**Development of Biopolymers as Binders for Feed for Farmed Aquatic Organisms**

Marina Paolucci1, Adele Fabbrocini2,

Maria Grazia Volpe3, Ettore Varricchio1 and Elena Coccia1

*1Department of Biological, Geological and* 

*Environmental Sciences, University of Sannio* 

*2C.N.R. National Research Council, I.S.MAR 3C.N.R. National Research Council, I.S.A.* 

*Italy* 

#### **1. Introduction**

Diets for aquatic animals are numerous. They differ from species to species and may change to meet varied nutritional requests during the life cycle, and may be designed for larvae, juveniles, adults and breeders. In this review we will focus on a particular aspect of aquaculture feed represented by binders. Binders can be liquids or solids with the capacity of forming bridges, coatings or films that make strong inter-particle bonding. Binders are used to improve feed manufacture and to stabilize diets in water. Differently from feed for livestock, feed for aquaculture requires an adequate level of processing to guarantee good stability in water, long enough for animals to consume it. For this reason the role of binder is crucial in determining variable levels of firmness adequate to specific feeding behaviour. Although the problem of feed stability is far more crucial with crustaceans than with fish, some fish are benthic and small pellets that sink rapidly to the bottom where they can be located and recognized by the chemoreceptors of the fish are highly sought. Usually commercial feed for fish is stable after extrusion and binders are not requested to improve water stability. In some recent experiments binders are included in practical diets for fish to generate firmer feces when emitted into water to reduce pollution (Brinker, 2007). Among crustaceans crayfish are slow feeders with a characteristic tendency, that they share with prawns and shrimps, to manipulate food using mouth appendages before ingestion (Holdich, 2002). Thus, in aquatic animal feed preparation, to stabilize feed pellets and to ensure minimum nutrient leaching and disintegration appear to be crucial. Feed stability is considered a crucial requirement also in the echinoculture. Indeed, sea urchin are grazers and need time to eat the offered feed, so that it must remain intact for several days, in order to limit the loss of nutrients and to make rearing structure management easier (Caltagirone et al., 1992; Mortensen et al., 2004; Pearce et al., 2004). In addition, prepared diets frequently lead to poor gonad quality in terms of texture, firmness, colour and taste (Pearce et al., 2002a), that means low marketability of the product. For these reasons research focused on the selection of appropriate binders to ensure consistence to the experimental feed must take into account their effects not only on feed stability but also on gonad yield and sensorial quality. Since a binder may not be optimal for all species, and even for the same species the feeding behaviour may change during the life cycle numerous studies have been conducted to evaluate the type of binder, the optimal level of inclusion and its effect on growth and digestibility in different species of aquatic animals.

In this review, feed will be examined from the point of view of binder selection, method of preparation, assessment of water stability and nutrient leaching and with reference to the binder effect on growth and utilization by the animal. Purposefully, this review does not deal with diet composition for aquatic animals. Research on nutritional requirements of aquatic animals is a highly competitive field in constant and rapid evolution and it is outside the scope of the present article. For readers interested in this topic we suggest referring to the numerous reviews and books available.

#### **2. Feed binders**

Food is the material which, after ingestion by the animal, is capable of being digested absorbed and utilized. However, not all components of food are digestible. The main components of food are as follows:

Nutrients are components of food which can be utilized by the animal either as energy sources or for metabolic processes. The energy source components are the proteins, lipids and carbohydrates, while the vitamins and minerals are need to be present in small quantities in the feed for metabolism and life maintenance. There are various factors influencing the choice of feed for farmed fish. Of these, the economic factor is usually given precedence. Feeds and feeding can represent about half the operating costs in farming. Other important factors in manufacturing aquatic animal feeds are the stability of the feed in water and its acceptability. Inclusion of a binder is a necessity to ensure water stable feed. Many substances have been selected for their binding properties. Numerous natural binders have been employed to manufacture hard pellets with the purpose of increasing water stability with a concomitant decrease in nutrient loss. Some of these binders, such as wheat gluten or starches, have a nutritive value for the animals, while others are inert raw materials without nutritional value (Sinha et al., 2011). More than 50 organic and inorganic binders have been employed in feed industry (Kalian & Morey, 2009). Among natural binders examined and employed by researchers, biopolymers are by far the most studied. The most common biopolymers derive mainly from plants and animals of marine and agricultural origin. Examples are cellulose, starch, pectin, chitin and proteins such as casein, whey, collagen and soy proteins. In this review we will focus on the most employed for aquatic feed.

According to De Silva and Anderson (1995) binders can be grouped in three different classes:

1. binders of protein origin

4 Aquaculture

feeding behaviour may change during the life cycle numerous studies have been conducted to evaluate the type of binder, the optimal level of inclusion and its effect on growth and

In this review, feed will be examined from the point of view of binder selection, method of preparation, assessment of water stability and nutrient leaching and with reference to the binder effect on growth and utilization by the animal. Purposefully, this review does not deal with diet composition for aquatic animals. Research on nutritional requirements of aquatic animals is a highly competitive field in constant and rapid evolution and it is outside the scope of the present article. For readers interested in this topic we suggest

Food is the material which, after ingestion by the animal, is capable of being digested absorbed and utilized. However, not all components of food are digestible. The main

Nutrients are components of food which can be utilized by the animal either as energy sources or for metabolic processes. The energy source components are the proteins, lipids and carbohydrates, while the vitamins and minerals are need to be present in small quantities in the feed for metabolism and life maintenance. There are various factors influencing the choice of feed for farmed fish. Of these, the economic factor is usually given precedence. Feeds and feeding can represent about half the operating costs in farming. Other important factors in manufacturing aquatic animal feeds are the stability of the feed in water and its acceptability. Inclusion of a binder is a necessity to ensure water stable feed. Many substances have been selected for their binding properties. Numerous natural binders have been employed to manufacture hard pellets with the purpose of increasing water stability with a concomitant decrease in nutrient loss. Some of these binders, such as wheat gluten or starches, have a nutritive value for the animals, while others are inert raw materials without nutritional value (Sinha et al., 2011). More than 50 organic and inorganic binders have been employed in feed industry (Kalian & Morey, 2009). Among natural binders examined and employed by researchers, biopolymers are by far the most studied. The most common biopolymers derive mainly from plants and animals of marine and agricultural origin. Examples are cellulose, starch, pectin, chitin and proteins such as casein, whey, collagen and soy proteins. In this review we will focus on the most employed for

digestibility in different species of aquatic animals.

referring to the numerous reviews and books available.

**2. Feed binders** 

aquatic feed.

components of food are as follows:


From a nutritional perspective a protein binder may be more appropriate than a carbohydrate binder for a carnivorous species, while the contrary holds true for a herbivorous species. Carbohydrates are often included in crustacean artificial diets for their protein-sparing effect (Shiau & Peng, 1992; Rosas et al., 2000). According to the theory of Minimal Total Discomfort (Forbes, 2001), the ingestion of carbohydrates of vegetal origin brings about a protein sparing effect in wild animals and therefore enhances growth without wasting proteins. In fact, the intake of food has physiological consequences via physical (e.g. distension) and chemical (e.g. glucose) stimulation of receptors in the viscera and, in the longer term, by changes in signals from adipose tissue (e.g. leptin), integrated by the CNS (Paolucci, 2010). These consequences are associated with the sensory properties of the food such that repeated exposure to a food generates a conditioned acceptance or rejection reflex with the physiological consequences of eating as the unconditioned stimulus (US) and the sensory characteristics of the food as the conditioned stimulus (CS). Such learnt preferences and aversions occur throughout the animal kingdom, from nematodes to human beings, with much of the research being carried out with insects, laboratory animals and farm animals. Preferences for and aversions to particular foods are manifested in non-random choices between two or more foods on offer but also influence the quantity eaten when only one food is available. These considerations have been developed into a theory of Minimal Total Discomfort (Forbes, 2001) which proposes that an animal experiments with the amount eaten per day, and its selection between different foods, until the total of the signals generated from excesses or deficiencies of food components is minimised. Changes in food composition and/or nutrient requirements can therefore be matched by appropriate changes in intake and selection.

#### **2.1 Carbohydrates**

Polysaccharides are natural biopolymers formed by high molecular weight carbohydrates (Aspinall, 1982). They are biodegradable, biocompatible and non-toxic polymers showing peculiar physical-chemical properties and environmentally sustainable features. The specific macromolecular structures, characterized by the presence of several polar functional groups, allow polysaccharides to retain significant amounts of water or biological fluids, thus providing the formation of hydrogels, i.e. three-dimensional, reliable networks, able to become water resistant throughout chemical or physical phenomena, such as gel formation, retrogradation process, pH-changing and cross linking processes (Coviello et al., 2007). The molecular diversity of carbohydrates allows a large array of functions of great significance. In the food industry, polysaccharides play important roles as phytocolloid as well as emulsifying agents (Farris et al., 2009). Non-digestible carbohydrates are insoluble, like cellulose, or soluble, like pectin, gum, β-glucan, mucilage, algal polysaccharides (Atkins, 1985; Clark & Ross-Murphy, 1987). Technologically, they are needed for altering the texture and consistency of foods (Drochner et al., 2004) and show properties that make them suitable for use in increasing food shelf-life (Volpe et al., 2010). Carbohydrates represent the most abundant biological molecules, covering a large array of fundamental roles in living things: from the reserve and transport of energy, (starch and glycogen), to the development of structural components (cellulose in plants, chitin in animals), to the linking between intercellular walls (hemicellulose) (Kennedy & White, 1983). The high molecular weight carbohydrates derived, are known as polysaccharides. They may be viewed as condensation polymers in which carbohydrates have been glycosidically joined, with the elimination of molecules of water, according to the empirical equation

$$\text{n C}\_6\text{H}\_{12}\text{O}\_6 \rightarrow \text{(C}\_6\text{H}\_{10}\text{O}\_5\text{)n} + (\text{n}-1)\text{H}\_2\text{O}\_4$$

The different macromolecular structures and chemical compositions of polysaccharides are responsible for the large array of their physical and biochemical applications. A wide range of polysaccharides, such as agar, alginate, chitin and pectin are able to hydrate in cold and hot water, thus giving rise to both viscous solutions or dispersions and gels. The great interest with these polysaccharides in aquatic animal feed is strictly related to their gelling properties.

#### **2.1.1 Agar**

Agar is a polymer extracts from agarophyte, a seaweed, typically a red alga (Usov, 1998). It consists of at least two separate polymers that could be fractionated into agarose andagaropectin. Agarose is high in molecular weight and low in sulphate. Agaropectin is low in molecular weight and high in sulphate. Agarose is the gelling fraction of agar. Agarose is a linear polymer structure consisting of alternating D-galactose and 3,6 anhydro-L-galactose as shown in Fig. 1.

Fig. 1. Molecular structure of agarose

The diverse forms of agarose determine the physicochemical characteristics of agar such as gelling and melting temperature and reactivity. Agar is a mixture of agarose and agaropectin in variable proportions depending on the original raw material and the manufacturing process employed. Agar gelation occurs only by its agarose content and is produced exclusively by hydrogen bonds. Agar does not need any other substance to gel, and therefore it has an enormous potential in applications such as foodstuff ingredient, for biotechnology uses, for cell and tissue cultures or as a support for electrophoresis or chromatography. Agarose produces "physical gels" which means that these aqueous gels are formed only by the polymer molecules united solely by hydrogen bonds. Due to this unique gelling property, such gels hold in the interior network a great amount of water which can move freely through the macroreticulum. Each molecule maintains its structure in complete independence so that the process is not a polymerization but a simple electrostatic attraction. Agar is actually a very complex polysaccharide and varies considerably depending on the source. Back in 1991, eleven different agarose structures were identified in different agar bearing weeds depending on gender, species environmental conditions and time of the year. Moreover, agar may be modified by substitution of sulphate, pyruvate, uronate or methoxyl groups. Modern alkali treatment methods tend to increase the level of anhydrous bridging in the molecule with subsequent improvement of gel strength.

#### **2.1.2 Alginate**

6 Aquaculture

suitable for use in increasing food shelf-life (Volpe et al., 2010). Carbohydrates represent the most abundant biological molecules, covering a large array of fundamental roles in living things: from the reserve and transport of energy, (starch and glycogen), to the development of structural components (cellulose in plants, chitin in animals), to the linking between intercellular walls (hemicellulose) (Kennedy & White, 1983). The high molecular weight carbohydrates derived, are known as polysaccharides. They may be viewed as condensation polymers in which carbohydrates have been glycosidically joined, with the elimination of

n C6H12O6→(C6H10O5)n + (n – 1)H2O

The different macromolecular structures and chemical compositions of polysaccharides are responsible for the large array of their physical and biochemical applications. A wide range of polysaccharides, such as agar, alginate, chitin and pectin are able to hydrate in cold and hot water, thus giving rise to both viscous solutions or dispersions and gels. The great interest with these polysaccharides in aquatic animal feed is strictly related to their gelling

Agar is a polymer extracts from agarophyte, a seaweed, typically a red alga (Usov, 1998). It consists of at least two separate polymers that could be fractionated into agarose andagaropectin. Agarose is high in molecular weight and low in sulphate. Agaropectin is low in molecular weight and high in sulphate. Agarose is the gelling fraction of agar. Agarose is a linear polymer structure consisting of alternating D-galactose and 3,6 anhydro-

The diverse forms of agarose determine the physicochemical characteristics of agar such as gelling and melting temperature and reactivity. Agar is a mixture of agarose and agaropectin in variable proportions depending on the original raw material and the manufacturing process employed. Agar gelation occurs only by its agarose content and is produced exclusively by hydrogen bonds. Agar does not need any other substance to gel, and therefore it has an enormous potential in applications such as foodstuff ingredient, for biotechnology uses, for cell and tissue cultures or as a support for electrophoresis or chromatography. Agarose produces "physical gels" which means that these aqueous gels are formed only by the polymer molecules united solely by hydrogen bonds. Due to this unique gelling property, such gels hold in the interior network a great amount of water which can move freely through the macroreticulum. Each molecule maintains its structure in complete independence so that the process is not a polymerization but a simple electrostatic attraction. Agar is actually a very complex polysaccharide and varies

molecules of water, according to the empirical equation

properties.

**2.1.1 Agar** 

L-galactose as shown in Fig. 1.

Fig. 1. Molecular structure of agarose

Sodium Alginate is the sodium salt of alginic acid, a complex mixture of oligo-polymers, polymannuronic acid (MM), polyguluronic acid (GG) and a mixed polymer (MG) where sequences like GGM and MMG co-exist (Smidsrod et al., 1966). The mannuronic acid forms β (1-4) linkages, so that M-block segments show linear and flexible conformation. The guluronic acid instead gives rise to α (1-4) linkages, introducing a steric hindrance around the carboxyl groups; for this reason the G-block segments provide a folded and rigid structural conformation, responsible for a pronounced stiffness of the polymer. M and G blocks of the alginic acid salts are reported in Fig. 2.

Fig. 2. Molecular structure of alginic acid

In a water solution and in the presence of divalent cations, such as calcium ions, the peculiar buckled backbone of G segments gives rise to water insoluble gel due to the strong interactions between the divalent cations and the COO- groups of the base residual of guluronic acid. Cations can be trapped in a stable, continuous and thermo-irreversible threedimensional network, whose conformation is typical of an "egg box" (Grant et al., 1973; Grasdalen, et al., 1981) as shown in Fig. 3.

Fig. 3. Alginic acid "egg box" structure.

As a consequence, calcium alginate gel does not dissolve in water. Nevertheless, a rehydration process occurs, allowing the gel to swell and to modulate the releasing of entrapped substances.

#### **2.1.3 Pectin**

Pectin is a polysaccharide extracted from plant cell walls and is used in many industrial applications as a thickener or gelling agent (Stephen, 1983). Pectin is a polymer of αgalacturonic acid with a variable number of methyl ester groups (Fig. 4).

Fig. 4. Molecular structure of pectin

Generally, pectin does not possess an exact structure, therefore, it does not adopt a straight conformation in solution, but is extended and curved with a large amount of flexibility. The methylation of the carboxylic acid groups forms methyl esters which are much more hydrophobic and consequently have a different effect on the structuring of the surrounding water. The properties of pectin depend on the degree of esterification, which is normally about 70%. Gel strength increases with increasing Ca2+ concentration but it reduces with temperature and acidity increase (pH < 3). In the absence of added cations, low methoxypectin (~35% esterified) gels by the formation of cooperative "zipped" associations at low temperatures (~10°C) forming transparent gels. This hydrogen-bonded association is likely to be similar to that of alginate. High methoxyl-pectin (> 43% esterified, usually ~67%) gels by the formation of hydrogen-bonding and hydrophobic interactions in the presence of acids (pH ~3.0, to reduce electrostatic repulsions) and sugars (for example, about 62% sucrose by weight, to reduce polymer-water interactions). While the gel formed by low methoxyl-pectin results as being thermoreversible and not stable in water, the gel formed by high methoxyl-pectin is thermoirreversible mostly in the presence of high content of sugar (sucrose) and at low pH. In these conditions, the process of re-hydration of pectin gel is partially avoided by the firm hydrophobic structural network.

#### **2.1.4 Chitosan**

Chitosan is a cationic carbohydrate biopolymer derived from chitin, the second most abundant polysaccharide present in nature after cellulose. The main sources of chitin are the shell wastes of shrimps, lobsters and crabs (Johnson & Peniston, 1982). Upon the removal of most of the acetyl groups of chitin by treatment with strong alkali, chitosan yields. So chitosan may be considered as a family of linear binary copolymers of (1→4)-linked 2 acetamido-2-deoxy-β-D-glucopyranose (GlcNAc) and 2-amino-2-deoxy- β-D-glucopyranose (GlcN) (Fig.5).

Fig. 5. Molecular structure of chitosan

8 Aquaculture

As a consequence, calcium alginate gel does not dissolve in water. Nevertheless, a rehydration process occurs, allowing the gel to swell and to modulate the releasing of

Pectin is a polysaccharide extracted from plant cell walls and is used in many industrial applications as a thickener or gelling agent (Stephen, 1983). Pectin is a polymer of α-

Generally, pectin does not possess an exact structure, therefore, it does not adopt a straight conformation in solution, but is extended and curved with a large amount of flexibility. The methylation of the carboxylic acid groups forms methyl esters which are much more hydrophobic and consequently have a different effect on the structuring of the surrounding water. The properties of pectin depend on the degree of esterification, which is normally about 70%. Gel strength increases with increasing Ca2+ concentration but it reduces with temperature and acidity increase (pH < 3). In the absence of added cations, low methoxypectin (~35% esterified) gels by the formation of cooperative "zipped" associations at low temperatures (~10°C) forming transparent gels. This hydrogen-bonded association is likely to be similar to that of alginate. High methoxyl-pectin (> 43% esterified, usually ~67%) gels by the formation of hydrogen-bonding and hydrophobic interactions in the presence of acids (pH ~3.0, to reduce electrostatic repulsions) and sugars (for example, about 62% sucrose by weight, to reduce polymer-water interactions). While the gel formed by low methoxyl-pectin results as being thermoreversible and not stable in water, the gel formed by high methoxyl-pectin is thermoirreversible mostly in the presence of high content of sugar (sucrose) and at low pH. In these conditions, the process of re-hydration of pectin gel is

Chitosan is a cationic carbohydrate biopolymer derived from chitin, the second most abundant polysaccharide present in nature after cellulose. The main sources of chitin are the shell wastes of shrimps, lobsters and crabs (Johnson & Peniston, 1982). Upon the removal of most of the acetyl groups of chitin by treatment with strong alkali, chitosan yields. So

galacturonic acid with a variable number of methyl ester groups (Fig. 4).

partially avoided by the firm hydrophobic structural network.

entrapped substances.

Fig. 4. Molecular structure of pectin

**2.1.4 Chitosan** 

**2.1.3 Pectin** 

Chitosan has a very diversified range of established and potential applications related to its polycationic properties, which are unique among polysaccharides and natural polymer in general (No & Meyers, 1995). It is well known that chitosan may complex with certain metal ions and this property is used for removal of traces of heavy metals or radio isotope in waste water. Another well documented application of chitosan is as a cholesterol-lowering agent but the much more controversial use of it, is as a weight reducing agent. It is also used as a potential vehicle for orally administered controlled-release drugs. So this material is proposed for preparing micro spheres and microcapsules (El-hefian et al., 2011). These are only few of the applications of chitosan. Although chitin is insoluble in most solvents, the properties of chitosan are related to its polyelectrolyte polymeric carbohydrate character. It is insoluble in water while it readily dissolves in dilute solutions of most organic acids such as acetic, citric, tartaric acids. Chitosan gel does not undergo a substantial swelling process when placed in water; it is instead stable because the polymer itself results as being insoluble in water (Cerver et al., 2004).

#### **2.1.5 Carrageenan**

Carrageenan is the hydrocolloid obtained from some red seaweeds by extraction with water or aqueous alkali (Murano et al., 1992; Kalia, 2005). Carrageenan consists chiefly of potassium, sodium, calcium, magnesium, and ammonium sulfate esters of galactose and 3,6-anhydrogalactose copolymers. These hexoses are alternately linked α-1,3 and β-1,4 in the polymer(Fig. 6). The threemain copolymers in the hydrocolloid are designated kappacarrageenan (k-carrageenan), iota-carrageenan (ι-carrageenan) and lambda-carrageenan (λcarrageenan). K-carrageenan is mostly the alternating polymer of D-galactose-4-sulfate and 3,6-anhydro-D-galactose. І-carrageenan is similar, except that the 3,6-anhydrogalactose is sulfated at carbon 2. Between k-carrageenan and ι-carrageenan there is a continuum of intermediate compositions differing in the degree of sulfation at carbon 2. In λ-carrageenan, the alternating monomeric units are mostly D-galactose-2-sulfate (1,3-linked) and Dgalactose-2,6-disulfate (1,4-linked). The ester sulfate content for carrageenan ranges from 18 to 40%. In addition, carrageenan contains inorganic salts that originate from the seaweed and from the process of recovery of the extract. Carrageenan is recovered by either alcohol precipitation, or drum drying, or freezing. Primary differences which influence the

Fig. 6. Molecular structure of carrageenan

properties of k-, ι- and λ-carrageenan are the number and the position of the ester sulfate groups on the repeating galactose units (Kennedy & White, 1983). Higher levels of ester sulfate lower the solubility temperature of the carrageenan and produce lower strength gel, or contribute to gel inhibition.

The comparative properties of the three types of carrageenan are reported in Table 1.


Table 1. Comparative properties of types of carrageenan

#### **2.1.6 Carboxymethylcellulose**

10 Aquaculture

properties of k-, ι- and λ-carrageenan are the number and the position of the ester sulfate groups on the repeating galactose units (Kennedy & White, 1983). Higher levels of ester sulfate lower the solubility temperature of the carrageenan and produce lower strength gel,

The comparative properties of the three types of carrageenan are reported in Table 1. **Kappa carrageenan Iota carrageenan Lambda carrageenan** 

> Dilute solutions exhibit thixotropic characteristics. Soluble in hot water; sodium iota carrageenan is soluble in

Free flowing, non-gelling pseudo-plastic solutions in

of polymer chains.

effect on viscosity.

Range from low to high

Compatible with water miscible solvents.

Insoluble in most organic

Stable over a wide range of temperatures, including freeze/thaw cycles.

Soluble in 5% salt solution,

Approximately 35% ester sulfate and little or no 3,6-AG. Typical use level — 0.1 to 1.0%.

Addition of cations has little

Partially soluble in cold water, fully soluble in hot water. No gel, random distribution

water.

viscosity.

solvents.

hot or cold.

cold and hot water.

temperatures.

syneresis. Clear gel.

solvents.

2.0%.

Table 1. Comparative properties of types of carrageenan

Freeze/thaw stable.

Insoluble in most organic

Approximately 32% ester sulfate and 30% 3,6-AG. Typical use levels — 0.2 to

The addition of calcium ions will induce the formation of a durable, elastic gel, and increase gelling and melting

Elastic gels, forms helix with Ca++. Limited aggregation contributes to elasticity, no

Fig. 6. Molecular structure of carrageenan

or contribute to gel inhibition.

Soluble in hot water.

clear with sugar.

solvents.

2.0%.

Approximately 25% ester sulfate and 34% 3,6-AG. Compatible with water miscible solvents.

Insoluble in most organic

Typical use levels — 0.02 to

The addition of potassium ions induces the formation of a durable, brittle gel; it also increases the gelling and melting temperatures. Strong, rigid gel, some syneresis, forms helix with K+ ions. Ca++ causes helices to aggregate and the gel to contract and become brittle. Slightly opaque gel. Becomes

Carboxymethylcellulose (CMC) is a derivative of cellulose formed by its reaction with alkali and chloroacetic acid. The CMC structure is based on the β-(14)-D-glucopyranose polymer of cellulose (Fig. 7).

Fig. 7. Molecular structure of carboxymethylcellulose.

CMC molecules are shorter than native cellulose with uneven derivatization giving areas of high and low substitution. Substitutions are mostly 2-O- and 6-O-linked, followed in order of importance by 2,6-di-O- then 3-O-, 3,6-di-O-, 2,3-di-O- lastly 2,3,6-tri-O-linked. It appears that the substitution process is slightly cooperative (within residues) rather than a random process, which gives slightly higher than expected unsubstituted and trisubstituted areas. CMC molecules are highly extended (rod-like) at low concentration, while at higher concentration the molecules overlap and coil up and then entangle, to become a thermoreversible gel. Both increasing ionic strength and reducing pH decrease the viscosity as they cause the polymer to become more coiled. CMC dissolves rapidly in cold water and is mainly used for controlling viscosity without gelling. As its viscosity drops during heating, it may be used to improve the volume yield during baking by encouraging gas bubble formation. Its control of viscosity allows its use as thickener, and phase or emulsion stabilizer (for example, with milk casein), and suspending agent. CMC can be also used for its water-holding capacity as this is high even at low viscosity particularly when used with Ca2+ salt (Arisz et al., 1995).

#### **2.1.7 Guar gum**

Guar has been cultivated in India since centuries. India accounts for 80% of the total guar produced in the world. Historically, guar has been an important source of nutrition for humans as well as animals. Guar gum (galactomannan) is a high molecular weight carbohydrate derived from the natural seed of guar plant (*Cyampopistetragonolobus*). Structurally, guar gum is a polysaccharide consisting of a mannose backbone with a galactose side chain. The galactose is randomly placed on the mannose backbone with an average ratio of 1:2 galactose to mannose. The polymeric structure of guar gum containing numerous hydroxyl groups, has long since been exploited by industry (Goldstein et al., 1973). In Fig. 8 the guar gum chemical structure is reported.

The most important property of guar gum is its ability to hydrate rapidly in cold or hot water to attain uniform and very high viscosity at relatively low concentration. Another advantage associated with guar gum is that it provides full viscosity even in cold water. Although guar gum is one of the most cost-effective stabilizers and emulsifiers, its applications in food industry are numerous, due to its capacity to enhance texture, mouth feel, and to control crystal formation due to superior water-binding properties (Castillo-Garcia et al., 2005). It is inert in nature and it is resistant to oil, greases, and solvents. It also has excellent synergy with several other hydrocolloids, particularly xanthan gum (Ahmed et al., 2005). It has a very high viscosity even when very little is used.

Fig. 8. Molecular structure of guar gum

#### **2.2 Proteins**

Among proteins, the mostly widely used as feed binders are urea formaldehyde, wheat gluten and gelatin. Gelatin-based binders are a good alternative to gluten and urea formaldehyde. Gelatin and gluten have the advantage of being fully digestible and contain proteins (De Muylder et al., 2008), however, gluten employment may have some drawbacks due to its antigenicity (Shewry, 2009).

#### **2.2.1 Gelatin**

Gelatin is a mixture of water-soluble proteins derived from collagen by hydrolysis. The protein fraction consists almost entirely of amino acids. These amino acids are joined by amide linkage to form a linear polymer ranging from 15,000 to 250,000 Mw. Gelatin is particularly attractive for forming hydrogel packaging because it is relatively inexpensive and biodegradable, and its structure enables multiple combinations of molecular interactions (Farris et al., 2009). There are two types of gelatins which are characterized by their mode of manufacture. Type A gelatin (pH 3.8–6.0; isoelectric point 6–8) is derived by acidic hydrolysis of pork skin and contributes to increasing the plasticity and elasticity to the blend. Type B gelatin (pH 5.0–7.4; isoelectric point 4.7–5.3) is derived by basic hydrolysis of bones and animal skin and contributes to giving high gel strength to the blend. Various purity grades of gelatin are sold commercially in the form of translucent sheets, granules, or powders. Gelatin is practically odorless and tasteless. It is insoluble in acetone, chloroform, ethanol (95%), ether, and methanol. It is soluble in glycerin, acids, and alkalis, although strong acids or alkalis cause it to precipitate. It swells and softens in water, gradually absorbing 5 to 10 times its own weight in water. It solubilizes in hot water. Upon cooling to 35–40 °C, it forms a jelly or gel. At temperatures ≥40 °C, the system exists as a sol. A gel of higher viscosity is formed in alkaline media as compared with acid media (Robinson et al., 1975). Since it is a protein, gelatin exhibits chemical properties characteristic of proteins (e.g., gelatin is hydrolyzed by most of the proteolytic systems to yield its amino components). Gelatin reacts with acids and bases, aldehydes and aldehydic sugars, anionic and cationic polymers, electrolytes, metal ions, plasticizers, preservatives, and surfactants.
