On the other hand alkaline hydrolysis of agar, doesn’t occur below 8 pH. However enzymatic hydrolysis isn’t relevant due to the very few agarases. Mainly bacillus marine bacteria like Esquizosaccharomycetes, synthesise agarases and these bacteria do not reside in food products.
Chaotropic Agents
Proton capturing chemicals compounds are called chaotropic agents, and they can disturb the agar gelation by stopping the hydrogen bridge formation between two subsequent agarose molecules. Since food products don’t carry any chaotropic agents like lithium perchlorate, lithium acetate, magnesium chloride, phenol, guanidinium chloride or 2-propanol etc, agar can be used in food stuffs.
Tannic Acid
Tannic acid is another potential inhibitor or agar gelation. Tannic acid is found in some fruits such as cranberries, apricots, peaches, grapes, etc. Glycerol can inhibit the reaction of tannic acid in agar, therefore adding some amount of glycerol during preparing food stuffs can avoid the gel breakage.
Sugar
The reaction between sugar molecule and agar can be observed in Graclaria agars. When these agars are dissolved into aqueous solution with the sugar concentration around 60%, the reaction between the hydrogen bonds of agarobiose and the molecules of sugar, takes place. The thread pitch of gelling helices causes the synergies in the agars of higher gel strength and lower sulfate content. Sugar reactivity usually occurs due to the presence of 3-6, anhydro bridges in agarose of higher molecular weight, around 140 kD. It should be considered here that L-galactose have tendency to aid the construction of hydrogen bridges in agars.
Agar–Locust Bean Gum (LBG) Synergies
It has been observed that agar–locust bean gum or LBG-synergies are only feasible in the agars of Gelidium and Pterocladia agarophytes. LBG-synergies increases the gel strength, transforms the gel texture in such a way that gel stiffness drops off and gel flexibility is elevated, making the gel less fragile. On the other hand, agars
