Chapter 2 REVIEW OF LITERATURE

Chapter 2 

REVIEW OF LITERATURE 

Xylooligosaccharides (XOS) are relatively new type of oligomers 

which have gained a lot of interest because of many technological and 

health benefits and lot of research is going on to explore their dietary and 

physiological roles. The production of XOS from agro-industrial wastes, 

evaluation of their health benefits and utilization in synbiotic products 

was studied in the present project. 

To support the study, literature has been reviewed under the following 

headings: 

2.1. Oligosaccharides (OS) 

2.1.1. Definition and Properties 

2.1.2. Types of Oligosaccharides 

2.1.3. Applications of Oligosaccharides 

2.2. Production of xylanases 

2.2.1. Microbial Production 

2.2.2. Trichoderma harzianum, as xylanase producing organism 

2.3. Xylooligosaccharides (XOS) 

2.3.1. Production of XOS from lignocellulosic biomass 

2.3.2. Refining

2.3.3. Applications of XOS 

2.3.3.1. Food Applications 

2.3.3.2. Pharmaceutical Applications 

2.3.3.3. Other Applications 

2.3.4. Health benefits of XOS 

2.3.4.1. Gastrointestinal effects 

2.3.4.2. Other health benefits 

2.4. Efficacy studies 

2.5. Synbiotic Foods 

2.5.1. Definition and Background of Synbiotic 

2.5.2. Yogurt as Synbiotic Food 

2.1. OLIGOSACCHARIDES (OS) 

2.1.1. Definition and Properties 

Oligosaccharides (OS) are an important group of polymeric 

carbohydrates that are found either in free or combined forms in all living 

organisms. According to the IUB-IUPAC nomenclature, oligosaccharides 

may be defined as oligomers which are composed of 2–10 monosaccharide 

residues structurally linked by glycosidic bonds that are readily 

hydrolyzed to their constituent monosaccharides either by acids or 

specific enzymes (Nakakuki, 1993). They are found naturally in fruits, 

vegetables, milk and honey. Most oligosaccharides have a mild sweet 

taste, and the mouth-feel they lend to food, that has drawn the interest of 

the food industry to use them as a partial substitute for fats and sugars in

foods. Moreover, oligosaccharides can be used as functional food 

ingredients that have a great potential to improve the quality of many 

foods. It has been reported that these have various physiological 

functions. Some of oligosaccharide properties are given in Figure 2.1 

(Nakakuki, 2002). 

Figure 2.1: Properties of oligosaccharides (adapted from Nakakuki, 2002). 

2.1.2. Types of Oligosaccharides 

Extensive research on production of oligosaccharides for use in 

food industry had been started. With the advancement in the field of 

enzymology, and assessment of their functional attributes all over the 

world, industrial production of oligosaccharides was started at a larger 

scale. Many oligosaccharides such as starch-related, sucrose-related, and 

lactose-related oligosaccharides have been developed, after which xylo- 

oligosaccharides, agaro-oligosaccharides, manno-oligosaccharides, and 

chitin/chitosan-oligosaccharides have been produced from various 

polysaccharides such as xylan, agar, mannan, chitin, and chitosan as the

aw materials. Different types of oligosaccharides along with their sources 

are given in Table 2.1.

Table 2.1: Dietary oligosaccharide with their natural sources and industrial production 

Type of 

processes (adapted from Murphy, 2001). 

Oligosaccharides 

Natural 

Occurrence 

Industrial Production 

Process 

Lactulose Cow milk Isomerization of lactose 

Lactosucrose, 

glycosucrose 

Isomalto- 

oligosaccharide 

Xylo- 

oligosaccharide 

Stacchyose, 

raffinose 

Palatinose- 

oligoaccharide 

Gentio- 


Beet Extraction and 

Soyabean, other 

sources of stach 

transglycosylation of 

sucrose 

Hydrolysis and 

transglycosylation of starch 

Soyabean Hydrolysis of polyxylans 

Beet, soyabean Synthesis from starch 

Soyabean 

Synthesis from starch 

Hydrolysis and 


Cyclodextrin Hydrolysis and 

Fructo- 

oligosaccharide 

Galacto- 



Fruits, vegetables Synthesis and extraction 

Human milk, 

cow milk 

from saccharose 

Enzymatic synthesis from 

lactose

On the basis of physiological properties, the oligosaccharides can 

be classified as digestible or non-digestible. The idea of non-digestible 

oligosaccharides (NDOs) was derived from the observation that the 

anomeric C atom (C1 or C2) of the monosaccharide units of some dietary 

oligosaccharides has a configuration that makes their osidic bonds non- 

digestible to the hydrolytic activity of the human digestive enzymes. The 

major classes of NDOs at present exist or in the process of development as 

food ingredients comprise carbohydrates in which the monosaccharide 

units are fructose, galactose, glucose and/or xylose (Roberfroid and 

Slavin, 2000). 

2.1.3. Applications of Oligosaccharides 

Foods for specified health use (FOSHU) legislated by government 

of Japan in the early 1990s include more than 200 food items incorporating 

oligosaccharides as the functional ingredient (Kalra, 2003). 

NDOs are fermentable substances which have significant effect on 

the large intestinal flora to contribute to the human health and are called 

‘prebiotics’ (Mussatto and Mancilha, 2007). Several NDOs have been 

launched as functional food ingredients during the last few decades and 

there is a continuous increase in their industrial applications. 

Among the main uses of OS, center of attention is in beverages 

including fruit drinks, coffee, cocoa, tea, soda, health drinks and alcoholic 

beverages and milk products e.g. instant powders, powdered milk and ice 

cream, fermented milks and probiotic yogurts based on live 

microorganisms that exert beneficial effects on the host. These effects are 

imparted due to improvement of the microbiological balance in the 

intestine. The synbiotic products containing a mixture of probiotics and 

prebiotics affect the host by improving the survival and implanting live

microbial dietary supplements in the gastrointestinal tract (Gibson and 

Roberfroid, 1995). Further, existing uses of the NDOs in the food industry 

take in desserts such as jellies, puddings and sherbets; confectionary 

products such as candy, cookies, biscuits, breakfast cereals; chocolate and 

sweets; breads and pastries; table spreads and spreads such as jams and 

marmalades; and meat products such as fish paste and tofu (Voragen, 

1998). 

Since the definite physico-chemical and physiological 

characteristics of the NDOs products vary with the type of mixture 

prepared, the most suitable oligosaccharide for a particular food 

preparation also fluctuates. Glacto-oligosaccharide, for instance, is a 

suitable OS for incorporating in bread because these are not broken down 

during yeast fermentation and baking, imparting good taste and texture. 

Some of industrial and pharmaceutical uses of oligosaccharides include 

feed, drug delivery agent, cosmetics and mouth washes (Crittenden and 

Playne, 1996). Lactulose, for example, is being used predominantly as a 

pharmaceutical product controlling constipation and portosystemic 

encephalopathy (Villamiel et al., 2002). 

A number of oligosaccharides have been studied by various in-vitro 

methods, animal models and human clinical trials. The most useful ones 

reported are the fructo-oligosaccharides (FOS), lactulose, galacto- 

oligosaccharides, xylo-oligoaccharides and inulin. These OS are being 

produced in large scale and added into various products in the food 

industry. Also, there are research trials (but in minor proportion) about 

newly developed OS like soybean-oligosaccharides, lacto-sucrose, 

isomalto-oligosaccharides, gluco-oligosaccharides and xylo- 

oligosaccharides, enclosing support in all of them of promoting the 

increase of intestinal microflora. Primarily, xylo-oligosaccharides have 

intestinal improving and hypolipidemic activities and antimicrobial

activity against some bacteria (Christakopoulos et al., 2003). It was 

observed that the in-vitro growth of Bifidobacterium spp is improved by 

using XOS which are as effective as raffinose and better than fructo- 

oligosaccharides (Vázquez et al., 2000), 

2.2. PRODUCTION OF XYLANASES 

2.2.1. Microbial Production 

Xylanases are widespread in nature. Microorganisms are primarily 

responsible for xylan degradation (Wong et al., 1988; Eriksson et al., 1990; 

Prade, 1996; Sunna and Antranikian, 1997; Subramaniyan and Prema, 

2002). They occur both in prokaryotes and eukaryotes and have been 

reported from marine and terrestrial bacteria, rumen bacteria, fungi, 

marine algae, snails, crustaceans, insects and seeds of terrestrial plants 

(Dekker and Richards, 1976). 

High level of induction of xylanase activity is often achieved using 

xylan, other hemicellulose-rich materials and low molecular weight 

carbohydrates such as xylose (Senior et al., 1989). Fungi produce xylanases 

extracellularly (into the medium), thus avoiding the need for cell lysis. 

Xylanases have been purified from large number of fungal and bacterial 

species (Sunna and Antranikian, 1997) and detailed studies on their 

biochemical properties have been carried out (Bastawade, 1992; Kulkarni 

et al., 1999). Fungal xylanases have attracted special attention first because 

of the growing interest on their potential use in paper and pulp industries 

(Viikari et al., 1994a, b) and secondly, because of their potential role in 

fungal pathogenicity (Walton, 1994). 

The optimum pH for xylan hydrolysis is around 5 for most fungal 

xylanases, whereas pH optima of bacterial xylanases are generally slightly 

higher. Most of the fungi and bacteria produce xylanases that tolerate

temperatures below 40-50°C. The enzymes that lack thermostability have 

disadvantages at industrial scale as they result in low hydrolysis 

efficiencies, high enzyme requirements and increased cost. The technical 

and economical feasibility of the hydrolysis process may be enhanced by 

use of thermostable xylanases to carry out enzymatic hydrolysis at 

elevated temperatures over prolonged periods of time (Yu et al., 1987). 

Thermostable xylanases are particularly useful in biobleaching as the 

cooking of wood is conducted at high temperatures (Bierman, 1993). 

Thermophilic fungi produce more thermostable xylanases as compared to 

the mesophilic fungi (Gomes et al., 1994). Thermophilic eubacteria and 

archaebacteria produce xylanases with much longer T1/2 (half life) at 80°C 

than thermophilic fungi (Dahlberg et al., 1993), but the levels of the 

enzymes produced by these bacteria are much lower than those of many 

fungi. Thermomyces lanuginosus produces thermostable xylanase with a 

T1/2 of 148 min at 75 °C (Lischnig etal., 1993). 

Production of xylanases by using cost effective agricultural 

residues through solid state fermentation and immobilized cell systems 

provide suitable means to reduce the manufacturing cost of biobleached 

paper and helps in achieving environment friendly technology in paper 

industry. Highly thermostable xylanase is produced by an extremely 

thermophilic Thermotoga spp. with a half-life of 20 min at 105°C (Bragger 

et al., 1989). 

Extremophilic xylanases (active under alkaline conditions and 

higher temperatures) have great potential for industrial application in 

bleaching process (Shoham et al., 1992). Duarte et al. (1999) produced and 

purified alkaline xylanases from different strains of Bacillus pumilus. A 

thermostable and alkalophilic xylanase from Thermoactinomyces thalophilus 

retained 50% of its activity at 65°C after its incubation for 125 min (Kohli et 

al., 2001). Thermostable xylanases have also been produced by

Thermoascus aurantiacus, B. stearothermophilus, Caldocellum saccharolyticum, 

Clostridium stercorarium, Thermomonospora spp. and Rhodothermus marinus 

(Buchert et al., 1994; Subramaniyan and Prema, 2002). Most of the 

xylanases reported in the literature contain significant cellulolytic activity 

that makes them less suitable for paper and pulp industry (Subramaniyan 

and Prema, 2002). 

2.2.2. Trichoderma harzianum, as Xylanase Producing Organism 

Fungi are the most potent producers of xylanases. Trichoderma 

species are free living fungi that are common in soil and root ecosystems. 

They are opportunistic, a virulent plant synbionts, as well as being 

parasites of other fungi. Trichoderma is a genus of asexually reproducing 

fungi that are often the most frequently isolated fungi; nearly all 

temperate and tropical soils contain 10 1 -10 3 culturable propagules per 

gram. They are prolific producers of extracellular proteins and are the best 

known for their ability to produce other useful enzymes that degrade 

cellulose and hemicellulose (Harrman and Kubicek, 1998). The taxonomy 

of the genus Trichoderma has been still under revision. However, the basis 

remain the work of Rifai (1969), assigning the Trichoderma strains to nine 

species aggregates (T. piluliferm, T. polysporum, T. hamatum, T. koningi, T. 

aureoviride, T. harzianum, T. longibrachitum, T. pseudokoningi and T. viride) 

differentiated primarily by patterns of conidiophores branching and 

conidium morphology. A series of revisions of genus Trichoderma by Bisset 

(1984; 1991 a, b, c) and Doi et al., 1987) introducing and differentiating new 

strain of Trichoderma has followed. 

The Trichoderma harzianum is a filamentous fungus which is very 

important from the biotechnological point of view. Filamentous fungi are 

particularly interesting producers of xylanases since they excrete the

enzymes into medium and the enzymes levels are much higher that those 

of yeast and bacteria (Kulkarni et al., 1999). Although, a plethora of 

xylanase producing strains have been described their use for commercial 

production at present is restricted mainly to Trichoderma sp and Aspergillus 

sp. (Haltrich et al., 1996). Most of industrial xylanase producing strains are 

species of Aspergillus and Trichoderma (Dobrev et al., 2007). 

Xylanase activity is found to be higher in fungi than in bacteria. 

Several strains of the soft rot fungus Trichoderma have been shown to be 

efficient producers of xylan degrading enzyme activity (Silveria et al., 

1997). Among fungi, the maximum xylanase activity reported is 3350 

IU/mL in Trichoderma reesei (Haapala et al., 1994). The species of the 

mesophilic fungus Trichoderma are reported to produce enzymes that are 

involved in degradation of hemicellulose to fermentable sugars (Bailey et 

al., 1993). 

Several Trichoderma strains have been reported to be effective in 

controlling plant diseases. Trichoderma harzianum, a potent antagonistic 

fungus in the control of plant pathogenic fungi, can be used as an 

alternative to chemical compounds. The antifungal activities of T. 

harzianum involve production of fungal cell wall degrading enzymes, 

antibiotics and toxins, and competition for key nutrients. Several 

Trichoderma strains with improved antagonistic activity have been 

detected and studied in protective field tests (Hjeljord and Tronsomo, 

1988). 

2.3. XYLOOLIGOSACCHARIDES (XOs) 

2.3.1. Production of XOS from lignocellulosic biomass 

Lignocellulosic (LCM) material is composed of three polymers 

namely, lignin of phenolic nature (Grima-Pettenati and Goffner, 1999), 

cellulose composed of glucose units linked through β1-4 linkage to form a

linear polymer (Gardner and Blackwell, 1974) and hemicelluloses, a 

variety of monomers including xylose, glucose, arabinose, rhamnose and 

mannose branched result in a heteropolysaccharide (Puls and Schuseil, 

1993). Source of substrate determines the nature of substantial part of 

hemicelluloses that can be a polymer of xylose (xylan), arabinose 

(arabinan) or mannose (mannan) (Huisman et al., 2000). Hemicellulose, the 

most abundant polysaccharide from plant source is a base material for 

preparation of several value added products. Hence, the use of 

agricultural by-products/wastes for xylooligosaccharides production is 

based on the philosophy called ‘biomass refinery’ which states 

fractionation of hemicellulosic component of these by-products is of 

interest to obtain separate streams useable for different product 

applications (Myerly et al., 1981). LCM affluent in xylan, such as 

agriculture residues (corncobs, bagasse, cornstalks, sunflower, rice hulls 

and almond shells), hardwoods, and algae can effectively be used to 

produce xylo-oligomers through fractionation processes (Nabarlatz et al., 

2004). 

Xylans are the second most plentiful hemicellulosic polymers, 

comprised of xylose units (Joseleau et al., 1992). Xylans correspond to an 

enormous resource of biopolymers for practical applications, accounting 

for 25–35% of the dry biomass of woody tissues of dicots and lignified 

tissues of monocots, and occur up to 50% in some tissues of cereal grains 

(Vazquez et al., 2006). Xylans are heteropolysaccharide composed of a 

chain of xylose units joined through β-(1→4) glycosidic linkages (Bakir et 

al., 2001), with branching of short chain heXOses/pentoses (D-glucuronic 

acid or its 4-O-methylether, L-arabinose and/or various oligosaccharides, 

composed of D-xylose, L-arabinose, D- or L-galactose and D-glucose) 

(Subramaniyan and Prema, 2002). Polymers like xylans can be classified 

into homoxylans and heteroxylans, that include complex heteroxylans,

glucuronoxylans, (arabino) glucuronoxylans, (glucurono) arabinoxylans 

and arabinoxylans (Shimizu, and Nemaliales with their backbone consists 

of xylopyranose (Xylp) residues linked by β-(1→ 3), or mixed β-(1→ 3, 1→ 

4) glycosidic linkages. 

There have been a number of approaches divulged for fractionation 

of LCM mainly: 

I. Isolation or solublization of xylan through chemical fractionation and 

enzymatic hydrolysis 

II. Direct enzymatic hydrolysis of xylan rich material 

III. Autohydrolysis: hydrolytic treatments based on aqueous/steam 

processing and chemical and/or enzymatic hydrolysis. 

The combined chemical and enzymatic extraction of XOS is 

performed in two stages i.e the xylan extraction from LCM and enzymatic 

hydrolysis of that xylan into xylooligomers (Figure 2.2). Isolation or 

solublization of xylan through chemical fractionation and enzymatic 

hydrolysis. The xylan fraction is usually treated with low concentrated 

solutions and mild conditions (Timell, 1967). This can be obtained by 

treatment of the LCM with dilute acids (Dominguez et al., 1997) alkali (i.e. 

solutions of ammonia, Ca(OH)2, NaOH, KOH or mixture of any of these) 

(Barl et al., 1991), the stable pH of xylan supporting this treatment. Feed 

stock material can be pretreated with oxidizing agents, alcohols or salts to 

remove lignin/pectic substances depending upon the nature of material. 

From the alkali treated LCM, recovery of degraded hemicellulosic by- 

products can be made by precipitation with organic compounds. Further 

degradation of isolated xylan can be achieved by hydrolysis with 

xylanases, which can be added directly to the reaction media, 

immobilized, or may be produced in situ by microorganisms. The 

xylanases play a key role in xylan hydrolysis to XOS (Collins et al., 2005; 

Dobrev et al., 2007).

Figure 2.2: General scheme of XOS production through chemical and enzymatic hydrolysis 

To avoid production of xylose (monomer) during enzymatic 

hydrolysis, enzymes with lesser exo-xylanase and/or β-xylosidic activity 

are required. Moreover, it helps to attain XOS with lower degree of 

polymerization (DP), which is an advantage for application in food (Loo et 

al., 1999). 

Corncob meal as a substrate was used by Pellerin et al (1991) for 

XOS production through highly specific endo-xylanse (E.C.3.2.1.8) 

isolated from Clostridium thermolacticum. The enzyme with 290U/mg 

protein employed for depolymerization of corncob xylan. Highly pure 

xylan was extracted using 24% KOH followed by filtration and 

precipitation with ethanol. The XOS produced through this process were 

absorbed on charcoal and stepwise elution with ethanol resulted in 

xylobiose, xylotriose and arabino-xylooligosaccharides. For effective 

enzymatic hydrolysis, the surface area available for enzyme adsorption 

and degree of delignification are important. Combined chemical and 

enzymatic treatment of corn husk that NaOH was more effective as

compared to H2SO4 and H3PO4 in increasing the husk susceptibility for 

enzyme action (Barl et al., 1991). 

Treatment of corn stover and corncobs with aqueous ammonia for 

enzymatic production of XOS was studied by Zhu et al (2006) in which 

food grade XOS were obtained. Pretreatment with aqueous ammonia 

helps in delignification and xylan rich substrate (in solid form) to be 

treated with enzyme, thus avoids the purification step otherwise involved 

to get soluble xylan. Glucan rich residue after xylanlytic activity is 

digested with the help of cellulose enzyme. Refining of xylooligomer 

solution is done by carbon adsorption followed by ethanol elution. In the 

same way, delignification of cotton seed residual cake is achieved with 

sodium hypochlorite (1% chlorine). This delignified material is extracted 

with 15 % NaOH to get xylan (Sun et al., 2002). Cotton stalks are employed 

by Akpinar et al (2007) for enzymatic production of XOS. In this study, by- 

products (xylose, furfural etc) formation is avoided by enzymatic 

hydrolysis. The reaction conditions of 40°C temperature and pH 4.5 are 

the optimum for enzyme activity. The xylooligomers obtained in the 

degree of polymerization (DP) range of 2-7 are refined/fractioned by 

ultrafiltration using 10, 3 and 1 kDa membranes. Purified oligomers via 

complete removal of enzyme and unhydrolyzed xylan are the final 

product. 

Pretreatment of the corncob for XOS production helps in breaking 

down the xylan-lignin complex, liberating xylan available for the action of 

enzyme. Soaking in dilute acid solution (1.0g/l H2SO4) for 12 h at 60°C, 

filtration and washing are performed before steaming the feedstock 

material at 135-140°C for 30 min. The resultant slurry facilitates enzymatic 

degradation of soluble xylan at pH 4.5. The main products of this reaction 

are xylan containing oligomers with minor quantities of arabinose, xylose 

and glucose (Yang et al., 2005).

Steam/water or 

acid solution 

Direct enzymatic production of XOS from xylan rich LCM should 

be executed from potential substrate that is prone to enzymatic action. 

Autohydrolysis or hydrothermal treatment is another approach which has 

quite frequently been used for XOS production. With optimum reaction 

conditions, lignocellulosic material is gradually broken down in a reaction 

with water or steam which is catalyzed by hydronium ions to form 

oligosaccharides and cellulose/lignin in solid form. The graphical scheme 

of this process is shown in Fig 2.3. Other side reactions e.g degradation of 

acetyl group to acetic acid adds to the generation of hydronium ions in 

later stages of this reaction. This process can be assisted by addition of an 

acidic solution in the reaction medium, but the XOS are intermediate 

products in this case and final products are monosaccharides. Some of the 

side-progressions during xylan degradation are ash neutralization, 

solubilization of acid soluble lignin and extractive removal contribute 

towards impurities in final product. Refining step in this case becomes 

quite crucial for attaining purified, food graded XOS. Feedstock can be 

pretreated before water treatment to simplify purification step in such 

circumstances. Mild operational conditions help to achieve the desired DP 

in final product (Suwa et al., 1999). 

Xylan rich 

Autohydrolysis 

Xylan Degradation 

Crude 

Xylooligomers 

Figure 2.3: General scheme of XOS production through 

autohydrolysis/hydrothermal treatment (Suwa et al., 1999) 

Substrate 

Pretreatment 

(optional) 

Cellulose/lignin 

(insoluble fraction)

A variety of substrates have been reported for the production of 

XOS through autohydrolysis. Among them crop residues (Endo and 

Kuroda, 2000), corncob (Garrot et al., 2002), sugarcane bagasse (Jacobsen 

and Wyman, 2002), barley hulls (Vegas et al., 2005), almond shells 

(Nabarlatz et al., 2005), brewry spent grains (Carvalheiro et al., 2004), corn 

stovers (Mosier et al., 2005), rice hulls (Vila et al., 2002), bamboo (Ando et 

al., 2003), hard woods (Teleman et al., 2000) and flax shive (Jacobs et al., 

2003) have been explored. 

Depending upon the required level of purity, a series of many 

physical/chemical steps might be involved in purification process after 

hydrothermal treatment. Vacuum evaporation is the prime step in refining 

process. It not only increases the concentration of the processed liquors, 

but also helps in removing the acetic acid and compounds responsible for 

off flavor (Eden et al., 1998). Some of the organic acids e.g. formic, 

propionic or acetic acid has been suggested to purify xylan degenerated 

products (Schweiger, 1973). Further, recovery of hemicellulose- 

degradation products can be made through other organic solvents such as 

alcohols and acetone (Sihtola, 1976). 

2.3.2. Refining of XOS 

Whether Xylooligosaccharides are produced by enzymatic 

hydrolysis or auto-hydrolysis, a variety of other compounds such as 

monosaccharides, and some nonsaccharide compounds like furfural, acid 

soluble lignin, and protein derived products appear in the final XOS 

solution. In order to have food/pharmaceutical grade XOS, these 

solutions need to be purified for the concentrated product with maximum 

XOS content. Commercially available XOS are reported to have purity of 

75-95%. The chemical and enzymatic hydrolyses produce final product

with lesser impurities because of previous chemical process and specific 

action of enzyme. 

Most of the refining processes start with vacuum evaporation, 

which not only increases the product concentration but also removes the 

volatile compounds. However, solvent extraction can be useful technique 

for eliminating non-saccharide compounds (Va´zquez et al., 2005). Use of 

ethanol, 2-propanol and acetone has been suggested by several 

researchers in solvent precipitation for XOS refining (Swennen et al., 2005; 

Va´zquez et al., 2005). However, recovery and purification depend on the 

nature of raw material and type of solvent used for purification. 

A number of adsorbents such as activated charcoal or carbon, 

titanium, acid clay, bentonite, diatomaceous earth, aluminium hydroxide 

or oxide, silica and porous synthetic materials have been used for 

purification of XOS liquors in a process known as adsorption. Depending 

upon the objective, adsorption might be used in combination with other 

treatments, either for the removal of undesired materials (Yuan et al., 2004; 

Mosier et al., 2005) or for the separation of oligos from monosaccharides 

(Ohsaki et al., 2003; Sanz et al., 2005). 

The membrane techniques are significantly being used in XOS 

processing for a number of purposes, including generation by enzymatic 

reactions (Freixo and de Pinho, 2002), removal of XOS with undesirable 

DP range (Dhara et al., 1991), refining and concentration. Swennen et al 

(2005) employed ultrafiltration as substitute to ethanol precipitation for 

separation of arabinoxylo-oligosaccharides from enzymatically 

hydrolyzed arabinoxylan, leading to fractions with similar DP and degree 

of substitution than the precipitated ones. Membranes are used for 

concentrating enzymatic hydrolyzates of lignocellulose pulp and Yuan et 

al. (2004) employed nanofiltration membranes for concentrating XO 

obtained by enzymatic breakdown of xylan from steamed corncobs,

whereas Akpinar et al (2007) reported use of 10 kDa membrane for total 

removal of enzyme and unhydrolyzed xylan without losing 

oligosaccharide having DP 5. 

Refining by chromatographic separation has been carried out for 

XO purification at an analytical level, which results in high purity 

fractions. For instance, the concurrent elimination of salts and coloring 

compounds from XOS solutions can be accomplished by chromatographic 

separation (Isao et al., 1989), hydrothermally treated LCM have been 

fractionated by anion-exchange chromatography and size-exclusion 

chromatography (Kabel et al., 2002), whereas size-exclusion 

chromatography has been used simultaneously with other techniques for 

purification of feruloylated oligosaccharides (Katapodis et al., 2003). 

Various purification steps might be needed for achieving high-purity 

XOS. 

Spray drying and lyphilization are said to be in use for the 

resurgence of hemicelluloses derived xylooligomers in powder form 

(Palm and Zacchi., 2004). 

2.3.3. Applications of XOS 

The XOS are sugar oligomers made up of xylose units, which 

appear naturally in bamboo shoots, fruits, vegetables, milk and honey. 

Since 1980’s, several oligosaccharides have got recognition as food 

ingredients in a short span of time, particularly in Japan and Europe, 

primarily because of the possible health effects associated to the 

consumption of these compounds. The budding commercial magnitude of 

these non-digestible oligosaccharides is supported for their beneficial 

health properties, particularly the prebiotic activity (Crittenden and 

Playne, 1996).

These XOS can be used in many fields including food industry as 

ingredients of functional foods, in cosmetics, pharmaceuticals or 

agricultural products. Figure 2.4 summarizes few of the applications of 

XOS. 

Figure 2.4: Main applications of Xylooligosaccharides 

2.3.3.1. Food Applications 

A food can be regarded as functional if it is satisfactorily 

demonstrated to affect beneficially one or more target functions in the 

body, beyond adequate nutrition, in a way that improves health and well- 

being or reduces the risk of disease (Gibson and Williams, 2000). 

According to above definition, the XOS are considered as potential 

ingredients in functional foods. There is an emerging market for such 

functional OS to be used in food. There are a number of factors

esponsible for application of XOS as functional ingredients e.g. 

consumers demand, scientific research supporting the fact that such foods 

help improving and maintaining overall health and well being, the role of 

XOS for self medication and prevention of disease. 

The XOS are advantageous over other non-digestible 

oligosaccharides in terms of both health and technological related 

properties. Besides their number of health effects, The XOS are 

characterized for interesting physico-chemical properties; they are 

moderately sweet, stable over a wide range of pH and temperatures and 

have sensory characteristics suitable for incorporation into foods (Alonoso 

et al., 2003). 

The XOS act as non-digestible dietary component that escape from 

stomach’s low pH and enzymes to colon and selectively stimulate the 

certain population of bacteria (Loo et al., 1999). This property of XOS 

makes them a useful ingredient in novel functional foods. A latent 

synergy between probiotics and prebiotics leads to development of foods 

containing both of them; such foods are referred as ‘synbiotics’ 

(Crittenden et al., 2001). ‘Bikkle’, is an example of such synbiotic food 

product being manufactured by Suntory Ltd. Japan since 1993, which is a 

drink comprised of bifidobacteria, xylooligosaccharides, oolong tea extract 

and whey minerals. With an acceptable odor, and non-cariogenic 

characteristics (Kazumitsu et al., 1997; Kazuyoshi; 1998) XOS are 

prospective food ingredients for special foods. These oligomers have low- 

calories, permitting their utilization in anti-obesity diets (Taeko et al., 

1998). 

From food processing point of view, the XOS demonstrate benefits 

over other OS and inulin in terms of resistance to both low pH and high 

temperature, thus can be used in carbonated drinks, low-pH juices and 

acidic foods (Modler, 1994).

In food industry, another application for XOS has been found in 

inference to the production of low calorie sweeteners as xylitol and 

antioxidant compounds. There are certain examples, like the production 

of detoxified fermentation media (xylose solutions for the fermentative 

production of xylitol), eliminating the polyphenols with antioxidant 

activity from hemicellulosic wood hydrolysates (Parajó et al., 1997; Moure 

et al., 2001). Doerr et al. (2002) reported the use of XOS as flavor enhancer 

in formulating a beverage. The XOS reportedly were found to have a 

positive impact on addition in a non-alcoholic carbonated drink with an 

intense sweetener (mixture of acesulfame K and aspartame). It has been 

found that by adding the XOS, full bodied character of beverage was 

significantly enhanced without any drawback of off flavor perception or 

mouth-feel. 

2.3.3.2 . Pharmaceutical Applications 

Certain oligosaccharides including XOS have found their 

application in pharmaceuticals, as in antiviral and antitumor drugs. 

Several polysaccharides are under investigation due to their suspected 

antitumoral activity. It was revealed in-vitro that a fraction of xylose, XOS 

and water soluble lignin has cytotoxic effects and reduced the viability of 

leukemia cell lines derived from acute lymphoblastic leukemia (Ando et 

al., 2004). 

Red seaweed Nothogenia fastigiata derived sulfated xylogalactans 

(Damonte et al., 1996) and a xylomannan from algal (Pujol et al., 1998) 

were found to exhibit antiviral activity against herpes simplex virus types 

1 and 2. A polysaccharide mixture (formed by glucose, xylose, mannose 

and glucuronic acid) acquired from marine algae has antiviral activity, 

and when it is bounded to a protein, both (the original polysaccharide and

this complex) act as a biological response modifier, exerting a great 

influence on the immune system. 

Bifidobacteria population is reported to flourish with the use of XOS, 

and on the basis of this fact a nutritional product (an oil blend with 

eicosapentaenoic acid or docosahexaenoic acid and a source of 

indigestible carbohydrate i.e XOS which is metabolized to short chain 

fatty acids by the microorganisms present in the human colon) has been 

developed for the people suffering from ulcerative colitis (DeMichele et al., 

1999). 

2.3.3.3. Other Applications 

In addition to food and pharmaceutical uses, ethers and esters have 

been produced from xylan and high molar mass XOS and are being used 

as thermoplastic compounds for biodegradable plastics, water soluble 

films, coatings, capsules and tablets (Glasser et al., 1995), and for the 

preparation of chitosan–xylan hydrogels as well (Gabrielii et al., 2000). 

There are reports on the XOS application in agriculture as ripening 

agent, growth stimulator or accelerator and yield enhancer on limited 

level (Katapodis et al., 2002). The XOS as feed of domestic animals and fish 

has also been stated by Smiricky-Tjardes et al. (2003). 

2.3.4. Health Benefits of XOS 

Several functional effects of NDOs being of prebiotic nature are 

found to improve health by researchers. Some of them are briefly enlisted 

in Table 2.2 

2.3.4.1. Gastrointestinal Effects 

Microbial population of human gut plays a vital role in maintaining 

health of an individual by improving GIT health and through systemic 

absorption of metabolites. It is generally believed that certain bacterial 

species among gastrointestinal flora advantageously affect the health such

as Bifidobacterium and Lactobacillus spp. These bacteria have been the focus 

of attention as increased population of them is an indication of health 

microbes (Ballongue, 1998). As the XOS fall in the category of NDOs, 

modulation of intestinal flora is associated with prebiotic character of 

XOS. The Xylooligosaccharides being NDO escape from low pH stomach 

fluids and digestive enzyme, and are metabolized in large bowel. 

Carbohydrates fermentation in large intestine is a complex process, 

because the final products of the metabolism of particular bacterial species 

can become the substrate for others, and some of the microbes may grow 

upon substrates which are not fermentable by them. 

Table 2.2: Health/biological effects of XOS produced by different processing 

techniques 

Substrate/Manufacture 

approach 

Biological effect 

Rice bran enzymatic processing Immunomodulatory action 

Hardwood pulp enzymatic and Bacteriostatic action against 

acid hydrolysis 

Vibrio anguillarum 

Birchwood xylan enzymatic 

Antimicrobial activity against 

processing to acidic OS 

gram positive 

Acidic XOS containing uronic Anti-allergy agents 

acid residues 

Histamine-release inhibitors 

Hyaluronic acid-formation 

promoters 

Anti-inflammatory activity 

Hair growth stimulation 

Algae enzymatic processing Cancer cell apoptosis inducers 

Pulp slurry chemical-enzymatic Therapeutic agents for 

processing 

osteoporosis 

Treatment 

Broadleaf pulp enzymatic 

Collagen production enhancer 

processing 

Hardwood pulp chemical and 

enzymatic processing 

Inhibition of melanin and 

inhibition of 

melanoma cell proliferation

Bamboo hydrothermal 

processing 

Selective cytotoxicity against 

acute lymphoblastic leukemia 

cells 

LCM enzymatic processing Hypolipemic activity (against 

cholesterol, 

phospholipid and 

Wheat flour arabinoxylan 

enzymatic hydrolysates 

Rice hulls hydrothermal 

treatment 

Rice bran enzymatic 

hydrolysates 

XOS mixture with tea 

catechins 

Bagasse hydrothermal 

processing and enzymatic 

processing 

Wheat bran enzymatic 

hydrolysis 

triglycerides) 

Prevention of atherosclerosis 

Antioxidant (DPPH-radical 

scavenging) 

Activity 

Antioxidant (DPPH-radical 

scavenging) 

Activity 

Cosmetics 

Prevention and control of 

anemia 

and arteriosclerosis 

Antioxidant activity 

Antioxidant activity 

(erythrocyte 

hemolysis assay) 

Feruloyl XOS enzymatic 

Protective effect against lipid 

reactions 

(LDL) 

Adopted from Moure et al. (2006) 

A study evaluating the in vitro fermentation of commercial 

prebiotic oligosaccharides shows that the XOS increased the number of 

Bifidobacteria, when compared to other oligosaccharides (Rycroft et al., 

2001). The XOS can selectively be utilized as a carbon source by beneficial 

bacteria, which was found in a study where many Bifidobacterium species 

and Lactobacillus brevis were able to grow to high yields using XOS. Some 

bacteroides isolates also efficiently fermented XOS except E. coli, 

Enterococci, Clostridium difficile or Clostridium perfringens (Crittenden et al., 

2002). The XOS with 2-10 DP range have got the ability to enhance the

Bifidobacterium population and to suppress the growth of Clostridium has 

been stated by Izumi and Azumi (2001). 

According to Zampa et al., (2004), favorable effects of the XOS are 

not only related to their capability of increasing the populations of 

Bifidobacteria and Lactobacilli, but also from the reduction in concentrations 

of secondary bile acids, that wield a negative action on colon and present 

a dose-dependent toxic potential related to their co-mutagenic and tumor- 

promoting attributes. Dietary supplements comprised of the XOS have 

been reported to suppress production of secondary bile acids and 

physiologically active fatty acids during the digestive process (Rull, 2006). 

Along with increased growth of beneficial microbes, the 

fermentation of XOS in colon results in the production of short chain fatty 

acids (SCFA) like acetate, propionate and butyrate, CO2, H2, and lactate. 

Systemic or local metabolism of them provides energy generation to the 

host. The SCFA production has been associated with a number of health 

effects, among which improvement in bowel function, lipid metabolism, 

calcium absorption and reduction of the risk of colon cancer (Scheppach et 

al., 2001), serving as fuel in different tissues and playing a potential role in 

the regulation of cellular processes (Blaut et al., 2002) are the promising 

ones. Concentrating on the XOS functions in gut, the studies in rats 

proved the ability of feruloyl oligosaccharides for stimulating the growth 

of Bifidobacterium bifidum (Yuan et al., 2005), and a greater bioavailability of 

bound ferulic acid in comparison with the free compound (Rondini et al., 

2004), providing grounds for positive effects on the intestinal 

microorganisms that might affect the growth of precancerous colonic 

lesions (Hsu et al., 2004). 

A study on fermentation of four different types of XOS (acetylated, 

linear, substituted with uronic acids and arabino-XO) by faecal inocula, 

showed that linear- and arabino-XO were fermented quickly than the

other substrates during the first 20 h, and that after this adaptation time, 

all the substrates tested degraded with same rate. Increased cell material 

and accumulation of relatively high substituted XOS with DP range of 5–7 

were the outcomes of fermentation. Formation of the SCFA and lactate 

happened during the fermentation and their concentrations rose with the 

consumption of total XOS. The fermentation took place in two stages; the 

first one leading mainly to the formation of acetate and lactate, and 

butyrate and propionate were the production from the second one (Kabel 

et al., 2002). 

2.3.4.2. Other health benefits 

There are several other health benefits of the XOS reported in 

literature. Oku and Sadako (2002) stated that these have non-cariogenic 

properties and save insulin secretion from the pancreas, thus stimulating 

intestinal mineral absorption. 

Cummings et al. (2001) studied that bowel norm can be altered 

through XOS as these OS are mildly laxative by stimulation of bacterial 

growth and fermentation. A satisfactorily elevated and regular ingestion 

may cause diarrhea because of osmogenic retention of fluid in both the 

small and large intestines. These symptoms are disappeared within a few 

days due to the increase in the population of intestinal microbes having 

ability to readily utilize these XOS. Though the maximum permissible 

dose varies from individual to individual, it has been calculated in 0.12 

g/kg body weight for male Japanese adults (Oku and Sadako, 2002). 

The XO intake has been reported as highly effective for reducing 

severe constipation in pregnant women without any adverse effects 

(Tateyama et al., 2005). This NDO might be a help to evade intestinal 

disorders such as constipation, inflammatory bowel disease, diarrhea and 

gastritis (Matsuoka, 2005). Nutritional formula for infants comprising XOS

has been claimed to improve gut barrier maturation and provides 

synergistic effects all along the intestinal tract (Garcı´a-Ro´denas et al., 

2004). 

2.4. EFFICACY STUDIES ON XOS 

The XOS behave as dietary fibre and since the dietary fibre has the 

properties mandatory for its consideration as an important ingredient in 

the preparation of functional foods, due to its beneficial effects such as 

increasing the volume of faecal bulk, decreasing the time of intestinal 

transit, cholesterol and glycaemia levels, trapping substances that can be 

dangerous for the human organism, stimulating the growth of the 

intestinal flora, etc. Furthermore, the XOS have been the latest and most 

exhilarating development, because even as they are already fermentable 

substances, their effects on bowel habit are minor and their contribution to 

the health relies on their noteworthy effects on the large intestinal flora. 

For this reason they are called “prebiotics” (Manning and Gibson, 2004). 

Various oligosaccharides such as fructo-oligosaccharides, inulin, 

lactulose and galacto-oligosaccharides have been checked through 

different in vitro methods, animal models and human clinical trials for 

their suspected health benefits. There are reports of similar studies for 

newly developed XOS, isomalto-oligosaccharides, gluco-oligosaccharides 

and soybean-oligosaccharides on minor scale. Increased awareness about 

OS as prebiotics and their fermentable nature in past couple of decades 

has occurred because of their proven health benefits. Efficacy studies on 

animal models have proven that indigestible oligosaccharides might exert 

many of health benefits related to them being prebiotics (Roberfroid, 

1993). 

A focused research on colon and the fermentation products in colon 

including the SCFA has been conducted. The most important products of

acterial fermentation are SCFA in the large intestine. Almost 95% of the 

produced SCFA including acetate, propionate and butyrate throughout 

carbohydrate fermentation could be taken up and consumed by the host 

(Cummings et al., 1987). Among these three SCFA, acetate and propionate 

are energy providers for brain, heart and muscle, whereas butyrate 

supplies about 50% of the daily energy required to the gastrointestinal 

mucosa (Roediger, 1989). Nevertheless, butyrate is an important fatty acid 

of particularly because of its known role in cellular differentiation and 

proliferation of colonic mucosa (Williams et al., 2003; Rafter et al., 2004). 

The SCFA are related to certain processes in the colon like colonocytes are 

supposed to be sustained by fermentation of the SCFA where oxidation of 

butyrate supplying about 70% of oxygen to be consumed by colonic 

tissues. Fractional information illustrates that colonic mucosa of rats 

prefers butyrate as its energy source (Roediger, 1980). 

Okazaki et al. (1990) reported that XOS are not degraded in the 

small intestine of humans but are expansively fermented in the large 

bowel to SCFA which can be further utilized by host. In-vivo 

consequences of different indigestible oligosaccharides including XOS in 

Sprague dawley rats was studied by Campbell et al. (1997) for the period 

of 14 days. It was found that total cecal SCFA pool was higher, with acidic 

pH of cecum in rats fed on the diet including XOS, fructo-oligosaccharides 

and oligo-fructose. Hence, being an indirect energy source of large bowel, 

these OS positively impacted the maintenance of mucosal cell 

differentiation and consequently enhanced gastrointestinal mucosal 

integrity. 

Fermentation of non-digestible materials by bacterial strains in 

large intestine is an important process of digestive system. Effect of fibre 

source on short chain fatty acid production was examined in another 

study by May et al. (1994) who concluded that XOS and gum arabic

among five fibre sources were fermented to greatest SCFA concentrations 

in an in vitro experiment. 

The gastrointestinal tract is a kind of intricate ecosystem having a 

variety of microflora and is reactive towards the input like ingested food 

or drug. Thus, the food can be used to manage the population densities of 

beneficial (Bifidobacteria, Lactobacilli) and harmful (Escherichia coli, 

Clostridium spp.) bacteria. There have been reports specifying that which 

strain of bacteria is responsive towards what type of non-digestible 

oligosaccharides. The increased awareness about composition of bacteria 

residing in GIT has led researchers to identify substrates that can be 

utilized by desired microflora. The known role of LAB in improving 

resistance to enteric pathogens is one key point. Influence of non- 

digestible substrates specifically NDO to alter the absolute or relative 

concentration of bacterial groups is also established. XOS’s prebiotic 

potential originates from their discerning fermentation by Bifidobacterium 

species. On these bases, they are sometimes referred to as bifidogenic 

factors (Gomes and Malcata, 1999). 

In a three week in-vivo study to verify the effects of XOS on the 

growth of Bifidobacteria by Okazaki et al. (1990) shows that these XOS have 

an encouraging effect on human intestinal flora. The analysis included 

intestinal flora, pH and volatile fatty acids. Although, there was no change 

in total counts during the study period but at the end of experiment, 

Bifidobacteria population was significantly higher (up to 24-32%) while 

decrease in count of Bacteroides and Clostridium was observed. Other 

suitable environmental conditions of intestine such as lower faecal pH and 

maintenance of faecal water content within normal range were the 

outcomes of XOS administration in same experiment. Similarly, 

population of cecal Bifidobacteria and total anaerobes was greater, while 

total aerobes were lower in rats fed oligosaccharide diets compared with

those fed the control diet (Campbell et al., 1997). In another study of mixed 

cultures with human faeces, it was observed that the sole carbon source 

XOS can significantly improve population of Bifidobacteria after 24 h 

(Rycroft et al., 2001). 

The Bifidobacteria are gram positive, anaerobes which naturally 

dwell in human and animal intestines. Production of metabolites like 

acetate and lactate by these bacteria lowers the colon pH and proliferation 

of pathogens is restrained in this way. This phenomenon is particularly 

evident in breast fed infants, whose microbial flora comprises just about 

utterly of Bifidobacteria (Ibrahim and Bezkorovainy, 1994). However, 

Bifidobacterium population declines in the adult’s intestine, though it stays 

relatively stable representing 3–6% of the faecal flora, till the later age 

when the population of Bifidobacteria becomes to be diminishing (Satokari 

et al., 2003). 

The dominance of Bifidobacterium among the gastrointestinal 

microbes has been allied with a number of health benefits. These bacteria, 

especially B. longum, have been shown to decrease the occurrence and 

duration of diarrhoea linked with antibiotics (Orrhage et al., 1994). In 

another study involving B. bifidum Plummer et al. (2004) advocates that 

this organism might play a positive role in toxin neutralization, thus 

reduce the incidence of antibiotic-associated diarrhea. The role of 

Bifidobacteria against certain microbial infections has been investigated. 

Production of various acids, hydrogen peroxide or bacteriocins, the 

competition for nutrients or adhesion receptors, anti-toxin action and 

immune system stimulation can be a proposed mechanism of Bifidobacteria 

against pathogenic organisms (Fooks and Gibson 2003; Rakoff-Nahoum et 

al., 2004). Clostridium spp. produces a variety of adverse substances 

including toxins and volatile amines. Experiments involving ingestion of 

live Bifidobactria in probiotic formulation have not only reduced the

clostridia population but also revealed to display inhibitory effects on 

many other pathogenic organisms (Asahara et al., 2004). 

There are several reports in literature revealing some other health 

related benefits wielded by Bifidobacteria. For instance, inflammatory 

bowel disease (IBD) is thought to be related with type of intestinal flora of 

an individual. According to Ouwehand et al. (2002), symptoms of this 

disease can be reduced by altering the proportion and activity of 

microflora residing the gut. Significant laxative action of bowel movement 

induced by Bifidobacteria is reported by Kleessen at al. (1997) in a study, 

thus alleviating constipation. 

There has been some oblique but suggestive data that these 

microbes can avert or delay the onset of certain cancer. The roots of this 

statement originate from the awareness that change in the intestinal 

microbial population due to a diet high in fat and meat and low in fibre, 

might consequently alleviate levels of putrefactive microorganisms with 

decreased levels of Bifidobacterium (Benno et al., 1991). Such modification 

in the intestinal population is escorted by increased activity of faecal 

enzymes e.g beta-glucuronidase, azoreductase, urease and nitroreductase. 

These enzymes contribute to an increased risk for cancer by converting 

pro-carcinogens into carcinogens (Hirayama and Rafter, 2000). 

Preliminary reports on fermented dairy products by some researchers 

show that strains of Bifidobacteria can lower the serum cholesterol levels, 

thus lowering the risk of cardiovascular diseases (Xiao et al., 2003). 

Whilst, there are a lot of health benefits accredited to Bifidobacteria, 

the need of improving the bifidogenic factors is well understood. XOS 

have been tested both in-vitro and in-vivo studies as a potential 

Bifidobacteria growth promoter. Rats were fed on four types of 

experimental diets for a period of 35 days in a study by Hsu et al. (2004). 

The treatment groups were comprised of a) control diet, b) diet which

included 1,2-dimethylhydrazine (DMH) 15 mg/kg body weight, c) DMH 

+ 60g FOS/kg diet, d) DMH + 60g XOS/kg diet. Xylooligosaccharides had 

greater effect on Bifidobacteria population than fructo-oligosaccharides 

(FOS). Similarly, a study reported by Howard et al. (1995) on mice and rats 

shows results in relatively different pattern. It was observed that dietary 

FOS, XOS and gum Arabic effected cecal and colonic microbiota variably. 

Findings reveal that concentrations of Bifidobacteria were greatest with 

FOS treatment among all, followed by XOS dietary treatment. Campbell et 

al. (1997) however, found that NDO like XOS significantly increase the 

cecal Bifidobacteria and total anaerobes as compared to control diet given 

to five rat groups in a 14 day experiment. 

Response of resident gut microbes towards fermentable fibre 

(oligo-fructose, inulin and lactosucrose) diet in comparison to less 

fermented cellulose diet was tested in 6 weeks study on mice. It is 

concluded that NDOs can be used well to manage proportion of intestinal 

microbes and growth of lactic acid producing bacteria (LAB) is higher 

than Entrics and E coli (Buddington et al., 2000). 

These XOS have also been known to lower the blood ammonia 

levels in patients suffering from liver cirrhosis by altering the intestinal 

environment. This was reported by Kajihara et al. (2000) who conducted a 

study on fourteen cirrhotic patients. Significant decrease in blood 

ammonia levels along with promoted growth of Bifidobacteria was 

observed when subjects were given 3 g XOS daily for a period of two 

weeks. These observations indicate that XOS might exert inhibiting effect 

on enteric colonization of ammonia producing microbes like bacteroids. 

Likewise, preventive effects of acidic XOS on contact hypersensitivity in 

mice were explored by Yoshino et al. (2006). It was stated after observation 

in mice that oral administration of acidic XOS might be beneficial for the 

prevention of contact hypersensitivity.

It has also been reported that XOS exhibit some other advantageous 

consequences on health. The XOS were given in experimental diet to 

diabetic rats, and certain metabolic parameters like blood glucose, serum 

and liver lipids were examined. The experiment showed that the XOS can 

improve growth retardation, hyperphagia, polydipsia and elevated levels 

of serum glucose. It was also stated that lowered liver triglycerides and 

reduction in desaturation index fatty acid composition of liver 

phosphatidylcholine were outcome of XOS diet. Concluding from above 

stated observation, it can be recommended that XOS have got properties 

to be used as sweetener for improving diabetic conditions (Imaizumi et al., 

1991). 

2.5. SYNBIOTIC FOODS 

2.5.1. Definition and Background of Synbiotic 

The functional foods type is growing immensely because of 

consumer’s awareness of diet and health as well as food processor’s 

consideration due to enhanced value of food with added ingredients. First 

generation of functional foods was probably the fortified cereals with 

trace minerals. Synbiotics are one of the newest forms of such products 

and are defined as combination of a prebiotic oligosaccharide along with 

live microbes (probiotic) in a food product for enhanced benefits. There is 

thus, a great flexibility in choice of a prebiotic with known health effects 

that can help probiotic bacteria to establish in colon environment (Rastall 

et al., 2000). For instance, “Bikkle” is a type of synbiotic product being 

produced and marketed by Suntory Ltd. since 1993, containing 

Bifidobacteria, XOS, whey minerals and oolong tea extract. Among other 

types of synbiotic foods, synbiotic dairy products are by now being 

marketed in Europe and Japan (Young, 1998). For the preparation of 

synbiotic fermented milks, several bacterial strains of Lactobacillus and

Bifidobacterium spp. have been recognized and are in use as probiotic, 

while fructo-oligosaccharides, galacto-oligosaccharides, lactulose, and 

inulin derived products are generally being employed as prebiotic 

(Ziemer and Gibson, 1998; Klaenhammer and Kullen, 1999). 

Bomba et al. (2002) in an experiment on weanling pigs reported a 

significant increase in the population of Lactobacilli and Bifidobacteria, 

when comparison was made between feeding on probiotic strain 

Lactobacillus paracasei and fructo-oligosaccharide in addition to the above 

stated strain. Some other studies demonstrate that doggedness of 

probiotic is observed in synbiotic preparation both with reference to 

location in the colon (Rastall and Maitin, 2002) and on the duration of 

healthy effects after termination of product use (Roberfroid, 1998). 

Moreover, two different portions i.e small and large intestine of 

gastrointestinal tract can be addressed with the target synbiotic food 

(Holzapfel and Schillinger, 2002). There are some reports stating that 

synbiotic preparations can particularly reduce the risk of colon cancer. In 

an efficacy study on rats, this claim was checked by Gallaher and Khil 

(1999) who studied if there was decrease in number of aberrant crypt foci 

(cancer precursors). It was found though fructo-oligosaccharides and 

Bifidobacteria fed alone did not show any affect but when synbiotic was 

administered, aberrant crypt foci were decreased in five out of six 

subjects. Fermia et al. (2002) has presented an example of synergistic effect 

of synbiotics in a study, where different elements acted on different sites 

with improved immune modulation. In another study to verify synbiotic 

effect, Lactobacillus acidophilus 74-2 was administered with fructo- 

oligosaccharide in a milk-based product, to the second vessel 

(duodenum/jejunum) of the SHIME (Simulator of the Human Intestinal 

Microbial Ecosystem) reactor, which is in-vitro imitation of the microbial 

ecosystem of human intestine. The results worth noticing were increase of

Bifidobacteria, when there was the addition of L. acidophilus 74-2 with 

fructo-oligosaccharides. Furthermore, key positive changes such as rise in 

the production of volatile fatty acids and a significant increase of β- 

galactosidase activity was observed, whilst the activity of β- 

glucuronidase, known to be detrimental, was reduced (Gmeiner et al., 

2000). 

There are several grounds for utilizing oligosaccharides as prebiotic 

in synbiotic preparations. To begin with, prebiotics are non-living food 

components, meaning thereby that they do not cause the troubles linked 

with the use of probiotic strains in foods, in the context of microbial 

survival. Correspondingly, since most of the prebiotics are carbohydrate 

in nature, they can be supplemented to a vast range of food products 

including confectionary and baked foods as well as more traditional 

fermented milk food products and fruit drinks. Keeping in view the above 

stated information, the synbiotic food in this study was XOS enriched 

yogurt in which no strain was externally incorporated, rather yogurt’s 

indigenous flora behaved as probiotic part. A synergistic effect is 

accomplished through the addition of XOS in yogurt for getting healthier 

yogurt. 

2.5.2. Yogurt as Synbiotic Food 

As reported by Codex Alimentarius, yogurt is a coagulated milk 

product that results from fermentation of lactic acid in milk by 

Lactobacillus bulgaricus and Streptococcus thermophilus (Meydani, 2000). It is 

one of most popular fermented dairy product having good quality 

proteins and live microbes which are indigenously present in it. It is 

widely accepted among consumers because of its flavor and aroma with 

nutritional value. Many studies show that most therapeutic and

immunological effects of yogurt are associated with presence of LAB in it. 

Improved number of LAB in the intestine may curb the growth of 

pathogenic bacteria (Gilliland et al., 1978; Prajapati et al., 1986), which in 

turn adds to reduce infection by acting as prophylaxis (Friend and 

Shahani, 1984). 

Generally, yogurt manufacture involves fortification of milk with 

dairy ingredients to raise the concentration of protein to 40–50 g protein 

per kg. Stabilizers are sometimes added to improve the texture. The milk 

is then homogenized and heated to 90°C for 10 min, cooled to the 

fermentation temperature (40-43°C) and inoculated. In literature, there has 

been the use of whey protein concentrate (WPC) reported besides the 

traditional use of skim milk powder to fortify milk (Sodini et al., 2005). 

Improved water holding capacity and significant rheological differences 

were observed, when comparison of WPC fortified yogurt was made with 

that of control treatment. In addition to imparting better physico-chemical 

properties, some additives might help in enhancing flavor, nutritional 

status and survival of probiotic culture. Besides conventional starter 

cultures, probiotic bacteria are now often included in yoghurts intended 

to contribute to the health through maintaining a valuable balance of 

intestinal bacterial populations. The probiotics bacteria are primarily 

selected from the genera Lactobacillus and Bifidobacterium, both are 

constituent of the normal human intestinal microflora (Mitsuoka, 1982; 

Tannock, 1995). Influence of whey powder, cysteine, whey protein 

concentrate, acid casein hydrolysates, or tryptone addition on the viability 

of Streptococcus thermophilus, Lactobacillus acidophilus, and Bifidobacteria was 

observed by Dave and Shah (1998). The viability of Bifidobacteria was 

superior to a variable extent by the addition of cysteine, whey protein 

concentrate, acid casein hydrolysates, or tryptone, but whey powder 

proved to be futile in improving their viability.

The use of prebiotics is another way to boost the population of 

probiotic bacteria amongst the intestinal microbiota. Prebiotics are non- 

digestible dietary components that selectively stimulate the growth 

and/or activity of indigenous probiotic bacteria in the intestinal tract 

(Gibson and Roberfroid, 1995). The well-known prebiotics so far have 

been carbohydrates like lactulose, inulin, and various oligosaccharides 

(Crittenden, 1999). Intestinal bacterial populations are altered through the 

consumption of these non-digestible ingredients, particularly promoted 

proliferation of Bifidobacteria (Fuller and Gibson, 1997). Further, in-vitro 

experiments by Wang et al. (1999) and some studies on rodents (Kleessen 

et al., 1997; Brown et al., 1998) including rats are associated with human 

gut microflora (Silvi et al., 1999), and in pigs (Brown et al., 1997), have 

illustrated that resistant starch can also encourage the Bifidobacteria 

proliferation in the intestinal tract, and consequently might have the 

potential to act as a prebiotic in humans. 

The evident potential for synergy between probiotics and prebiotics 

is the basis for developing a synbiotic yogurt containing XOS as a 

prebiotic component and indigenous cultures of yogurt, serving as 

probiotic ingredients.

Chapter 2 REVIEW OF LITERATURE

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