6

CHAPTER 2

REVIEW OF LITERATURE

This chapter deals with the review of pertinent literature on the lactulose,

process technologies of lactulose production and its application in food and

pharmaceutical industries.

2.1 Lactulose

Lactulose is a non-caloric, synthetic disaccharide formed from one molecule

each of fructose and galactose. Technologically, lactulose can be produced by the

isomerization of lactose molecule in which a new ketose sugar from aldose by

regrouping the glucose residue to the fructose molecule (Aider and Damien, 2007). It is

widely used in pharmaceutical and food industries because of its beneficial effects on

human health. Lactulose is non-digestible that may beneficially affect the host by

selectively stimulating the growth of health promoting bacteria such as such as

Bifidobacterium bifidum, B. adolescentis, Lactobacillus acidophilus, L. casei and

Bacteroides vulgatus in the colon and can improve health of the host.

The demand for lactulose is expected to increase, as rated by different surveys.

The interest in lactulose has increased considerably in recent years, as they are potential

candidates for many commercial applications in food and pharmaceutical sectors. With

increasing demand, the annual production of lactulose increases to fulfill the

requirement of consumers. The total annual global productions of lactulose in

1994/1995 are estimated to be 20,000 t and it increase upto 40,000 t in 2004 and

7

45,000 t in 2009. However, a highest estimate of lactulose production of over 50,000 t

annually has also been reported (Playne and Crittenden, 2009).

Two major manufacturers of lactulose are Solvey Pharmaceuticals and Morinaga

Milk Industry. Solvey group of manufacture are more concentrated towards

pharmaceutical application, while Morinaga’s production emphasizes mainly in food

market (Playne and Crittenden, 2009). Commercially lactulose is available either in

dried form or as syrup of 50-72% (w/v) lactulose. The Solvay Group of manufacturer

has been producing lactulose for over 40 years. They were the first lactulose producing

company in the world, and are today, with a market share of around 50%, the world's

largest manufacturers with production facilities in the Netherlands and Canada

(http://www.lactulose.com/). Fresenius Kabi Austria is also one of the world’s top

manufacturers and marketers of liquid and crystalline lactulose into pharmaceutical

application (http://www.lactulose.eu/).

2.2 Process Technologies for the Production of Lactulose

Lactulose can be produced by the isomerization of lactose by regrouping the

glucose residue to the fructose molecule (Kochetkov and Bochkov, 1967). The large

number of complex reagents, alkalies or enzyme can be used as catalyst for the

isomerization of lactose to lactulose. An ideal catalyst must have low cost, easy to

remove from the medium, environment friendly, safe and non-toxic. The methods used

for lactulose production can be divided into two principal groups; chemical and

enzymatic method.

2.2.1 Chemical Synthesis of Lactulose

Technology of lactulose production is mainly based on the isomerization

reaction of lactose in alkaline media. The process includes expensive separation and

8

purification steps to remove byproduct. The large number of chemical reagents such as

calcium hydroxide, sodium hydroxide, tertiary amines, aluminate, borate, etc. has been

used for the isomerization of lactose to lactulose. The catalyst used for lactulose

synthesis must have an attractive features like maximum level of isomerization with a

low reaction by-products, to be environmentally safe and non toxic, the cost of the

catalyst must be as possible low, but available in great quantity, must be easy to remove

from the medium, to give repetitive results of isomerization (Aider et al., 2007).

Whereas, the catalysts used for the isomerization of lactose have both positive and

negative aspects. The catalysts for chemically synthesis of lactulose could be divided

into three principal groups; alkalizing catalysts, complexing agents and ion-mediated

processes (Table 2.1).

Table 2.1 Different processes for the isomerization of lactose

Type of catalysis Example(s)

Alkaline Addition of alkaline catalyst like calcium, sodium or potassium

hydroxide, triethylamine, magnesium oxide and sodium

hydrosulfite, heterogenous catalysts

Complexing

Addition of complexing agents like borate or aluminate in

combination with alkaline catalyst, sodium aluminate

Ion-mediated Employment of electroactivation and ion exchange

chromatography

(Source: Schuster-Wolff-Bühring et al., 2010)

2.2.1.1 Alkalizing catalysts

In alkaline condition, lactulose is synthesized from lactose

(4-O-β-D-galactopyranosyl-D-glucose) via the Lobry de Bruyn-Alberda van Ekenstein

molecular rearrangement in which the glucose moiety of lactose is isomerised to

fructose (Andrews and Prasad, 1987; Andrews, 1989). It was first synthesized by

9

heating a solution of lactose solution and lime water, prepared from calcium hydroxide

and heated at 35 °C for several days (Montgomery and Hudson, 1930). After that, a new

method has been developed, in which isomerization of lactose solution (60%) was

carried out in presence of calcium hydroxide (0.1%) under the temperature of

100-102 °C and reaction time of 15 min (Yakovleva, 1963; Matvievsky, 1979; Russian

Patent No 1392104, 1985).

In addition to above, other alkaline catalysts like triethylamine, sodium

hydroxide, magnesium oxide and sulfites have also been employed successfully for the

isomerization of lactose at pH 10-12 of the reaction mixture (Parrish, 1970; Carobbi et

al., 1985; Zokaee et al., 2002). At elevated reaction temperatures (70-100 °C), a

maximum lactulose production and lactulose yields has been achieved within few hours

of reaction time, after that a subsequent drop in lactulose concentration was observed.

Furthermore, to maintain maximum yield of lactulose both isomerization and

degradation were stopped by cooling and lowering the pH.

Lactulose has also been synthesized using milk ultrafiltrate as source of lactose

and egg shell as catalyst, is proposed as an alternative means for the lactulose

production by utilizing these industrial wastes (Montilla et al., 2005a). The optimal

production of lactulose (1.18 g/100 mL) was reached at 98 °C within 60 min of reaction

time with low levels of secondary products (epi-lactose, galactose and organic acids).

Further, egg shell powder has been replaced by other effective calcium carbonate-based

catalysts oyster shell powder and limestone for lactose isomerisation. The maximum

yield of 18-21% lactulose was achieved under the reaction conditions of 12 mg/mL

catalyst loading, reflux time of 120 min at 96 °C (Paseephol et al., 2008). A simplified

scheme of lactose alkaline isomerisation was shown in Figure 2.1.

10

Figure 2.1 Alkaline isomerization of lactose (Source: Montilla et al., 2005a)

The kinetics for the isomerization of lactose to lactulose in presence of sodium

hydroxide at constant and variable pH conditions has been studied by Hashemi and

Ashtiani (2010). The effect of different parameters including temperature, pH and time

on the reaction rate, conversion, sugar concentration and maximum conversion of

lactose to lactulose were investigated. The optimal process conditions for lactulose

synthesis has been observed as temperature of 70 °C, pH 11 and reaction time of about

60 min.

2.2.1.2 Complexing Agents

Lactulose has been prepared by employing the equimolar amounts of lactose and

boric acid, as a complexing reagent in water with triethylamine as catalyst, which

favour to form ketose sugars and restrict degradative side-reactions. A maximum yield

of 87% has been achieved with boric acid by adding triethylamine to a lactose solution

up to pH 11 and keeping the solution for 4 h at 70 °C (Hicks and Parrish, 1980).

11

Lactulose has also been synthesized by the isomerization of lactose present in

cheese whey was carried out by the addition of boric acid into the ultrafiltrate permeate

(Hick et al., 1984). The kinetics of lactulose formation in presence of borate and sodium

hydroxide has been studied to determine the optimum reaction parameters. The

optimum operating conditions were found to be pH 10.5-11.5, a temperature of

70-75 °C and a molar ratio of lactose to boric acid of 1.0. Under these conditions for the

isomerization, equilibrium was about 75%, which is lower than with the use of

triethylamine as a catalyst (Kozempel and Kurantz, 1994a). A continuous processing of

lactulose has also been investigated by Kozempel and Kurantz (1994b), in which the

integration of the LA-rearrangement with borate and sodium hydroxide and an average

yield of 78% have been achieved with a 20% (w/w) lactose solution. A comparative

study has been performed for the formation of lactulose using three different catalytic

systems, i.e. sodium hydroxide, sodium hydroxide and boric acid and sodium aluminate

(Zokaee et al., 2002). Under similar reaction conditions, high lactulose were formed

with hydroxide and boric acid, whereas in case of aluminate less lactulose was formed

and its degradation was increased.

2.2.1.3 Ion-Mediated Processes

Ion-mediated processes are based on the indigenous generation of alkaline

conditions without the addition of further catalysts. As mentioned above, the production

of lactulose has been completed in two steps: use of chemicals (hydroxide, borates,

aluminates, etc.) for the isomerization of lactose and additional step, i.e. purification by

ion exchange resins. An anion exchange resins have been used to increase the level of

lactose isomerization by exploiting OH- ions exchange between solution in reaction and

resins and to simplify the process of lactulose production using the same resins for

demineralization of the end product (Russian Patent No 2101358, 1994).

12

Lactulose has also been synthesized from the isomerization of whey lactose by

ion exchange resin method. For this, alkalization of whey has been carried out by the

dissociation of H2O molecules into hydroxide ions and oxonium ions in an electric cell,

separated through a semipermeable membrane into anode and cathode chamber. The

rates of isomerization of lactose to lactulose as well as the temperature were apparently

influenced by the settings of voltage (Khramtsov et al., 1999; Khramtsov et al., 2003).

Another technology for the alkalization of whey was achieved by circulation over strong

ion exchange resins activated with hydroxide ions. The exchange of ions like chloride

and the release of hydroxide ions from the resin increased the pH of whey and this

caused the LA-transformation of lactose into lactulose (Khramtcov et al., 2004).

2.2.2 Enzymatic Methods of Lactulose Production

Lactulose can also be produced by enzymatic methods, but these methods are

not being currently used for commercial production. Enzymatic synthesis of lactulose is

commonly carried out with two enzymes: β-galactosidase and glycosidase.

β-Galactosidase (EC.3.2.1.23) is well known biocatalyst for trans-galactosylation

reaction and for the synthesis of lactose based derivatives including galacto-

oligosaccharides (Panesar et al., 2006; Panesar et al., 2010). Free β-galactosidases as

well as whole cell and immobilized form can be used for lactulose production.

The enzyme β-galactosidase (EC.3.2.1.23), most commonly known as lactase,

which hydrolyses lactose into its monomers, i.e. glucose and galactose has potential

applications in food processing industry. Due to its hydrolytic activity, it is extensively

used by the dairy industry, mainly for reducing lactose content or improving storage and

processing characteristics. Later, it was found that this enzyme also formed glycosidic

bonds between two saccharides by removing a water molecule in a reaction called

transgalactosylation. As a result of this, β-galactosidase has also been used to synthesize

13

galacto-oligosaccharide (GOS) and lactulose (Lee et al., 2004; Otieno et al., 2010). The

hydrolysis reaction and reaction pathway for transglycosylation using β-galactosidase

are shown in Figure 2.2 and Figure 2.3, respectively.

O

OH

HO

OH

CH2OH

OO

HO

OH

CH2OH

OH

HOO

HO

OH

CH2OH

OH

O

OH

HO

OH

CH2OH

OH

Lactose

-D-Galp-(1 D-Glc

D-GlcD-Gal

-D-Galactosidase

Figure 2.2 Hydrolysis of lactose to glucose and galactose by -galactosidase

The enzyme β-galactosidase catalyzes not only the hydrolysis of β-glycosidic

linkages of lactose, i.e. transferring galactose to a hydroxyl group of water and resulting

in the liberation of D-galactose and D-glucose, but also a transgalactosylation reaction,

i.e., transferring galactose to the hydroxyl groups of the D-galactose or the D-glucose

moiety in lactose (Jorgensen et al., 2001; Prenosil et al., 1987a; Zarate and Lopez-

Leiva, 1990). In transgalatosylation reactions, the linkage between the galactose units,

the transgalatosylation efficiency and the final product formed, depend on the reaction

conditions and source of enzyme used. It has been observed that β-1,4 bonds are

generally formed by enzymes from B. circulans (Mozaffar et al., 1984), Cryptococcus

laurentii (Ozawa et al., 1989), Bifidobacterium bifidum (Dumortier et al., 1994) and

Kluyveromyces lactis (Lee et al., 2004), whereas, β-1,6 bonds are formed by enzymes

from A. oryzae and S. thermophilus (Matsumoto, 1990; Sako et al., 1999). Additionally,

14

the proportion of transgalactosylation to hydrolysis reactions also varies with the

sources of enzymes used. β-Galactosidase from E. coli or A. niger, strongly promote the

hydrolytic activity, whereas, a strong transglycosylation activity were shown by

β-galactosidase from Aspergillus oryzae or Bacillus circulans (Mahoney, 1998).

Figure 2.3 Reaction pathway for transglycosylation and hydrolysis using

β- galactosidase (Source: Neri, 2008)

The transglycosylation reactions are more preferably carried out at high

substrate concentrations but the low solubility of lactose is the main hinder for the

efficiency of the reaction. To overcome this, high temperature was used for

transglycosylation reactions. Increased temperature favors high substrate solubility,

resulting in increased transgalactosylation activity (Hung and Lee, 2002), due to the

presence of excess glycoside acceptor at high concentrations of substrate, that compete

with water for the galactosyl-enzyme intermediate (Lee et al., 2004). In the

transgalactosylation reaction, galactose acts as a non-competitive inhibitor of

Galactose Acceptor

Sugar Enzyme Galactose

Acceptor

Sugar

Enzyme Lactose + Enzyme Lactose

Glucose

Enzyme Galactose

Galactose

Lactose Hydrolysis

Acceptor

Sugar

H2O

Transgalactosylation Reaction

15

β-galactosidase as it competes with lactose for the active site (Shukla and Chaplin,

1993).

2.2.2.1 Sources of β-Galactosidase

The enzyme can be obtained from a wide variety of sources such as micro-

organisms, plants and animals (Table 2.2), however, according to their sources, their

properties differ markedly (Finocchiaro, 1980; Richmond et al., 1981; Mahoney, 1998).

Micro-organisms offer various advantages over other available sources such as easy

handling, higher multiplication rate and high production yield. As a result, micro-

organisms have been most preferred as a source of β-galactosidases.

The microbial β-galactosidase can be obtained from various sources like

bacteria, yeast and fungi. The enzymes available commercially are derived from safe

sources, principally, the yeast Kluyveromyces fragilis, K. lactis and Candida

pseudotropicalis, the fungi Aspergillus niger and A. oryzae and a Bacillus species

closely related to Bacillus stearothermophilus (Panesar et al., 2006). The most widely

used microbial sources are Kluyveromyces sp. and Aspergillus sp.

Bacterial Enzymes: The enzyme β-galactosidase can be produced by a large

number of bacteria but Streptococcus thermophilus and Bacillus stearothermophilus are

considered as potential bacterial sources. The enzyme from Escherichia coli serves as a

model for understanding the catalytic mechanism of -galactosidase action, but it is not

considered suitable for use in foods due to toxicity problems associated with the host

coliform (Mahoney, 1997). Hence, the -galactosidase from E. coli is generally not

preferred for use in food industry (Joshi et al., 1989; Stred’ansky et al., 1993; Mahoney,

2003).

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Table 2.2 Sources of -galactosidase

Plants Animal organs Bacteria Fungi Yeast Peach, Apricot, Almond, Kefir

grains, Tips of

wild roses, Alfalfa seeds,

Coffee beans

Intestine, Brain, Skin tissue, Bovine liver

Alicyclobacillus acidocaldarius subspp. rittmannii, Arthrobacter spp. Bacillus acidocaldarius, B. circulans, B. coagulans, B. subtilis, B. megaterum, B. stearothermophilus, Bacteriodes polypragmatus Bifidobacterium bifidum, B. infantis Clostridium acetobutylicum, C. thermosulfurogens Corynebacterium murisepticum Enterobacter agglomerans, E. cloaceae Escherichia coli, Klebsiella pneumoniae Lactobacillus acidophilus, L. bulgaricus, L. helviticus, L. lactis, L. kefiranofaciens, L. sporogenes, L. themophilus, L.delbrueckii Leuconostoc citrovorum Pediococcus acidilacti, P. pento Propioionibacterium shermanii Pseudomonas fluorescens Pseudoalteromonas haloplanktis Streptococcus cremoris, S. lactis, S. Thermophius, Sulfolobus solfatarius Thermus rubus, T. aquaticus, Trichoderma reesei, Vibrio cholera, Xanthomonas campestris

Alternaria alternate, A. palmi Aspergillus foelidis, A. fonsecaeus, A. carbonarius, A. oryzae Auerobasidium pullulans Curvularia inaequalis Fusarium monilliforme, F. oxysporum Mucor meihei, M. pusillus Neurospora crassa Penicillum conescens, P. chrysogenum, P. expansum Saccharopolyspora

rectivergula Scopulariapsis sp. Streptomyces violaceus

Bullera singularis Candida sp. pseudotropicalis Saccharomyces anamensis, S. lactis,

S. fragilis Kluyveromyces

bulgaricus, K. fragilis, K. lactis,

K. marxianus

(Source: Brandao et al., 1987; Richmond et al., 1981; Adams et al., 1994; Berger et al., 1995; Mahoney, 1997; Nagy et al., 2001; Cho et al., 2003; El-Gindy, 2003; Hoyoux et al., 2001; Gul-Guven et al., 2007; Tang et al., 2011)

17

The activity and the stability of the partially purified β-galactosidases from

Thermus sp strain T2 and K. fragilis have been compared (Ladero et al., 2002). Both

enzymes showed a remarkable hydrolytic activity and a weak transgalactosylation

activity, even in the presence of high concentrations of lactose. The media and process

conditions of Bifidobacterium longum CCRC 15708 have been optimized and a

maximum β-galactosidase activity of 18.6 U/mL has been obtained after 16 h

(Hsu et al., 2005). It has been observed that the growth and production of

β-galactosidase by Bifidobacterium longum CCRC 15708 in a fermenter was influenced

by cultivation temperature, pH and agitation speed (Hsu et al., 2007). A maximum

β-galactosidase activity of 36.7 U/mL was achieved in 10 h in a jar fermenter having the

pH, temperature, and the agitation speed controlled at 6.5, 37 °C, and 100 rpm,

respectively.

The efficiency of different cell disruption methods on the extraction of

intracellular β-galactosidase enzyme from Streptococcus thermophilus and

Lactobacillus delbrueckii subsp. thermophilus has been tested (Tari et al., 2010).

Lysozyme enzyme treatment was determined as the most effective method, which

resulted in approximately 1.5 and 10 times higher enzyme activity than glass bead and

homogenization treatment, respectively. The recombinant β-galactosidase (BgalC), a

β-galactosidase gene of Thermotoga maritima has been cloned and expressed in

Escherichia coli exhibited maximum activity at an optimal pH of 5.5 and an optimum

temperature of 80 °C (Katrolia et al., 2011). The enzyme has showed important

properties like stability over a broad pH range of 5.0-9.0 and thermostability up to

75 °C. The effect on the growth and β-galactosidase activity of Propionibacterium

acidipropionici Q4 has been studied by the sequential addition of lactose and lactate as

first or second energy sources (Zarate and Chaia, 2012). The highest β-galactosidase

18

activity has been observed in a medium supplemented with lactate only, whereas, higher

final biomass has been obtained in a medium with lactose. A marked increase of the

intracellular pyruvate level was observed in case of lactate as a second energy source,

followed by lactate consumption and increase of specific β-galactosidase activity

whereas lactose consumption became negligible. The study on the optimization of

various physical (incubation time, temperatures and pH) and chemical parameters

(carbon, nitrogen and metal ions) have also been carried out to maximize the production

of β-galactosidase from isolated Bacillus subtilis (Gouripur and Kaliwal, 2013). The

production of β-galactosidase was found to be optimum with xylose, yeast extract and

MgSO4.7H2O ion at temperature of 37 ºC and pH 7.0 after 48 h of incubation.

Fungal Enzymes: The optimum pH range for the fungal enzyme is 2.5-5.4,

which makes them suitable for processing of acid whey and its ultrafiltration permeate.

The optimum temperatures for these enzymes are high and can be typically used at

temperatures up to 50 °C. The purification of -galactosidase from different fungal

sources has been carried using a variety of purification techniques.

β-Galactosidase produced by submerged culture of Aspergillus japonicus

showed 2.95 U/mg protein specific activities with an approximate molecular weight of

27 kDa (Saad, 2004). Hypocrea jecorina has ability to grow on lactose due to the

formation of an extracellular glycoside hydrolase family 35 β-galactosidase encoded by

the Bga1 gene. Further, it was observed that the induction level of Bga1 was influenced

by the different growth rates attainable on these carbon sources (lactose, D-galactose

and galactitol) and it was founded that galactitol is the actual inducer of Bga1 formation

during growth on D-galactose in H. jecorina (Fekete et al., 2007).

The solid state fermentation (SSF) for the production of β-galactosidase by

isolated Aspergillus oryzae has also been studied and the maximal growth and

19

β-galactosidase production by A. oryzae has been observed with wheat bran and rice

husk in 1: 1 ratio, glucose (12.5%, w/w), sodium nitrate (1%, w/w), moisture content of

90%, pH 5.0 at 30 ºC for 7 days using 10 mL spore suspension (1x107 spores/mL) as

inoculum’s size (Nizamuddin et al., 2008). A marine fungal isolate Aspergillus flavus

was screened and grown in the media containing lactose for the production of

intracellular β-galactosidase by permeabilization of the cell membrane with different

chemical reagents for the extraction of β-galactosidase (Anumukonda and Tadimalla,

2009). The specific activity of purified enzyme was found to be 18.96 U/mg. The

fungus Aspergillus flavus MTCC 9349 has also been studied for β-galactosidase

production. The effect of media and process parameters were studied by applying

statistical methods. Under the optimized process conditions, the yield of enzyme was

found to be 499.63 U/g dry cell weight (Anumukonda and Tadimalla, 2010).

The β-galactosidase has been efficiently purified from an acidophilic fungus,

Teratosphaeria acidotherma AIU BGA-1, using affinity chromatography (Isobe et al.,

2013a). The enzyme exhibits high activity from an extremely acidic pH region (1.5) to

neutral pH region (7.0) and the optimal activity were observed at pH 2.5-4.0 and 70 °C.

Furthermore, it was observed that the isolated strain Teratosphaeria acidotherma AIU

BGA-1 produced four intracellular β-galactosidases with different pH activity profiles,

in which three forms were found to be acidophilic and stable from extremely acidic to

neutral pH region. However, the forth one was alkalophilic and unstable at acidic pH

region (Isobe et al., 2013b).

Yeast Enzymes: Yeast has been considered as an important source of

-galactosidase from industrial point of view. With neutral pH optima, these are well

suited for hydrolysis of lactose in milk and are widely accepted as safe for use in foods.

Much work has been carried on the production of β-galactosidase from different yeast

20

strains for its potential use. The most commercially available yeast -galactosidase

under the trade name of Maxilact (DSM Food Specialties, Delft, The Netherlands) and

Lactase (SNAM Progetti, Italy) are preparations extracted from Kluyveromyces lactis,

and LactozymTM

(Novo, Nordisk A/S, Bagsvaerd, Denmark) from Kluyveromyces

fragilis (German, 1997; Roy and Gupta, 2003; Mahoney, 2003).

Various strains of Kluyveromyces sp. from kefir has been tested for the

production of enzyme in lactose and natural whey-based medium (Thigiel and Deak,

1989). K. marxianus and K. fragilis strain have shown the maximum enzyme yield with

specific activities of 1.90 U/mg and 1.65 U/mg, respectively. Production of enzyme

using hybrid strain of K. marxianus and Candida macedoniensis has been reported by

Molitor et al. (1990). The optimal enzyme production has been reported at 28 °C, pH

3.0 and using an inoculum of 4% (v/v) within 32-38 h of incubation period.

The production of the enzyme from K fragilis using cheese whey or

deproteinized cheese whey at pH 5.5 and 30 °C has been carried out by Nunes et al.

(1993). The maximum enzyme activity was observed between the pH ranges of 6.6-6.8

at 30-37 °C. The study has also been reported on the use of solid state fermentation for

the production of lactase from corn grits or wheat bran moistened with deprotienized

milk whey by K. lactis using full factorial design (Becerra and Siso, 1996).

The optimization of pH, temperature and inoculum ratio for the production of

β-galactosidase by Kluyveromyces marxianus CDB 002 were carried out using

sugarcane molasses (100 g/L) in a lactose-free medium. The optimum conditions for

β-galactosidase synthesis using sugar cane molasses were temperature of 30-34 °C and

an inoculum ratio of 1% (v/v), an initial pH of 5.5 (Furlan et al., 2001). The

optimization of β-galactosidase production by K. lactis, using deprotienized whey as

fermentation medium has been reported. The optimized condition for the enzyme

21

production was reported as: temperature 30.3 °C, pH 4.68, agitation speed 191 rpm and

fermentation time 18.5 h (Ramirez-Matheus and Rivas, 2003).

The study on the effect of oscillating dissolved oxygen tension on metabolism of

Kluyveromyces marxianus was carried out in terms of productivity of β-galactosidase. It

was observed that the oscillations at the shortest period (300 s) resulted in higher

specific enzyme activity (2800 U/g) and volumetric enzyme activity of 31,700 U/L

(Cortés et al., 2005). Response surface methodology has also been applied for the

optimization of β-galactosidase production using K. lactis NRRL Y-8279 (Dagbagli and

Goksungur, 2008) and maximum specific enzyme activity of 4218.4 U/g was obtained

at pH 7.35, after incubation period of 50.9 h.

The optimization of process conditions has also been carried out for the optimal

production of β-galactosidase from whey using Kluyveromyces marxianus NCIM 3551

and the optimum β-galactosidase production was obtained with 16 h old culture at the

inoculums size of 10% over the incubation period of 20 h at pH 5.0 at 25 °C (Gupta and

Nair, 2010). Isolated psychrotolerant yeast Guehomyces pullulans 17-1 has ability to

produce both extracellular and cell-bound β-galactosidase. The isolated yeast strain

produced over 17.2 U/mL of β-galactosidase activity within 120 h at the flask level,

whereas, β-galactosidase activity over 25.3 U/mL within 144 h during the 2 L

fermentation at the optimum cultivation conditions (Song et al., 2010).

Further, Guehomyces pullulans 17-1 cells were mutated by using

nitrosoguanidine in order to enhance β-galactosidase production (Xu et al., 2011).

Under the optimized medium and cultivation conditions, the mutant produced

29.2 U/mL and 48.1 U/mL at the flask level and 2 L fermentor, respectively. The study

on optimization of medium composition and growth conditions were carried out to

achieve increased production and secretion of β-galactosidase enzyme by probiotic

22

yeast spp. (Meera et al., 2013). The increase in concentration of β-galactosidase was

observed with an increased lactose concentration upto 7%, the optimum pH and

temperature was found to be 5.0 and 35 °C, respectively.

β-Galactosidase produced by micro-organisms is both intracellular and

extracellular in origin. Moreover, in case of intracellular enzyme, especially in case of

yeast cells, tough cell wall is the main barrier to release the enzyme from the cells. To

overcome this permeability barrier, the permeabilization technologies can be applied.

2.2.2.2 Permeabilization of Microbial Cells for β-Galactosidase Activity

The permeabilization methods are simple, rapid and allow the assay of enzymes

under the natural environment of the cell. Thus, cell permeabilization can be used as an

important tool in the biotransformation processes that can be an inexpensive alternative

to purified enzyme systems. In this, cell structure is altered to make it porous to allow

small molecules, such as substrates or products, to cross freely and the cells are spared

from the harsh treatment associated with disruption of cells (Naglak et al., 1990).

Different chemical agents (Such as organic solvents and detergents) have been applied

to increase lactose permeability of the microbial cells for β-galactosidase activity

(Table 2.3).

Organic Solvents: Permeabilization of yeast cells for the release of

β-galactosidase has been carried out with the solvent concentrations of 20 to 95%.

Different solvents (isopropanol, ethanol and methanol) have been found to be effective

for the release of enzymes into buffer (Fenton, 1982). The permeabilization of

Kluyveromyces cells for β-galactosidase activity has been reported by using various

organic solvents (Decleire et al., 1987 a). Different concentrations of solvents such as

acetone, ethanol, propanol, isopropanol, toluene/ethanol, n-butanol, sec-butanol,

tert-butanol, isobutanol and dimethylsulphoxide have been compared with respect to

23

their permeabilization efficiencies. The minimum concentration of solvents to be used

to obtain a good permeabilization was 10% n-butanol, 20% propanol, 30% isopropanol,

30% tert-butanol, 40% ethanol, 40% acetone and 70% dimethylsulphoxide.

Furthermore, the treatment of cells with surfactant Brij 35 with an addition of small

amount of toluene in ethanol (4: 96) resulted a high enzymatic activity. The mixture of

chloroform and ethanol has also been found effective in the permeabilization of

Kluyveromyces cells (Champluvier et al., 1988a).

The effect of incubation time, incubation temperature and the concentration of

both cells and solvents (chloroform, toluene, and ethanol) of the permeabilization of

Kluyveromyces lactis (CBS 683) cells in terms of β-galactosidase activity have also

been studied for know the efficiency of these solvents. Maximum enzyme activity has

been achieved with chloroform or with a mixture of chloroform and ethanol at 5-37 °C.

However at temperature of 37 °C, the process was rapid and permeabilization occurred

in 5 min or less. Similar results have also been observed with toluene and a mixture of

toluene and ethanol (Flores et al., 1994).

Permeabilization with ethanol increased the intracellular β-galactosidase activity

up to 240-fold as compared to that of untreated cells. Permeabilized immobilized cells

hydrolyzed milk whey lactose more rapidly than untreated cells and more than 90%

milk whey lactose was hydrolyzed in a packed-bed bioreactor at 37 °C (Gonzalez-Siso

and Suarez-Doval, 1994). The permeabilization of S. thermophilus and L. delbrueckii

sub sp. bulgaricus cultures was carried with ethanol. Upon the treatment of cells with

30-55% (v/v) ethanol, a 100% loss in viability was observed and upto 15-fold increase

in measurable β-galactosidase activity in both the cultures (Somkuti et al., 1998). The

ethanol permeabilized cells have also been used for the saccharification of milk whey in

packed bed bioreactors (Bacerra et al., 2001).

24

Table 2.3 Different permeabilizing agents used for -galactosidase

Micro-organism(s) Permeabilizing agent(s) Reference(s)

Kluyveromyces bulgaricus

Propanol, isopropanol, n-butanol, tert-butanol, ethanol,

acetone, dimethylsulphoxide

Decleire et al. (1987a)

K. fragilis Cetyltrimethylammonium bromide (CTAB)

Joshi et al. (1987)

K. fragilis Digitonin Gowda et al. (1988)

K. lactis Chloroform-ethanol mixture Champluvier et al. (1988)

K. fragilis Digitonin Joshi et al. (1989)

K. fragilis Isoamyl alcohol Castillo and Casas (1900)

K lactis Digitonin, isopropanol, ethanol,

tert- butanol, ethanol: toluene, chloroform,

Siso et al. (1992)

K. fragilis Ethanol Gonzalez-Siso and Suarez-

Doval (1994)

Streptococcus thermophilus

Sodium dodecyl sulphate (SDS), Sodium deoxycholate

Triton X-100,

Somkuti and Steinberg (1994)

K. lactis Chloroform, ethanol and toluene

Flores et al. (1994)

K. fragilis Cetyltrimethylammonium

bromide

Bachhawat et al. (1996)

S. thermophilus Ethanol Somkuti et al. (1998)

Lactobacillus delbrueckii Ethanol Somkuti et al. (1998)

K. lactis Ethanol Becerra et al. (2001)

K. marxianus Ethanol, propanol, isopropanol, n-butanol, dimethylsulphoxide,

toluene, acetone, choloroform,

CTAB, digitonin

Panesar (2004)

K. lactis Ethanol Lee et al. (2004)

K. marxianus Mixture of ethanol and toluene Kumari et al. (2011)

The permeabilization of K. lactis cells has been carried out with 50% ethanol

concentration and these permeabilized cells were further used for the production of

lactulose from lactose and fructose and upto 1.3- and 2.1-fold increases in lactulose

concentration and productivity was observed as compared with untreated washed cells

(Lee et al., 2004). Various process parameters (solvent concentration, incubation time,

incubation temperature) have been optimized for the permeabilization of K. marxianus

using Response Surface Methodology (RSM) to get maximum optimal β-galactosidase

activity. The optimum operating conditions for permeabilization process were 49.6%

25

(v/v) ethanol concentration, 23 °C temperature and process duration of 18 min.

(Panesar, 2008).

Detergents: The permeabilization of Kluyveromyces fragilis cells to lactose

with cetytrimethylammonium bromide resulted in the increase of enzyme activity by

480 fold with a detergent concentration of 0.1% at 4 °C in 5 min (Joshi et al., 1987).

The yeast, K. fragilis was permeabilized using a low-molecular-weight substrates using

digitonin. β-Galactosidase of yeast has been found to be much higher in the

permeabilized cells as comparison to that of untreated cells (Gowda et al., 1988). The

maximum permeabilization was found at 37 °C for 15 min with 0.1% digitonin in 0.1M

potassium phosphate buffer.

Digitonin has also been used as a permeabilizing agent for the permeabilization

of Kluyveromyces fragilis to increase the enzyme activity of the yeast cells. The optimal

β-galactosidase activity was observed when the cells were treated with 0.1% digitonin at

room temperature for 30 min. The activity measured in these permeabilized cells was

400- to 500-fold greater than in the untreated cells and 25% more than in the cell-free

extract prepared by toluene autolysis (Joshi et al., 1989). The efficiency of various

permeabilizing agents (sodium dodecyl sulfate, Triton X-100, sodium deoxycholate, and

one commercial bile acid) has been tested for β-galactosidase in Streptococcus

thermophilus (Somkuti and Steinberg, 1994). Among these permeabilizing agents, cells

that exposed to oxgall or triton X-100 shown 15 times higher levels of β-galactosidase

activity than control cells and permeabilized cells released 87% of glucose available in a

5% lactose solution within 10 min at 50 °C. Bachhawat et al. (1996) has reported the

permeabilization of K. fragilis using cetyltrimethylammonium bromide (0.1%) at

24-26 °C.

26

In addition to permeabilization technology, the use of immobilization

technology can also make the process economical due to its several importances.

2.2.2.3 Immobilization of β-Galactosidase

The immobilization of β-galactosidase is an area of great interest because of its

potential benefits. The use of immobilization technology is of significant importance

from economic point of view since it makes reutilization of the enzyme in continuous

operation and also precludes the need to separate the enzyme from the reaction mixture

(Panesar et al., 2006). It can also help to improve the enzyme stability. Nowadays,

immobilized β-galactosidase is intensively being used for the production of prebiotics

like lactulose and galacto-oligosaccharides. The enzyme has been immobilized by

various methods such as physical absorption, entrapment, and covalent binding method

(Table 2.4).

Physical Adsorption: Physical adsorption is considered as the simplest method

of immobilization in which an enzyme is immobilized onto a water-insoluble carrier and

the biocatalysts are held to the surface of the carriers by physical forces (van der waals

forces). Frequently, however, additional forces are involved in the interaction between

carrier and biocatalyst principally hydrophobic interactions, hydrogen bridges and

heteropolar (ionic) bonds (Hartmeier, 1986). The advantage of this method is that, it is

simple to carryout and has little influence on the conformation of the biocatalyst.

However, the disadvantage of this technique is the relative weakness of the adsorptive

binding forces. Different inorganic (alumina, silica, porous glass, ceramics,

diatomaceous earth, clay, and bentonite) and organic (cellulose, starch, activated carbon

and ion-exchange resins, such as amberlite, sephadex, and dowex) support materials can

be used for enzyme adsorption. Further adsorption of enzyme may be stabilized by

glutaraldehyde, but this treatment can denature some proteins.

27

Table 2.4 Different sources of β-galactosidase and methods of immobilization

Immobilization

Method (s)

Source of

β-galactosidase

Immobilizing agent (s) Reference (s)

Physical

adsorption

A. oryzae Phenol-formaldehyde resin Yang and Okos (1989)

E. coli Chromosorb-W Bodalo et al. (1991)

B. circulans Polyvinyl chloride and Silica Bakken et al. (1992)

B. stearothermophilus Chitosan Di Serio et al. (2003)

A. niger Porous ceramic monolith Papayannakos and Markas

(1993) K. lactis CPC-silica and agarose Giacomini et al. (1998)

Thermus sp. T2 PEI- sepabeads, DEAE-

agarose

Pessela et al. (2003)

K. fragilis Cellulose beads Roy and Gupta (2003)

A. oryzae Celite and chitosan Gaur et al. (2006)

A. oryzae zinc oxide nanoparticles Husain et al. (2011)

A. oryzae Concanavalin A-Celite 545 Ansari and Husain (2012)

Entrapment K. bulgaricus Alginate using BaCl3 Decleire et al. (1987b)

E. coli Polyacrylamide gel Khare and Gupta (1988)

A. oryzae Nylon-6 and zeolite Pietta et al. (1989)

Thermus aquaticus Agarose bead Berger et al. (1995)

A. oryzae Spongy polyvinyl alcohol Cryogel

Rossi et al. (1999)

Penicillium expansum Calcium alginate El-Gindy (2003)

S. cerevisiae

A. oryzae

A. oryzae

Calcium alginate Alginate-gelatin beads and

crosslinked with glutaraldehyde

Haider and Husain (2007)

Freitas et al. (2011)

Covalent Binding Lactobacillus bulgaricus Egg shells Makkar and Sharma (1983)

A. oryzae Silica gel activated with TiCl3

and FeCl3

Kozhukharova et al. (1991)

E. coli (Recombinant

β-galactosidase)

Cyanuric chloride-activated

cellulose

Kery et al. (1991)

K. lactis Corn grits Gonzalez-Siso and Suarez-Doval (1994)

K. lactis Thiosulfinate/thiosulfonate Ovsejevi et al. (1998)

B. circulans Eupergit C (Spherical acrylic

polymer)

Hernaiz and Crout (2000)

K. fragilis Silica-alumina Ladero et al. (2000)

K. lactis Graphite surface Taqieddin and Amiji (2004)

A. oryzae Chitosan bead and nylon

membrane

Portaccio et al. (1998)

A. oryzae Cotton cloth and activated

with tosyl chloride

Albayrak and Yang (2002)

A. oryzae Amino-epoxy sepabead Torres et al. (2003)

A. niger Magnetic polysiloxane-polyvinyl alcohol

Neri David et al. (2009)

A. oryzae Polyvinylalcoheol hydrogel and

magnetic Fe3O4-chitosan as

supporting agent

Hronska et al. (2009)

Bacillus circulans Epoxy-activated acrylic support Torres and Batista-Viera

(2012)

Bacillus circulans Hierarchical macro-mesoporous

silica

Bernal et al. (2012)

28

Immobilization of -galactosidase on hydrophobic cotton cloth indicated that the

enzyme adsorbed on the cloth was about 50% active as free enzymes (Sharma and

Yamazaki, 1984). The immobilization of -galactosidase active yeast Kluyveromyces

fragilis and Kluyveromyces lactis onto chitosan showed an enzyme activity of

0.9-2.2 U/mg dry cell weight (Champluvier et al., 1988 b). Enzyme activity of

immobilized enzyme from K. fragilis was higher but the operational stability of

A. oryzae enzyme was 5-14 times higher depending upon the mode of immobilization

(Kminkova et al., 1988). When adsorption method was used, the highest activity was

obtained with yeast enzyme and support Ostsorb-DEAE. The enzyme from A. oryzae

immobilized on polyvinyl chloride (PVC) and silica gel membrane has been used for

the hydrolysis of lactose in skim milk in an axial-annular flow reactor (Bakken et al.,

1990). Further, maximum immobilization occurred at pH 5.5 and optimal results were

obtained with citrate/phosphate buffer during immobilization of -galactosidase from

E. coli by physical adsorption on chromosorb-W (Badalo et al., 1991). A novel reactor

consisting of -galactosidase from B. circulans immobilized on a ribbed membrane

composed of PVC and silica has also been used for skim milk lactose hydrolysis

(Bakken et al., 1992). The immobilization of partially purified Bullera singularis

β-galactosidase in Chitopearl BCW 3510 bead (970 GU/g resin) by simple adsorption

has also been carried out (Huyn-Jae et al., 1998).

The studies on the kinetic behaviour of β-galactosidase from Kluyveromces

marxianus (Saccharomyces) lactis, immobilized on to different oxide supports, such as

alumina, silica, and silicated alumina indicated that the immobilized enzyme activity

strongly depends on the chemical nature and physical structure of the support (Di Serio

et al., 2003). In particular, when the particle sizes of the support are increased, the

enzymatic activity strongly decreases. Immobilization of -galactosidase from Thermus

29

sp. preceded very rapidly onto PEI-Sepabeads and conventional DEAE-Agarose.

However, the adsorption strength was much higher in the case of PEI-Sepabeads

(Pessela et al., 2003).

A recombinant thermostable Bacillus stearothermophilus β-galactosidase was

immobilized onto chitosan using Tris (hydroxymethyl) phosphine (THP) and

glutaraldehyde, and a packed bed reactor was utilized to hydrolyze lactose in milk. The

thermostability and enzyme activity of THP-immobilized β-galactosidase during storage

was superior to that of free and glutaraldehyde-immobilized enzymes. The

THP-immobilized β-galactosidase showed greater relative activity in the presence of

Ca2+

than the free enzyme and was stable during the storage at 4 °C for 6 weeks,

whereas the free enzyme lost 31% of the initial activity under the same storage

conditions (Chen et al., 2009). Aspergillus oryzae β-galactosidase was immobilized on

an inexpensive bio-affinity support, concanavalin A-cellulose. Concanavalin A-

cellulose adsorbed and cross-linked β-galactosidase preparation retained 78% of the

initial activity. The optimum temperature was increased from 50 to 60 °C for the

immobilized β-galactosidase. The cross-linked adsorbed enzyme retained 93% activity

after 1 month storage while the native enzyme showed only 63% activity under similar

incubation conditions (Ansari and Husains, 2010).

The immobilized Aspergillus oryzae β-galactosidase on native zinc oxide (ZnO)

and zinc oxide nanoparticles (ZnO-NP) has been applied for the hydrolysis of milk and

whey lactose. It was observed that 54%, 63% and 71% milk lactose was hydrolyzed by

soluble, ZnO adsorbed and ZnO-NP adsorbed β-galactosidase in batch process after 9 h,

however, whey lactose was hydrolyzed to 61%, 68% and 81% under similar process

conditions, respectively (Husain et al., 2011).

30

Covalent Binding Method: Covalent binding is a conventional method for

immobilization. It can be achieved by direct attachment with the enzyme and the

support material through covalent bond formation (Trevan, 1988). Enzyme molecules

bind to support material via certain functional groups such as amino, carboxyl,

hydroxyl, and sulfydryl groups. These functional groups must not be in the active site. It

is often advisable to carry out the immobilization in the presence of its substrate or a

competitive inhibitor so as to protect the active site. Functional groups on support

material are usually activated by using chemical reagents such as cyanogen bromide,

carbodimide and glutaraldehyde.

Eggshells ground into pieces can be good carrier for immobilization of

-galactosidase because of its low cost, good mechanical strength and resistance to

microbial attack (Makkar and Sharma, 1983). Fungal enzyme from Aspergillus oryzae

has been immobilized onto powdered nylon-6 and zeolite (Pietta et al., 1989). Zeolites

were non-ideal since its coupling yield was low whereas, nylon resulted in a stable

matrix. The derivatives obtained either by diazo or by carbodiimide coupling showed

the highest activities during immobilization of the enzyme on glycophase-coated porous

glass (Dominguez et al., 1988). E. coli -galactosidase immobilized onto gelatin using

chromium (III) acetate and glutaraldehyde retained the relative activities of 25% and

22% for glutaraldehyde and chromium (III) acetate immobilized enzyme, respectively

(Ladero et al., 2000).

The heat stability of lactase can be increased through immobilization (Dekker,

1989; Kery et al., 1991; Chen et al., 2002). The effect of temperature and pH on the

catalytic activity of immobilized -galactosidase from K. lactis indicated that the

temperature-activity curves are similar for both the free and immobilized enzyme (Zhou

and Chen, 2001). However, the maximum activity of the immobilized enzyme was

31

shifted from 40 °C to 50 °C compared with the free enzyme. The comparison of a new

and commercially available amino-epoxy support (amino-epoxy-sepabeads) to

conventional epoxy supports to immobilize -galactosidase from A. oryzae showed that

the enzyme stability can be significantly improved by the immobilization on this

support, suggesting the promotion of some multipoint covalent attachment between the

enzyme and the support (Torres et al., 2003). The immobilization of thermophilic

-galactosidase on sepabeads for lactose hydrolysis showed decrease in product

inhibition, which can be helpful in improving the industrial performance of the enzyme

(Pessela et al., 2003). Alginate-chitosan core-shell microcapsules have also been used

for the immobilization of -galactosidase (Taqieddin and Amiji, 2004). The rate of

2-nitrophenyl -galactopyranoside conversion to 2-nitrophenol was faster in the case of

calcium alginate-chitosan microcapsules as compared to barium alginate-chitosan

microcapsules. Barium alginate-chitosan microcapsules, however, did improve the

stability of the enzyme at 37 °C relative to calcium alginate-chitosan microcapsules or

free enzyme.

Encapsulation of β-galactosidase from Aspergillus oryzae based on “fish-in-net”

approach with three different models of without protection, protection of protective

agent and molecular imprinting technique. Among these models, protective agents and

molecular imprinting technique pretreatment accomplished for the encapsulation of

β-galactosidase, the highest enzymatic activity of enzyme was obtained with molecular

imprinting technique (Wu et al., 2010). The free lactase has been cross-linked into

Fe3O4-chitosan magnetic microspheres for lactulose synthesis by dual-enzymatic

method in organic-aqueous two-phase media using lactose and fructose as the raw

materials (Hua et al., 2010). The organic-aqueous media can significantly promote the

transglycosidation activity of lactase and therefore improve the lactulose yield. Bacillus

32

circulans β-galactosidase has also been immobilized onto the epoxy-activated acrylic

supports sepabeads EC-EP and sepabeads EC-HFA to achieve protein immobilization

yields of 100% and enzyme activity yields of 83%.

Entrapment Method: The different methods include physical entrapment

within porous matrix, encapsulation, adsorption or attachment to a pre-formed carrier

and crosslinking have been used for the immobilization of microbial cells. Among

these, entrapment is the most commonly used method for the immobilization of

microbial cells (Song et al., 2005).

In entrapment method of immobilization, microbial cells are enclosed in a

porous polymeric matrix, which allow the diffusion of substrates to the cells and of

products away from the cells. The polymers frequently used for the entrapment are

alginate, carrageenan, xanthan, gelatin and chitosan, where as polysaccharides,

polyvinyl alcohol, polyethyleneimine, polyacrylamide and glass beads are current

synthetic polymers (Panesar et al., 2006). This entrapment method of microbial cells

immobilization offer several advantages including low cost, immobilization of a

mixture of cells, reuse of cells, higher cell densities in bioreactors, high yields of

immobilization and easy recovery of reaction products (Svec and Gemeiner, 1995;

Fukuda et al., 2001; Călinescu et al., 2012). These advantages make this method more

attractive and economic for industrial processes. The major advantage of the entrapment

technique is the simplicity by which spherical particles can be obtained by dripping a

polymer-cell suspension into a medium containing positively charged ions or through

thermal polymerization (Hartmeier, 1986). Further, beads formed particularly from

alginate are transparent and generally mechanically stable. The major limitation of this

technique for the immobilization of enzymes is the possible slow leakage during

continuous use in view of the small molecular size compared to the cells.

33

Fungal -galactosidase immobilized in polyvinyl alcohol gel was more

thermostable than free enzyme, and retained 70% of activity after 24 h at 50 °C and 5%

activity at 60 °C (Batsalova et al., 1987). The glutaraldehyde treated Kluyveromyces

bulgaricus cells having -galactosidase were entrapped in alginate using BaCl2 solution

(Decleire et al., 1987b). The alginate beads obtained after treatment with

polyethyleneimine followed by glutaraldehyde solution were stable. E. coli

-galactosidase has been immobilized in polyacrylamide gels and through the

preparation of cross-linked derivatives of E. coli -galactosidase by treating the enzyme

with bisimidoesters. The combination of three protective agents, viz., bovine serum

albumin, cysteine, and lactose, during immobilization gave an increased yield of 190%

in the case of dimethyladipimidate (DMA) cross-linked preparation (Khare et al., 1988).

Kluyveromyces marxianus cells having lactase activity were entrapped in calcium

pectate gel (CPG) and in calcium alginate gel (CAG) hardened by polyethyleneimine

and glutaraldehyde. Permeabilized cells entrapped in CPG hydrolyzed lactose more than

80% in semi-continuous and continuous processes (Tomaska et al., 1995).

The comparison of the various methods of immobilization of -galactosidase

from Thermus aquaticus indicated that immobilization by cross-linking followed by

entrapment in agarose beads can be beneficial for high enzyme loading with good

activity yield (Berger et al., 1995). The entrapment of A. oryzae -galactosidase in a

spongy polyvinyl alcohol cryogel increased the stability towards temperature, pH and

ionic strength than the free enzyme (Rossi et al., 1999). The fibers composed of alginate

and gelatin hardened with glutaraldehyde retained 56% relative activity of

-galactosidase for 35 days without any decrease. Moreover, the optimum conditions

were also not affected by immobilization (Tanriseven and Dogan, 2002). The conditions

for polyethyleimine (PEI)-coating of agarose supports to achieve a β-galactosidase

34

derivative has been optimized that allows a high lactose conversion from whey in a

steady bed-reactor with no enzyme leakage, together with good elution properties

(Gonzalez-Siso-Suarez-Doval, 2004). Another approach for immobilization of

β-galactosidase is the use of liposomes and in this direction response surface

methodology was applied to optimize the entrapment of the enzyme in liposomes by

dehydration-rehydration vesicle method, which resulted in an entrapment efficiency of

28% (Rodriguez-Nogales and Delgadillo-lopez, 2006).

Entrapment of concanavalin A-β-galactosidase complex preparation was found

to be more superior in the continuous hydrolysis of lactose in a batch process as

compared to the other entrapped preparations because it retained 95% activity after

seventh repeated use and 93% of its original activity after 2 months storage at 4 °C

(Haider and Husain, 2007). A. oryzae β-galactosidase was immobilized on the surface of

a novel bioaffinity support: concanavalin A layered calcium alginate-starch beads. The

maximum activity of the immobilized β-galactosidase has been obtained at 60 °C,

approximately 10 degrees higher than that of the free enzyme. It has been also observed

that the immobilized β-galactosidase exhibited significantly higher stability to heat,

urea, MgCl2, and CaCl2 than the free enzyme (Haider and Husain, 2009a).

The immobilized β-galactosidase, entrapped in alginate-gelatin beads calcium

alginate were used for the hydrolysis of lactose from solution, milk and whey in batch

processes as well as in continuous packed bed columns. The efficiency of columns,

containing calcium alginate entrapped soluble and crosslinked concanavalin A-complex

of β-galactosidase has been studied. From the results, it has been found that the

entrapped crosslinked Con A-β-galactosidase was more efficient in the hydrolysis of

lactose present in milk (77%) and whey (86%) in batch processes as compared to the

entrapped soluble β-galactosidase (Haider and Husain, 2009b). A comparative studied

35

were performed on the properties of free and immobilized β-galactosidase (Aspergillus

oryzae), entrapped in alginate-gelatin beads and cross-linked with glutaraldehyde. The

maximum enzymatic activity of the soluble form of β-galactosidase was obtained at pH

4.5 and 55 °C, whereas, the immobilized form was most active at pH 5.0 at 60 °C

(Freitas et al., 2011).

2.2.2.4 Biotransformation of Lactose to Lactulose Using β-Galactosidase

Biotransformation of lactose to lactulose has been carried out by using different

form of enzyme like free, immobilized, whole cells.

Free Enzyme: Biotransformation of lactose to lactulose has been carried out

using enzyme from different sources. The production of lactulose by enzymatic

transgalactosylation was carried out from lactose and fructose and the productivity of

lactulose was found to be 30 mmol/L (30% relative to lactose) for Aspergillus oryzae

β-galactosidase (Mayer et al., 2004). Further, gene encoding a thermostable

β-galactosidase from Sulfolobus solfataricus has been cloned and expressed in

Escherichia coli for lactulose production. The transgalactosylation reaction caused by

the β-galactosidase was applied to produce lactulose using lactose as a galactose donor

and fructose as an acceptor (Kim et al., 2006).

The enzymatic synthesis of lactulose from dairy waste (i.e. whey) can be carried

out using enzyme (β-galactosidase) in the presence of fructose. The controlled

enzymatic transgalactosylation of lactose in whey ultrafiltration permeate improve the

efficiency of lactulose synthesis. The factors that influenced the lactulose synthesis

efficiency were β-galactosidase preparation, substrate concentration and, also by the

ratio of lactose and fructose added to the reaction mixture (Adamczak et al., 2009;

Jaindl et al., 2009).

36

The enzymatic production of lactulose from lactose, as a single substrate, by a

thermostable recombinant cellobiose-2-epimerase from Caldicellulosiruptor

saccharolyticus were determined to be pH 7.5, temperature 80 °C, 700 g/L lactose, and

150 U/mL of enzyme (Kim and Oh, 2012). Under the optimum condition, the yield and

productivity of both lactulose and epilactose from lactose were 74% and 258 g/L/h,

respectively. Further, the production of lactulose from lactose in presence of borate to

increase the production was carried out using an enzyme cellobiose 2-epimerase from

Caldicellulosiruptor saccharolyticus (Kim et al., 2013). The maximum production of

lactulose has been observed at 1:1M ratio of borate-lactose. The lactulose concentration

of 614 g/L was achieve from 700 g/L lactose after incubation at pH 7.5 and temperature

80 °C for 3 h, with a productivity of 205 g/L/h.

Immobilized Enzyme: Biotransformation of lactose to lactulose has also been

carried out using enzyme immobilized on different matrices. Lactulose has also been

successfully synthesized by dual-enzymatic method in organic-aqueous two-phase

media using lactose and fructose as the raw materials. The dual-enzymatic system

consisted of immobilized lactase and immobilized glucose isomerase. Immobilized

lactase prepared by cross-linking the free lactase into Fe3O4-chitosan magnetic

microspheres (Hua et al., 2010). The continuous enzymatic process has been developed

for the production of prebiotic disaccharide lactulose through transgalactosylation using

free and immobilized β-glycosidase from Pyrococcus furiosus (Mayer et al., 2010).

The synthesis of lactulose from whey lactose using immobilized β-galactosidase

has been carried out in batch and continuous systems (Song et al., 2013a). From the

kinetic study, it was observed that galactose and glucose cause an inhibitory effect,

which was significantly higher for the transgalactosylation reaction to that of lactose

hydrolysis. Additionally, the immobilization decreased the inhibitory effect on the

37

enzyme by the inhibitors. The concentration of lactulose synthesized in continuous

packed-bed reactor was found to be 19.1 g/L lactulose at a flow rate of 0.5 mL/min.

Lactulose has also been synthesized by whey lactose using immobilized β-galactosidase

and glucose isomerase in absence of fructose. The optimal reaction conditions for

lactulose synthesis were 12 U/mL of immobilized β-galactosidase and 60 U/mL of

immobilized glucose isomerase using 20% (w/v) whey lactose in 100 mM sodium

phosphate buffer of pH 7.5 and temperature of 53.5 °C. Under these conditions, the

lactulose concentration and specific productivity were found to be of 7.68 g/L and 0.32

mg/U h, respectively (Song et al., 2013b).

Moreover, the cost of β-galactosidase production is the main hurdle for the

commercialization of the enzymatic method and several attempts have been made to

develop cost-effective system. The use of intracellular β-galactosidase as a whole cell

biocatalyst is also an effective way to lower the β-galactosidase production cost because

complex purification is not necessary.

Whole Cells: Whole cells can also be used as biocatalyst for the production of

lactulose because it has several advantages over purified enzymes. However, the

permeability barrier of the cell envelope for substrates and products often causes very

low reaction rates in whole cells, especially yeast cells. In order to reduce the

permeability barrier and prepare whole cell biocatalysts with high activities,

permeabilized yeast cells can be used (Panesar et al., 2006). The permeabilized

Kluyveromyces marxianus cells as a source of β-galactosidase were used to overcome

the problem of enzyme extraction and poor permeability of cell membrane. Ethanol

permeabilization of yeast cells has been shown to be an economical, easy, convenient,

and safe process for enzymatic bioconversion and product formation (Joshi et al., 1989;

Lee et al., 2004; Panesar et al., 2007). Among the commercial β-galactosidases

38

(Escherichia coli, Aspergillus oryzae and Saccharomyces fragilis), the enzyme from

Kluyveromyces lactis exhibited the highest lactulose productivity. The reaction

conditions for lactulose production were optimized using cells that had been

permeabilized by treatment with 50% (v/v) ethanol (Lee et al., 2004). Permeabilized

cells resulted in 1.3 and 2.1 fold increase in lactulose production as compared to

untreated washed cells.

2.3 Determination of Lactulose

The development of the analytical technologies for determination of lactulose

has become more and more rapid and precise. A range of methodologies have been

reported to determine the lactulose including, gas-liquid chromatography (GC), thin-

layer chromatography (TLC) and high-performance liquid chromatography (HPLC) and

other related techniques such as: capillary electrophoresis, differential pH methods, flow

analysis methods (Table 2.5).

Lactulose has been detected in milk and in sugars mixture by diluting the sample

ten times with acetone and successive thin-layer chromatography with acetonitrile:

water mixture. The method can detect even up to 0.02% lactulose on total carbohydrate

(Martinez- Castro and Olano, 1981). Lactulose can also be measured by thin layer

chromatography using silica gel 60 plates and propanol-borate solvent system (Flick et

al., 1987).

For the analysis of lactulose preparation, spectrophotometric methods were

applied to sugar mixtures produced during isomerization of lactose.

A spectrophotometric method was used to quantify galactose, tangatose, lactose and

lactulose. The method developed for determining lactose with methylamine at 65 °C

and pH 12.7 was suitable for combine measurement of lactose and lactulose. A high

39

pressure liquid chromatography was also used for the separation of galactose, tangatose,

lactose and lactulose with a commercial carbohydrate analysis column (Parrish et al.,

1980). Furthermore, a simple spectrophotometric assay was also developed for the

quantification of lactulose in pharmaceutical preparations. This method is based on

hydrolysis of lactulose under acidic conditions. The hydrolyzed product reacts with

resorcinol, giving absorption peaks at 398 and 480 nm. Both absorption wavelengths

can be used for the determination of lactulose. The limit of detection of lactulose at 398

nm and 480 nm was 0.075 μg/mL and 0.65 μg/mL, respectively (Khan et al., 2006).

Table 2.5 Determination of lactulose by using different methods

Method (s) Reference (s)

Capillary electrophoresis Montero et al. (2004); Paroni et al. (2006)

Flow analysis Method Mayer et al. (1996); Moscone et al. (1999)

Gas Chromatography D'Eufemia Celli et al. (1995); Martinez-Augustin

et al. (1995); Abazia et al. (2003); Montilla et al.

(2005a); Montilla et al. (2005b); Shippee et al.

(2005); Ruiz-Matute et al. (2007)

Gas Chromatography-Mass Spectra Rodriguez et al. (2009)

High Performance Liquid Chromatography Fleming et al. (1990); Dendene et al. (1995);

Bao et al. (1996); Cox et al. (1997); Marsilio et

al. (1998); Brands and Boekel (2003);

Cha´vez-Servı´n et al. (2004); Kim et al. (2006);

Nelofar et al. (2010)

Liquid Chromatography-Mass Spectra Lee et al. (2004); Thanawiroon et al. (2004);

Lostia et al. (2008)

Nuclear magnetic resonance Jayalakshmi et al. (2003); Mayer et al. (2004)

Spectroscopic method Adhikari et al. (1991); Nagendra and Rao (1992)

Amine et al. (2000a); Marconi et al. (2004);

Khan et al. (2006); Zhang et al. (2010)

Thin Chromatography Martinez-Castro and Olano (1981); Flick et al.

(1987)

A simple and rapid flow system was developed for the determination of

lactulose in milk samples, which is based on the hydrolysis of lactulose to galactose and

fructose by the enzyme β-galactosidase immobilized in a reactor (Moscone et al., 1999).

40

The amount of fructose produced was measured with an electrochemical biosensor

based on the fructose dehydrogenase enzyme, potassium ferricyanide (K3 [Fe (CN)6]) as

mediator and a platinum based electrochemical transducer. Furthermore, an enzymatic

spectrophotometric assay has also been developed for the determination of lactulose in

milk samples by the hydrolysis of lactulose to fructose and galactose, and fructose

dehydrogenase reacts with fructose in presence of a tetrazolium salt ([3-(4,5-

Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide]), giving a coloured

compound, which can be detected spectrophotometrically at 570 nm (Amine et al.,

2000b).

Lactulose during the reactions were analyzed by a High-performance liquid

chromatography (HPLC) system equipped with Shimadzu RID-10A detector with a

high performance carbohydrate cartridge column. The column was eluted at 40 °C with

80% (v/v) acetonitrile at a flow rate of 1mL/min. The lactulose produced in reactions

was identified (Lee et al., 2004; Kim et al., 2006) and the kinetic studies of

bioconversions (lactose to lactulose) has been carried with HPLC with a 300 mm × 7.8

mm i.d. Rezex Ca2+

column (Phenomenex) at 85 °C and a flow rate of 0.8 mL/min

(Mayer et al., 2010).

2.4 Applications of Lactulose

Lactulose has a number of applications in both food and pharmaceutical

industries, which are shown in Figure 2.4.

2.4.1 Food Industry

Lactulose is applied in a wide variety of foods as a bifidus factor or as a

functional ingredient for intestinal regulation. In addition to providing useful

41

modifications to food flavour and physicochemical characteristics, many of these sugars

possess properties that are beneficial to the health of consumers.

Figure 2.4 Potential applications of lactulose

As Prebiotics: Lactulose serves as a dietary carbohydrate that has a selective

microbial metabolism in the gut, directed towards health beneficial bacteria includes

bifidobacteria and/or lactobacilli. Lactulose is shown to be an effective food-grade

prebiotic for healthy adults particularly in the community with low bifidobacterial

populations (Tuohy et al., 2002). It has also been reported to improve the survival of

available probiotic strains in yoghurt (Tabatabaie and Mortazavi, 2008). The utilization

of lactulose by intestinal microflora is given in Table 2.6.

Food Additive: Lactulose can be used as a sweetener for diabetics, as a sugar

substitute in confectionery products, beverages, infant milk powders, bakery products,

yoghurts, dairy desserts and in various liquid or dried food preparations which are

routinely manufactured for old people (Crittenden and Playne, 1996; Strohmaier, 1998).

Lactulose also has some properties with desirable effects in food products such as

Lactulose

Food

Industries

Pharmaceutical

Industries

Prebiotics

Food Additive

Treatment of Constipation

and Hepatic Encephalopathy

Inflammatory Bowel Disease

Anti-endotoxin Effect

Blood Glucose and Insulin

Colon Carcinogenesis

Tumour Prevention and

Immunology

42

flavour enhancing properties, favourable browning behaviour, excellent solubility in

water, etc. Many tests have been performed on yoghurt, cookies, cake, chocolate, etc. to

find the change in behaviour of lactulose during processing of products (Battermann,

1997).

Table 2.6 Utilization of lactulose by the intestinal microflora

Micro-organism Growth

Bacteroides fragilis C-2

Bacteroides vulgatus E 1

Bifidobacterium adolescentis ATCC 15705

Bifidobacterium bifidum S 28

Bifidobacterium breve S1

Bifidobacterium infantis S12

Bifidobacterium longum E 194

Clostridium perfringens ATCC 13124

Clostridium ramosum ATCC 13937

Escherichia coli No. 28

Lactobacillus acidophilus ATCC 4356

Lactobacillus casei 8138

Lactobacillus salivarius ATCC 11741

Streptococcus faecalis ATCC 19434

Positive growth

Bacteroides melaninogenicus NCTC (9338)

Clostridium difficile No. 55

Eubacterium lentum ATCC 25559

Kebsiella pneumoniae PCI 602

No growth

Salmonella enteritidis

Salmonella typhimurium

Shigella flexneri

Shigella sonnei

Negative growth

(Source: Bovee-Oudenhoven et al., 2003; Cox et al., 2008; Tamura et al., 1993; Levine and Hornick,

1975)

2.4.2 Pharmaceutical Industry

In the pharmaceutical field, lactulose can be used for the treatment of

constipation, hepatic encephalopathy, tumour prevention, immunology, anti-endotoxin

effects, maintain blood glucose and insulin level (Schumann, 2002).

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Constipation and Hepatic Encephalopathy: Lactulose is physiological therapy

successfully useful in treatment of constipation. Due to its efficacy profile and safety,

lactulose is used in all age groups, from infants to the elderly patient, and regarded as

the drug of choice. Lactulose is widely used in the treatment of hepatic encephalopathy

(Sharma et al., 2009).

Inflammatory Bowel Disease: Oral administration of lactulose abolishes and

prevents systemic endotoxemia of gut origin. Therefore, lactulose may be used for

treatment of inflammatory bowel disease as bacteria and its endotoxin have an

important role in the pathogenesis of this disease. Obstructive jaundice is often

accompanied by bacterial translocation and subsequent sepsis and the administration of

lactulose may prevent systemic endotoxaemia and the subsequent inflammatory

response in an experimental model of obstructive jaundice (Koutelidak et al., 2003).

Fermentation of lactulose by gastrointestinal tract bacteria can produce considerable

amount of mobilizes endogenous hydrogen, which is protective for DSS-induced colitis

as a unique antioxidant, which can reduce oxidative stress and ameliorate symptoms of

inflammatory bowel disease in human beings (Chen et al., 2011).

Anti-endotoxin Effect: Lactulose treatment before operation can prevent

endotoxin-dependent complications such as renal dysfunction (Pain et al., 1991; Özçelik

et al., 1997). The anti-endotoxin effect of lactulose has also important medical

applications in metabolic diseases like the hepatorenal syndrome (Kramer, 1988),

exocrine pancreatic dysfunction (Mack et al., 1992), diabetes mellitus (Yelich et al.,

1992; Tabatabaie et al., 1997) and hypercholesterolemia (Liao and Florin, 1995).

Blood Glucose and Insulin: Lactulose has also an important application in

lowering blood glucose level (Bianchi et al., 1994). Cornell (1985) also suggested that

44

endotoxin reduce the pancreatic insulin production and thus lactulose shows anti-

diabetic effect.

Colon Carcinogenesis: The occurrence of colon cancer is the result of

biochemical processes, which may take place in lumen, mucosa and adjacent tissues of

the large intestine. Colonic microflora and its resulting metabolism products are prone

to influence colon carcinogenesis. Probiotic bacteria and prebiotic substances like

lactulose prone to reduce the risk of colon cancer (Wollowski et al., 2001).

Tumour Prevention and Immunology: Bifidobacteria play an important role

in tumor prevention. The anti-tumour and immunologic effects by bifidobacteria can be

enhanced by intake of lactulose (Schumann, 1997). Preoperative oral treatment with

lactulose is used to prevent complications in patients who underwent surgery of

obstructive jaundice (Greve et al., 1990).