Chapter I Introduction -...

Chapter I Introduction

2

1.1 Introduction

Carbohydrates are naturally occurring and well distributed, and are the most

important building blocks of the biosphere. These evolutionary and biologically

important organic compounds are present on Earth in different forms. Traditionally,

on the basis of the number of sugar units, carbohydrates are classified into three

groups: monosaccharides, oligosaccharides, and polysaccharides. The natural

macromolecules composed of several monosaccharide units (more than ten) are

known as polysaccharides and are synthesized at different stages of life cycle of

every living organisms for different purposes. The monosaccharide units of

polysaccharides are joined to each other by an acetal linkage. These acetal linkages

are formed by the reaction of a hemiacetal hydroxyl group of one unit with an

alcohol group of another unit which liberates water to give a glycosidic bond.

Polysaccharides not only have different sequences of monomeric units but also have

different sequences of glycosidic linkages and different types of branching. They

may be amorphous or even insoluble in water. All these factors together give

polysaccharides a great diversity of structure, property, and functions.

Polysaccharides can be classified into two separate groups based upon their

monomeric composition. Homopolysaccharides contain only one type of sugar

moiety, while heteropolysaccharides contain two or more (Sutherland 1982; Purama

et al., 2009; Sutherland 2007).

In recent years, the increased demand for natural polymers or biopolymers for

various industrial and biotechnological applications has led to a renewed interest in

exopolysaccharides or extracellular polymeric substances (EPS) production by

microorganisms as soluble or insoluble polymers. Different types of polysaccharides

produced by plants (cellulose, pectin and starch), algae (agar, alginate and

carrageenan) and bacteria (alginate, dextran, gellan, pullulan and xanthan gum) are

commonly used as food additives for their gelling, stabilizing or thickening

properties (Sutherland 1998). These EPS produced by both prokaryotes (eubacteria

and archaebacteria) and eukaryotes (phytoplankton, fungi, and algae), have a great

deal of research interest (Kumar et al., 2007). The growing environmental concerns

and increasing demands from end-use sectors are expected to increase the global

market for microbial products to about 250 billion US dollars by 2016 (McVilliams


3

2011). Exopolysaccharides are the metabolic product that accumulate on the cell

surface and play roles in various biological mechanisms such as immune response,

adhesion, infection, and signal transduction (Sutherland 1998; Kumar et al., 2007).

Table 1.1 shows the biological functions of microbial exopolysaccharides.

Table 1.1 Biological functions of EPS (Wingender et al., 1999; Wolfraardt

et al., 1999)

Microbial polysaccharides are high molecular weight carbohydrate polymers

present either at the outer membrane as lipopolysaccharides (LPS) that mainly

determine the immunogenic properties or secreted as capsular polysaccharides

(CPS) forming a discrete surface layer (capsule) associated with the cell surface or

excreted as EPS that are only loosely connected with the cell surface (Cuthbertson et

al., 2009). Whereas CPSs are assigned with functions directly related with

pathogenicity like resistance to specific and nonspecific host immunity, and

adherence (Taylor and Roberts 2005).

An important distinction of polysaccharide is based on their charge properties

they may be as naturally anionic and neutral. Microbial EPS like xanthan,

Function Relevance

Adhesion to surfaces Initial step in surfaces colonization, accumulation of

bacteria on nutrient-rich surfaces

Protective barrier Resistance to non-specific and specific host defense,

resistance to certain biocides including disinfectants

and antibiotics

Cell-to-cell recognition Symbiotic relationships with plants and animals,

initiation of pathogenic processes

Structural elements of

biofilms

Mediation of biofilms mechanical stability,

determination of the shape of EPS structure (capsule,

slime, sheath)

Retention of water Prevention of desiccation under water-deficient

conditions

Sorption of exogenous

organic and inorganic

compounds

Scavenging and accumulation of nutrients from the

environment, sorption of xenobiotics and toxic metal

ions (detoxification).

Promotion of polysaccharide gel formation

Interaction with enzyme

and enzymatic activities

Accumulation/retention and stabilization of secreted

enzymes.

Digestion of exogenous macromolecules for nutrient

acquisition


4

phosphomanan an alginate belongs to anionic group while EPS like levan,

scleroglucan pullulan and dextran are belongs to neutral group. Some

polysaccharides have anionic properties and they contain acidic groups, such as

carboxyl, phosphate or sulfate.

1.2 Exopolysaccharide producing microorganisms

The diversity of various EPS produced by microorganisms is often stressed. At

present a considerable number of bacteria Lactic Acid Bacteria (LAB), higher

basidiomycetes, lower filamentous fungi and yeasts from different ecological niches

are known for their ability to synthesize EPS in nature as well as in laboratory

culture system. However, many still remain uninvestigated or unexplored. For a

better observation at a glance a list of EPS producers is represented in Table 1.2.

1.2.1 Basidiomycetes fungi

Fungi from the division basidiomycota have been producer of one of the

most powerful secondary metabolites, and these metabolites have been researched

and developed into therapeutic agents. (Gan et al., 1998a; Eo et al., 1999b; Hatvani

2001).

1.2.1.1 Fungi derived β-glucans

Glucans are polysaccharides that contain glucose as a sole monomer unit

(Murray et al., 2002). This group of polysaccharides involves glycogen, cellulose,

and dextran. Their general formula is (C6H12O5)n (Duchon 1985). Polysaccharides

called β-glucans as well as β-1,3-D glucans or β-1,4-D-glucans (earlier, also

lichenins) are present in the cell walls of higher plants and also in the seeds of some

cereals (e.g., barley and oats). Related polymers, which are also called β-glucans

and/or β-1,3-D glucans and β-1,6-D-glucans, are synthesized by fungi, molds, and

yeasts.


5

Table 1.2 List of principal bacterial and fungal exopolysaccharide producing

microorganisms

Exopolysaccharides Organisms

Bacteria

Alginate Pseudomonas aeruginosa (Hay et al., 2009), Azotobacter

vinelandii (Gaona et al., 2004)

Cellulose Gluconabacter xylinus (Nguyen et al., 2008)

Curdlan Alcaligenes faecalis (Matsushita 1990)

Cellulomonas fauigena (Kenyon and Buller 2002)

Dextran Leuconostoc mesenteroides (Sarwat et al., 2008)

Gellan Sphingomonas paucimobilis (Bajaj et al., 2007)

Hyaluronan Pseudomonas aeruginosa (Bonde 1957)

Pasteurella multocida (DeAnglis et al., 1998)

Levan Bacillus subtilis (Kang et al., 2009)

Zymomonas mobilis (Poli et al., 2009)

Xanthan Xanthomonas campestris (Palaniraj and Jayaraman 2011)

Fungi

Pullulan Aureobasidium pullulans (Singh et al., 2008)

Scleroglucan Sclerotium glutanicum (Schmid et al., 2011)

Schizopyllan Schizophyllum commune (Kumar and Singhal 2011)

Lentinan Lentinula edodes (Zhang et al., 2007)

Grifolan Grifola frondosa (Tada et al., 2009)

Pleuran Pleurotus ostreatus (Hozova et al., 2004)

Krestin Coriolus versicolar (Ooi and Liu 2000)

Ganoderan Ganoderma lucidum (Volman et al., 2008)

Natural products containing β-glucan have been consumed for thousands of

years. Anecdotally specially in china and Japan for their healing powers as well as to

increase human longevity (Borchere 1999). Today, fungi are appreciated and

consumed primarily for their sensory and nutritional properties and are also used

increasingly in medicine and pharmaceutical industry. Fungi show favorable dietetic

properties with respect to their low fat and caloric value, and high levels of proteins,

minerals, and certain polysaccharides.


6

Higher Basidiomycetes represent a taxonomically, ecologically, and

physiologically extremely diverse group of eukaryotic organisms. Medicinal

mushrooms belonging to Basidiomycetes are abundant yet largely untapped source

of useful natural products with various biological activities (Wasser and Weis 1999;

Reshetnikov et al., 2001; Kirk et al., 2001; Xu et al., 2011). It is estimated that

about 650 mushrooms possess medicinal properties, but only several edible

(Flammulina velutipes, Grifola frondosa, Hericium erinaceus, Lentinus edodes,

Pleurotus spp., and Tremella spp.) and non-edible mushroom species (Ganoderma

lucidum, Schizophyllum commune and Trametes versicolor) have been investigated.

Presently, increasing attention is being paid to polysaccharides, which are an

integral component of fungi (Wasser and Weis 1999). The cell walls of fungi

contain two polymers, chitin and β-glucan. Glucans β (1→ 3), β (1→ 4), and β (1→

6) are a key reason that fungi are used as food additives and in pharmacology and

they have also shown beneficial effects when used in the treatment of various

diseases (Pelley and Strickland 2000). The medicinal properties of theses fungi are

due to the secondary metabolites which have been isolated and identified from the

fruiting bodies, mycelia and culture broth of mushrooms. These biologically active

molecules (BAM) belong to various functional polysaccharides, proteins and their

complexes and low molecular weight metabolites such as phenolic compounds,

polyketides, triterpenoids, steroids, alkaloids, nucleotides, lactones, and fatty acids

(Lindequist et al., 2005; Quang et al., 2006; Wasser 2010). Some of these

compounds have cholesterol-lowering, anti-diabetic, antioxidant, antitumor,

immunomodulating, antimicrobial, and antiviral activities ready for industrial trials

and further commercialization while others are in various stages of development.

The best example is Ganoderma lucidum, which contains more than 400 different

biologically active molecules (Kim and Kim 1999; Zhou and Gao 2002). Ohno et

al., (2005) reported the promising results for oncological treatments using β-glucan

isolated from Agaricus brasiliensis. β-glucan found in fungi differ in structure, water

solubility, molecule size and molecular mass. These features lead that not all β-

glucan in fungi show equally strong healing property (Zhang et al., 2007). Wasser

(2002) reported high molecular weight β-glucan as more effective than low

molecular mass. Ohno (2005) also reported high healing activity of high molecular

mass scleroglucan from genus Sclerotium. However, by Zhang et al., (2007)


7

reported higher antitumor activity by lentinan (Lentinula edodes) with a low

molecular weight. Furthemore, Wasser (2011) reported that efficiency of β-glucans

is influenced by length of side-chain, number of backbone branches and ratio of

(1,4) bonds to (1,6) and (1,3). Soluble β-glucans are strong immunomodulators than

the insoluble ones, was reported by Ishibashi et al., (2001) and Wasser (2011). The

antitumor activities of β-glucans refered by (1-3)/(1-6)-β form are and further related

to their ability to neutralize free radicals are considered to be vital reasons of cancer

occurrences (Chen and Seviour 2007). Jeurink et al., (2008) revealed in clinical

trials that stronger antitumor activitiy was shown by β-glucans linked with protein

than free β-glucans. Antibacterial, antiviral and antiallergic activities in fungi

derived β-glucans have also reported by Kumar et al., (2004).

The solubility of β-g1ucans is associated with the degree of polymerization

(DP). β-glucans are completely insoluble in water when DP>100. Solubility

increases as DP decreases. β-glucans can be classified according to their solubility

properties: (a) alkali-insoluble, acetic acid insoluble (1-3)-β-g1ucan; (b) alkali-

soluble (1-3)-β-g1ucan; and (c) highly branched (1-6)-β-g1ucan (Zekovic et al.,

2005). Among the natural β-g1ucans of clinical interest are lentinan, schizophyllan

and krestin (PSK).

1.2.1.2 Fungi derived β-glucans characteristic

Lentinan, produced from Shiitake mushroom, Lentinus edodes, is a β (1-3), β

(1-6) glucan (Fig. 1.1). Chihara et al., (1970) isolated lentinan and demonstrated its

antitumor effects were greater than other mushroom polysaccharides. It has a

molecular weight ranging from 400-1000 kDa (Kidd 2000) and has proved

successful in prolonging the overall survival of cancer patients, especially those with

gastric and colorectal carcinomas (Furue et al., 1981; Taguchi et al., 1985 a,b).

Adotey et al., (2011) lentinan showed also immunostimulative effect in case of

AIDS patients.


8

Fig. 1.1 Structure of Lentinan

Another important glucan is schizophyllan, produced and derived from

Schizophyllum commune, which has a β-g1ucopyranosyl group linked (1-6) to every

third or fourth residue of the main chain (Fig. 1.2). It has a triple-helix structure and

molecular weight of approximately 450 kDa. Schizophyllan also has shown to

increase overall survival of patients with head and neck cancers (Kimura et al.,

1994).

Fig. 1.2 Structure of Schizophyllan

Pleuran, is isolated from oyster mushroom Pleurotus ostreatus. This

compound is built of molecules of glucose linked by (1-3)-β bounds (Fig. 1.3). Such

backbone with line structure is linked with side-chains built of glucopyranose

molecules (Hozova et al., 2004). It has molecular mass between 600-700 kDa.

Pleuran demonstrates antitumor properties, lowers concentration of lipids in blood,

and antifungal properties (Chu et al., 2005).


9

Fig. 1.3 Structure of Pleuran

Krestin (PSK), is a β-glucan/protein compound consisting of 25-38% protein

residues and (1-4)-β-g1ucan with (1-6)-β-glucopyranosidic lateral chains (Fig. 1.4).

It is produced from Coriolus versicolus and has a molecular weight of 94 kDa (Ooi

and Liu 2000).

Fig. 1.4 Structure of Krestin

Grifolan, is derived from Grifola frondosa and commonly named GRN (Fig.

1.5). This glucan includes (1-3)-β linkages and has a molecular weight

approximately 450 kDa (Tada et al., 2009). It is present in both mycelium and

fruiting bodies of G. frondosa (Minato 2010). Grifolan is a polysaccharide that

demonstrates high biological activity like immunomodulator and antitumor agent

(Nie et al., 2006). Deng et al., (2009) reported that polysaccharides content from G.

frondosa confirmed immunomodulative properties in clinical trials of patients with

breast cancer. Grifolan is also used in treatment of HIV, hyperlipidaemia,

hypertension and virus hepatitis (Mayell 2001).


10

Fig. 1.5 Structure of Grifolan

1.3 Biosynthesis of polysaccharide in mushrooms

EPS production can be influenced by growth phase, medium composition

(carbon source nitrogen source), pH and temperature (De Vuyst and Degeest 1999;

Petry et al., 2000). The biosynthesis of EPS is related to the primary carbohydrate

metabolism of the producing cells (De Vuyst and Degeest 1999; Levander et al.,

2002). In general EPS production is expected to take place during active sugar

consumption, as it requires large numbers of activated nucleotide sugars, energy for

building the repeating units, for polymerization and transmembrane translocation. It

has been reported that EPS is a growth-associated product.

Although structure and functions of polysaccharides have been well known,

biosynthesis pathway of polysaccharide has not been fully understood, especially in

mushrooms. Sutherland (1977) and Sutherland (1994) suggested a general pathway

for the biosynthesis of extracellular polysaccharides in three major steps: (i)

substrate uptake, (ii) intracellular formation of polysaccharide, and (iii) extrusion

from the cell. A variety of enzymes are involved in the overall synthesis, each with

specialized functions in the process are shown in Figure 1.6.

According to Sutherland (1982), the enzymes involved in exopolysaccharide

biosynthesis may be classified into four types:

GROUP 1- those enzymes which are involved in initial metabolism of the substrate,

such as hexokinase.


11

GROUP 2- enzymes responsible for the synthesis and inter-conversion of sugar

nucleotides, (examples include the enzymes UDP-glucose pyrophosphorylase and

UDP-glucose dehydrogenase.

GROUP 3- transferases, which are enzymes responsible for formation of repeating

monosaccharide unit attached to the carrier lipid.

GROUP 4- translocases or polymerases which form the exopolysaccharide

biopolymer molecule.

Similarly, Fig. 1.7 depicts, the intracellular biosynthesis of bacterial EPS

regulated by enzymes located in various regions of the cell (Kumar et al., 2007)

Fig. 1.6 Biosynthesis of an exopolysaccharide (1) Hexokinase,

(2) Phosphoglucomutase, (3) Phosphoglucoisomerase (Wang and McNeil 1996)


12

Fig. 1.7 Catabolic mechanism representing the sugar nucleotide synthesis and

the interconversion of various monosaccharides through epimerization,

dehydrogenation and decarboxylation, occurred in the cell cytoplasm (Kumar

et al., 2007)

1.4 Fermentative production of exopolysaccharides

Fermentation for EPS production are batch, fed batch or continuous

processes depending on the microbial system used. In view of the importance of

EPS attempts have been made to produce them under submerged cultivation. Many

investigators have tried to cultivate mushrooms on solid artificial media for fruiting

body formation in order to obtain polysaccharides (Bae et al., 2000; Bhargava et al.,

2002). Furthermore, the cultivation of mushrooms for fruiting bodies production is a

long-term process needing from one to several months for the first fruiting bodies to

appear, depending on species and substrate (Pfefferle et al., 2000).

1.5 Factors influencing exopolysaccharide production

Fermentation is a very versatile process technology for producing value

added products such as microbial biopolymers. The fermentation parameters have a

high impact upon the viability and economics of the bioprocess, their optimization

holds great importance for process development. The production of microbial

polysaccharide production is greatly influenced by fermentation conditions such as


13

pH, temperature, oxygen concentration and agitation as well as by the composition

of the culture medium (Sutherland 2007; Nicolaus et al., 2010; Kazak et al., 2010).

The production of EPS is tightly regulated and is a carbon and energy-intensive

process resulting in a wide range of nutritional and environmental requirements.

Generally, media containing high carbon to limiting nutrient ratio (often

nitrogen) is favored for polysaccharide production. Conversion of 60–80 % of the

utilized carbon source into crude polymer is commonly found in high yielding

polysaccharide fermentations.

The optimal design of the medium is very important in the growth of

microorganisms, stimulating the formation of products and providing the necessary

energy for metabolic purposes. The nutrients required by a fungus include

macronutrients such as carbon, oxygen, nitrogen, phosphorus, sulphur, potassium

and magnesium, which comprise an average 98 % of dry cell mass of fungi

(Sutherland 1982).

1.5.1 Carbon source

Exopolysaccharides can be formed from a variety of carbon substrates. It is

widely accepted that EPS yields vary based upon carbon substrate utilized (De and

Basu 1996; Datta and Basu 1999; Degeest and De Vyust 2000). However, the

impact of carbon substrate on the chemical composition of exopolysaccharides

formed is a point of contention. Many studies have been conducted, utilizing a wide

variety of bacterial species, to evaluate the role of carbon source in

exopolysaccharide chemical composition. Several studies have found

exopolysaccharide composition to be unchanging with different carbon substrate

utilization (Breedveld et al., 1993; Degeest and De Vyust 2000), while others have

found exopolysaccharide composition to be variable upon utilization of different

carbon substrates (Petit et al., 1991; Pirog et al., 2003).

In general, glucose, sucrose, maltose, lactose, fructose, galactose, xylose,

cellobiose, sorbitol, xylitol, mannitol, and different types of agricultural byproducts

are used as a carbon source in the culture medium. In most of the cases glucose,

sucrose, and maltose have been selected as the most influential carbon sources for


14

the production of fungal EPSs. However, reasons are not clearly understood as in

most cases fungal growth and EPS production are not directly proportional.

Elisashvili et al., (2009) studied eight different basidiomycetes for EPS production,

and reported that G. lucidum, Inonotus levis, and Phellinus robustus produced

maximum EPSs in media containing glucose as a carbon source. Elisashvili et al.,

(2004) reported an interesting observation that L. edodes and Pleurotus spp. strains

produced maximum EPSs in culture media containing sodium gluconate as a carbon

source but the reasons were not clearly understood. The concentration of selected

carbon source in the culture media is another critical factor for EPS production. In

most of the findings, carbon at a concentration of 30-60 g/l was suggested to support

EPS production from fungi, although a few exceptions were also reported by Farina

et al., (1998), Sudhakaran and Shewale (1988), Xu et al., (2003), and Tavares et al.,

(2005).

1.5.2 Nitrogen source

Nitrogen comprises 8–14 % of the dry cell mass of bacteria and fungi. It is a

component of proteins and enzymes, and is necessary in cellular metabolism. EPS

producing microorganisms can utilize a variety of organic and inorganic nitrogen

sources. Among the various inorganic nitrogen sources ammonium chloride,

ammonium sulfate, sodium nitrate, potassium nitrate, urea, and di-ammonium

oxalate monohydrate are commonly used. However, high amount of EPS is

produced when or organic sources peptone, yeast extract poly peptone, martone A-1,

Soybean meal, and corn steep powder are supplemented. Many observations

suggested that in the presence of inorganic nitrogen sources, fungi produce less

EPSs in comparison to organic nitrogen supplements. Among the inorganic nitrogen

sources, ammonium salts are found to be efficient than other inorganic salts.

Sutherland (1996) reported that EPS production generally occurred in

nitrogen limiting conditions. Generally, the addition of extra nitrogen favors the

biomass concentration, but diminishes glucan formation. In the case of a glucan such

as pullulan, the production of polysaccharide is stimulated by depletion of nitrogen

source. Similarly, high concentrations of nitrogen in the form of ammonium

sulphate have been reported to reduce the scleroglucan production (Farina et al.,

1998).


15

1.5.3 Carbon/Nitrogen ratio

Carbon and nitrogen are both required for normal cellular metabolism, the

ratio in which they occur influences polysaccharide production. The maximum EPS

production is favored under conditions of nitrogen limitation (high C/N ratios)

(D’Haeze et al., 2004; Quelas et al., 2006). Under such nitrogen-limited conditions,

any excess sugars remaining can be used specifically for polysaccharide synthesis.

Generally exopolysaccharide yields can be maximized when the ratio of carbon to

nitrogen set as 10:1.

1.5.4 Medium pH

The pH of culture broth influences the physiology of a microorganism

significantly by affecting nutrient solubility and uptake, enzyme activity, cell

membrane morphology, byproduct formation and oxidative reductive reactions.

Culture pH can have profound effects on both the rate of production and the

synthesis of polysaccharides. However, the effect of pH on the biosynthesis of

exopolysaccharides and cell growth varies with different microorganisms (Lee et al.,

1999), operational conditions (Eugenia et al., 1995) and medium compositions

(Lacroix et al., 1985). In case of xanthan production, Moraine and Rogovin (1971)

observed that culture pH influenced polysachharide production more than cell

growth. Shu and Lung (2004) developed a two-stage processes, with the first stage

designed for optimal culture growth and the second stage for maximum

polysaccharide production.

1.5.5 Temperature

The internal temperature of the microorganism must be equal to that of its

environment. Like many chemical reactions, the microbial activity is sensitive to

environmental temperature. Temperature is crucial parameter that affects both

culture growth and polysaccharide production. The optimal temperature for cell

growth and EPS formation are predominantly different, especially in non growth

associated product, secondary metabolites (Wang and McNeil 1996). Temperature

can affect the availabilities of oxygen and substrate due to solubility changes in the

medium.


16

1.5.6 Aeration and agitation

Functions of aeration and agitation in fermentation can be described as

follows: (1) to provide adequate mixing of the contents, sufficient mass and heat

transfer rate, (2) to increase the transfer rate of O2 from gas bubbles to the cells, (3)

to increase the nutrient uptake rate and release rate of metabolic products from cells

to medium, (4) to promote CO2 removal from medium. Therefore, the rates of

aeration and agitation greatly influence the cell growth and EPS formation (Wang

and McNeil, 1996).

Availability of oxygen is limited by the rate of diffusion from the atmosphere

and is dependent upon the solution’s viscosity. During the polysaccharide

production the fermentation medium becomes more viscous and shows non-

Newtonian (pseudoplastic) behavior. However, mycelial pellet formation may occur

at low agitation rates and result in restriction of mass transfer regarding nutrient,

oxygen, product penetration and toxic metabolic products. Consequently, substrate

diffusion may be the rate-limiting process in filamentous fungi fermentation with the

form of pellets. The optimum agitation rate represents a balance between oxygen

transfer into the medium and shear stress, both of which increase with increasing

agitation rate. Higher shaking speeds favor EPS production because they decrease

the adsorption of the secreted extracellular polysaccharides on the cell wall,

providing stimulus for further EPS synthesis.

1.5.7 Metal ions

Phosphorus is an important element for secondary metabolism in fungi and

also regulates lipid and carbohydrate uptake by the cells. Phosphate salts, such as

K2HPO4 or KH2PO4, notably serve as a pH buffer in the fermentation medium and

they are also reported to be most efficient PO4 supplement (Dube 1983). Farina et

al., (1998) indicated an increase of total phosphorus from 0.12 to 0.28 g/l improved

the scleroglucan production (from 4 to 5 g/l). Potassium is the principal inorganic

cation in the cell; it is usually added as an inorganic salt (e.g. K2SO4, K2HPO4, or

KH2PO4). Potassium is a co-factor for some enzymes, required in the carbohydrate

metabolism and in many transport processes. Magnesium is also required by fungi, it

functions as an enzyme cofactor, and is present in cell walls and membranes. It is


17

usually supplied as MgSO4·7H2O. Pilz et al., (1991) noticed that thiamine and zinc

addition in a defined mineral medium could replace yeast extract. Their results

indicate the importance of meeting the zinc requirement of the microorganisms.

1.6 Recovery of exopolysaccharide

The extraction procedure and the method used for the purification of the

obtained fractions of active polysaccharides depend in many aspects on the type of a

fungus and physical properties of polysaccharides, such as solubility, molecule

conformation, branching level, molecular weight. The selection of extraction

methods includes concentration, isolation and purifications. The extraction of

polysaccharides is commonly performed in aqueous solutions. Water or basic

solutions may not be strong enough to separate water insoluble polysaccharides, for

this purpose acidic solutions may be employed. The extraction may also include

successive extraction with water at room temperature followed by centrifugation.

The main objectives of the recovery process are:

Concentration of fermentation broth or extract to a form, usually solid which

is microbiologically stable, easy to handle, transport and store and can be

readily redissolved or diluted for use in particular application.

Purification to reduce the level of non-polymer solids, such as cells or salts,

and to improve the functional performance, color, odor, or taste of the

product.

Deactivation of undesirable contaminating enzymes, such as cellulases,

pectinases, etc.

Modification of the chemical properties of the polymer to alter either the

functional performance, the solids handling properties of the dried product or

the dispersion and solution rate characteristics.

Isolation of polysaccharide is achieved by lowering the solubility of the polymer.

It results in either a precipitate or a phase consisting of a concentrated solution, by

either addition of a water miscible solvent, (e.g. methanol, ethanol, isopropanol,

acetone) or salt or acid. The method of choice for isolation of polysaccharide is

determined by economics, practicability and the final specification of the product,

with alcohol precipitation being widely favored.


18

Polysaccharides extracted from the source material are generally dissolved in

water or aqueous solutions. Nevertheless, other macromolecules, such as proteins,

may be also present in the medium. Therefore, several purification steps must be

carried out to remove other substances. The samples may be subjected to a previous

step consisting of a methanolic extraction in order to remove phenolic compounds,

monosaccharides, amino acids and other related molecules. The elimination of

phenolic derivatives is successful (Palacios et al., 2011), and increases the

effectiveness of extraction (Park et al., 2009). Proteins can be removed by

precipitation with trifluoroacetic acid (20%, w/v) or by treatment with the enzyme

protease at 40 °C for 1 h (pH 7.5). Then, proteins are separated by centrifugation.

After protein removal, polysaccharides are precipitated from the supernatants by the

addition of ethanol in 2:1 ratio (v/v). Concentrated sodium chloride solutions can be

added to favor the precipitation, and the solid can be washed with organic solvents,

such as acetone or ethanol.

Size exclusion chromatography (SEC) allows the separation of polysaccharides

according to their size, and the subsequent determination of the molecular weight.

Columns composed of hydroxylated polymers with residual carboxyl functionality

are usually employed for the separation of polysaccharides. Elution is performed

with diluted NaCl, NaNO3 or NaH2PO4 solutions at low flow rates. Polysaccharide

detection is achieved by refractive index detectors or by post column derivatization

with calcofluor (Bao et al., 2001; Ye et al., 2009). Table 1.3 depicts the common

methods used for isolation and purification of EPS.


19

Table 1.3 Common methods for isolation and purification of EPS

1.7 Characterization of exopolysaccharides

Exopolysaccharides are defined in terms of composition (type and relative

abundance of monomers), structure (relative distribution of monomers and type of

chemical bounds between them), conformation (arrangement of monomers chains

and bounds between them), relative molecular mass and type and arrangement of

substitutes (Morin 1998). Table 1.4 depicts the various characteristics and methods

of analysis of exopolysaccharides. These informations are used to analyze the

functional properties of polysaccharides, like solubility in water, relative viscosity

Methods Applied Contaminants Removed

Dialysis or dialfiltration

against distilled water

Any contaminants with molecular weight less

than membrane cut off

Trichloroacetic acid

precipitation (final

concentration ranging from 4

to 14%)

Amino acids, peptides, proteins. Some

polysaccharides might also be co-precipitated.

Enzyme digestion Proteins and nucleic acids by protease and

nuclease, respectively

Alcohol (e.g., ethanol,

methanol, acetone, etc.)

precipitation

Small molecules like ions could be eliminated,

but proteins and other macromolecules could also

be co-precipitated.

Anion exchange

chromatography

Any macromolecules with surface net charges

different from target compounds

Single dimension gel

electrophoresis (i.e.,

isoelectric focusing)

Any macromolecules with isoelectric point


Two-dimension SDS-PAGE Any macromolecules with surface net charges

and molecular masses different from target

compounds

Size exclusion

chromatography

Any contaminants with molecular weights



20

Table 1.4 Principal characteristics of microbial polysaccharides and analysis

methods

Characteristics Analysis method

Quantitative analysis of polysaccharides Gravimetric methods

Colorimetric methods

Quantitative and qualitative analysis of

monosaccharides components of EPS

High-Performance

Liquid Chromatography(HPLC)

-Reverse- phase HPLC

-Ion exchange HPLC

EPS structure and conformation

Nuclear magnetic resonance (NMR)

Differential Scanning Calorimetry

(DSC)

X- ray diffraction

Rheological analysis

Quantitative and qualitative analysis of

substitutes

Ion exchange HPLC

NMR

Infrared spectroscopy IR

EPS form and dimension

Dynamic light Scattering or Static

Light Scattering

and rheological behaviour (Stokke et al., 1998), ions binding capacity (De Philippis

and Vincenzini 1998). Relationship exists between the glycoside linkage, geometry

of polysaccharide and their conformation. Polysaccharides undergo transition from

an ordered state at lower temperature in the presence of ions, to a disordered state at

elevated temperature under low ionic environments (Nisbet et al., 1984). In case of

some polymers, this represents conversion from a gel to a sol state. The side-chains

attached to many linear polysaccharides promote conformational disorder and

inhibit any ordered assembly resulting in solubility in aqueous solutions. Thus,

xanthan, which possesses a cellulose backbone with trisaccharide side-chains on

alternate glucose residues, has been described as a natural water-soluble cellulose

derivative. Charged residues, found on the exterior of the extended molecules, may

readily promote interaction with ions and macromolecules. This may account for the


21

extensive binding of heavy metal cations ascribed to Zoogloea flocs and

polysaccharide (Friedman et al., 1968). Conformation refers at the form of

polysaccharide chains; it depends on the monomers and their position and bound

types in the polymeric chains (Belitz and Grosch 1999).

The main conformations adopted by EPS are:

1) Ribbon-type chains, like cellulose or alginate;

2) Helix conformation, founded to lichenin;

3) Combined conformation i.e. heteroglycans.

1.8 Functional properties of exopolysaccharide

The functional properties are defined as the properties measurable using

instruments and correlated with the final product characteristics through these

measurements. Linden and Lorient (1999) identified functional properties of a

compound on the basis of the following characteristics:

1) Rheological behavior (flow and viscosity functions, viscoelasticity)

2) Behavior towards water, depending on hydrogen and van der Waals interactions

3) Interactions of macromolecules with other macromolecules (properties of

polymerization through intermolecular ionic, hydrophobic or covalent associations)

4) Interactions with small molecules, with molecules having little polarity

(properties at the interfaces, formation of polydispersed systems).

1.9 Rheological properties of polysaccharides

The rheological behavior of polysaccharides solutions and the influence of

physical or chemical factors on the rheological properties are important because they

offer informations on the bioprocess, the biopolymer quality, the relations between

microstructure and physical properties (Pelletier et al., 2001), textural analysis

(Moreno et al., 2000). Rheological characteristics depend on a large number of

factors, as observed in Figure 1.8.


22

Fig. 1.8 Factors influencing the rheological characteristics of biopolymeric

aqueous solutions (Steffe 1996)

For example, if the polysaccharide is a polyelectrolyte, viscosity can be

controlled through electrostatic repulsion, ionic strength or addition of di and

polyvalent cations (making possible the formations of gel through ionic bonds). The

molecules size influences the rheological behavior at various shear stress. For

example, due to the length and rigidity of hydrated alginate molecules, the aqueous

solutions of this polysaccharide are shear-thinning or pseudoplastic (Imeson 2002).

The alginate molecules are disordered at small shear rates, whereas at high shear

rates the parallel orientation of polymeric chains occurs and the apparent viscosity

decreases (Aguilera and Stanley 1999). For the rheological analysis of EPS, two

measuring systems are recommended: parallel plate and cone-plate geometries

(Steffe 1996). Using these geometries, steady-state and oscillatory (or dynamic)

measurements can be performed; the first one type of analyses gives informations on

the flow behavior of polysaccharides and the second one characterizes the

viscoelastic behavior (Hochstein 2005).

1.10 Association properties of polysaccharides

The tendency of molecules to associate when the solutions are destabilised is

due to the destruction of the equilibrium between the attraction and the rejection

forces given by changes in medium (pH, ionic strength, temperature) and to the


23

formation of new bonds. For example, polysaccharides form gels in the presence of

water and under temperature influence; the gelling process implies interactions

between polymer chains with formation of complex structures (double helix) where

the solvent molecules are entrapped (Fig. 1.9). Some EPS form gels in the presence

of ions, like alginates at the interaction with calcium ions (Bracccini and Perez

1999) (Fig. 1.10)

Salts ions added in solutions act as flocculants by neutralizing the repulsive

charge at the surface. The flocculation efficiency is given by the critical coagulation

concentration of counter-ions, but is also dependent from the atomic number (Belitz

and Grosch 1999).

Fig. 1.9 Schematic representation of gel formation process

(Belitz and Gorsch 1999)

Fig. 1.10 Representation of coordinative bounds between Ca+2

and

alginate (Bracccini and Perez 1999)


24

1.11 Applications

Microbial exopolysaccharides including fungal EPS have gained an

importance due to their wide range of potential applications in various industries and

notably in therapeutics, food, petroleum, agronomy and pharmaceuticals.

1.11.1 EPS in food applications

EPS may function as viscosifying agents, stabilizers, emulsifiers, gelling

agents, or water-binding agents in food (Van den Berg et al., 1995). Majority of

polysaccharides used in foods are of plant origin. Most of them are chemically or

enzymatically modified in order to improve their rheological properties, e.g.

cellulose, starch, pectin, alginate and carrageenan. EPS produced by microorganism

have unique rheological properties. Dextran is the first industrial polysaccharide

produced by lactic acid bacteria. Dextran may be used in confectionary to improve

moisture retention, viscosity and inhibit sugar crystallization. In gum and jelly

candies it acts as gelling agents. In ice-cream it inhibits crystal formation, and in

pudding mixes it provides the desirable body and mouth feel.

1.11.2 Cosmetics

Glucan, mainly scleroglucan may be used in hair control compositions and in

various skin care preparations, creams and protective lotions. Polysaccharide

extracts of fungi with antioxidative properties have commercial application in

creams that provide protection from ultraviolet rays and from other detrimental

effects caused by activities of free radicals that lead to damages and aging of the

skin. Hyaluronan from G. frondosa used as ingredients for enhancing collagen

biosynthesis in skin cells (Kougias et al., 2001).

1.11.3 Emulsifiers

Surfactants and emulsifiers from the microbial sources have received

attention due to their biodegradability and possible production from renewable

resources. The polysaccharide, Emulsan produced by Acinetobacter calcoaceticus

stabilized emulsion more effectively than other commercial gums such as arabic,

tragacanth, karaya and xanthan (Ashtaputre 1995).


25

1.11.4 Biomedical applications

Antitumor, antiviral and immunomodulatory activities of polysaccharides

have been reported from fungi and bacteria.

1.11.4.1 Antitumor activity

Polysaccharides from fungi have been widely employed in tumor therapies

due to their properties as immunological enhancers. for instance, Agaricus,

Calocybe, Ganoderma, Grifola, Inonotus, Lentinus, Phellinus, Pholiota, Pleurotus,

etc. are different mushrooms genera capable of providing antitumor activity (Hsieh

and Wu 2011; Lee et al., 2011; Selvi et al., 2011; Zhang et al., 2011). The mitogenic

activity of fungal polysaccharides involves several mechanisms, such as the

antiproliferation of cancer cells and induction of apoptosis, regulation of the immune

system and the antimetastatic effects. Polysaccharide can depress the E-selectin

protein and gene expression which in turn inhibit tumor cell to cell adhesion (Yue et

al., 2012). Fungal polysaccharide widely employed as co-adjuvant in tumoral

therapies. Lentinan, a β-(1→3),(1→6)-linked from Lentinus edodes, has been used

in a combined treatment of patients with advanced or recurrent gastric or colorectal

cancer.

1.11.4.2 Antioxidant activity

Oxidative damage caused by free radicals may be related to prevalence of

common diseases such as aging and chronic degenerative diseases, such as diabetes,

cancer, arthritis, cardiovascular disease, Alzheimer, Parkinson, Down syndrome and

multiple sclerosis. An antioxidant has been defined as any substance that when

present at low concentrations compared to those of an oxidizable substrate,

significantly delays or prevents oxidation of the substrate. Polysaccharides from

different mushrooms showed free radical scavenging activity (Asantiani et al.,

2007), superoxide radical scavenging activity, reducing properties, lipid

peroxidation inhibition, suppression of proliferation and oxidative stress, etc.

(Asatiani et al., 2007). These activities are not only dependent on fungal species but

also on the chemical structure and arrangement of active polysaccharides (Heleno et

al., 2012; Wang et al., 2012). Similarly, mixed carbohydrates, such as

polysaccharide–peptide complexes, have also shown a potent antioxidant activity


26

(Li et al., 2012). The most widely used synthetic antioxidants are butylated

hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and tert-butylated

hydroxyquinone (TBHQ), which are applied in fat and oily foods to prevent

oxidative deterioration (Grice 1988; Kahl and Kappus 1993).

1.11.5 Other pharmaceutical applications

Fungal exopolysaccharides are effective in promoting the treatment of

diseases like microbial infections, diabetes and hyper-cholesterolemia. Curdlan is

used to lower the cholesterol concentration in liver. Gellan gum is used in oral drug

deliver, and in nasal spray pumps. Pullulan is also being investigated for its drug

delivery applications, hydrophobized pullulan as drug delivery carriers. Dextran is

also explored as potential plasma expanders. Scleroglucan include the use in tablet

coating, ophthalmic solutions, injectable antibiotic suspensions and calamine lotion.

1.11.6 Microbial Enhanced oil recovery

In oil recovery, scleroglucan increases the viscosity, hence the hydraulic

pressure of sea water or brine used to extract oil. Scleroglucan lubricates the drill

and controls the backpressures created during drilling. In addition, scleroglucan

exhibited high thermostability in drilling fluids when it was associated with

polyglycols through intermolecular interactions (Mahapatra and Banerjee 2013).

1.11.7 Bioremediation and waste water treatment

An expanding area of biotechnology is the application of EPS producing

microorganisms in the remediation of environmental effluents (Iyer et al., 2006;

Valenzuella et al., 2006). Biofilm-mediated bioremediation has been found to be a

more effective and safer alternative to bioremediation with planktonic bacteria

(Donlan 2002). The pressure of biofilm helps maintain optimal chemical and

physiological conditions, localized solute concentrations and redox potential,

allowing cells to improve mineralization processes (Singh et al., 2006).

Fungi have been proved to be efficient and economical or the removal of

toxic metals from dilute aqueous solutions by biosorption because fungal biomass

offers the advantage of having a high percentage of cell wall material, which shows

excellent metal binding properties (Das et al., 2008). Moreover, large quantity of


27

fungal biomass is available from antibiotics and food industries. Ultimately, the

biosorption results not only in metal removal but also provides an eco-friendly

environment. A. niger has been found capable of removing heavy metals like Pb,

Cd, and Cu (Kapoor and Viraraghvan 1995). Agaricus macrospores efficiently

extract Cd, Hg and Cu from contaminated wastes. Huang et al., (1988) also studied

removal of Cd using nine different species of fungi both in batch and continuous

reactors. Table 1.5 presents the exhaustive list of microorganisms used for the

removal for heavy metals.

Table 1.5 Biosorbent uptake of metals by microbial biomass

Metal Biomass Type Biomass class Reference

Ag

Freshwater alga Biosorbent Brierley and Vance 1988

Streptomyces noursei Filamentous fungus

bacteria Mattuschka et al., (1993)

Sacchromyces

cerevisiae Yeast Brady and Duncan (1993)

Au

Sargassum natans Brown alga Volesky and Kuyucak (1988)

Aspergillus niger Fungus Kuyuack and Volesky (1988)

Bacillus subtilis Bacteria Cell wall Beveridge (1986)

Cd

Penicillium

spinulosum Fungus Townesley et al., (1986)

Spirulina sp. Blue green algae Chojnacka et al., (2005)

Co

Ascophyllum

nodosum

Brown marine

algae Kuyucak and Volesky (1989)

Ulva reticulata Marine green

algae Vijayaraghavan et al., (2005)

Enterobacter

cloaceae Marine bacterium Iyer and Jha (2005)

Cr

Bacillus biomass Bacterium Brierley and Brierley (1993)

Pantoea sp. TEM 18 Bacteria Ozdemir et al., (2004)

Spirulina sp. Cyanobacteria Chojnacka et al., (2005)

Spirogyra sp. Filamentous algae Gupta et al., (2001)

Cu

Pencillium

chrysogenum Fungus Niu et al., (1993)

Ulva reticulata Marine green alga Vijayaraghavan et al., (2005)

Spirulina sp. Blue green algae Chojnacka et al., (2005)

Fe Bacillus subtillis Bacterial cell wall Beveridge (1986)

Sargassum fluitans Brown alga Figueira et al., (1995)

Hg

Pencillium

chrysogenum Fungus Nemec et al., (1977)

Cystoseira baccata Marine alga Herrero iet al., (2005)

Chlamydomonas

reinhardtii Algae Tuzun et al., (2005)

Ni Ulva reticulata Marine green algae Vijayaraghavan et al., (2005)

Polyporous versicolor White rot fungus Dilek et al., (2002)


28

Aim and scope of the study

During the past three decades many biological active compounds like

polysaccharides and polysaccharide-protein complexes have been isolated from

fungi, algae, lichens and plants. Furthermore most of them which have various

biological activities were originated from fungi, especially mushrooms. Mushrooms

in submerged culture are more preferably used to produce EPS which is gradual

substitute for solid culture in recent years. It takes several months for the solid-

culture mushrooms to grow into the fruiting bodies on solid substrates. Moreover,

submerged culture gave rise to many potential advantages of higher mycelial

biomass or EPS production in a compact space and shorter time with less chances of

contamination. Exopolysaccharides from mushrooms have found a wide range of

applications as thickeners, bioadhesives, stabilizers, probiotic, and gelling agents in

food and cosmetic industries. The use of β-(1,3)-D-glucans produced from

mushrooms have attracted attention in the last few years with the exploitation of

their biological activities i.e. antitumor, immunomodulating, antibacterial, antiviral

and antioxidative properties. The use of white rot fungi for the removal of heavy

metals also deserves great attention. Hence, the techniques such as submerged

cultivation and biosorption have been used by various researchers for the treatment

of heavy metals contaminated water.

Many microbial EPS provide properties that are almost identical to the gums

currently in use. With innovative approaches, efforts are underway to supersede the

traditionally used plants and algal gums by their microbial counterparts. β-glucans

have been shown to possess various industrial applications. This finding calls for

future perspectives into wide production of EPS using white rot fungi for their

application in many quite distinctive areas, such as food industry, biomedicine,

cosmetology, agriculture, environmental protection and waste water management.


29

Objectives of the present study

The objectives of the present study were following:

Isolation and screening of potential EPS producing white rot fungi.

Optimization of various physico-chemical parameters for EPS production

using one-factor-at-a-time method and statistical methodology under

submerged cultivation of S. commune AGMJ-1.

Purification and characterization of EPS.

Removal of hexavalent chromium from fortified solution by S. commune

AGMJ-1 and phytotoxicity study.

Biosorption of Cr(VI) using dried EPS and mycelial biomass of S. commune

AGMJ-1

Biosorption of Cr(VI) using pretreated wet mycelial biomass of S. commune

AGMJ-1

Assessment of antioxidant properties, antiproliferative activity and

genotoxicity study of EPS.

Chapter I Introduction -...

Documents

Transcript of Chapter I Introduction -...