Received: 4 June 2017 | Revised: 13 February 2018
DOI: 10.1111/jfbc.12564
REVIEW
b-Galactosidase: Biotechnological applications
in food processing
Janifer Raj Xavier1 | Karna Venkata Ramana1 | Rakesh Kumar Sharma2
1
Food Biotechnology Division, Defence
Food Research Laboratory, Defence
Research and Development Organization,
Mysore, Karnataka, India
2
Defence Food Research Laboratory,
Defence Research and Development
Organization, Mysore, Karnataka, India
Correspondence
Mrs. Janifer Raj Xavier, Food Biotechnology
Division, Defence Food Research
Laboratory, Defence Research and
Development Organization, Mysore
570011, Karnataka, India.
Email: janifer@dfrl.drdo.in/jennipal@gmail.
com
Funding information
Defence Research and Development
Organisation
J Food Biochem. 2018;e12564.
https://doi.org/10.1111/jfbc.12564
| Accepted: 14 February 2018
Abstract
b-Galactosidases produced by microorganisms are being used in food technology for hydrolysis of
lactose in milk and milk by-products. The enzyme has attracted much attention in view of lactose
intolerance in human population and due to importance of milk in human diet. b-Galactosidases
hydrolyze b-galactopyranosides, that is, lactose, and form a range of trans-galactosylation products
or galactooligosaccharides (GOS) capable of providing several health benefits as prebiotics. In addi-
tion, the enzyme also finds applications in production of lactose based sweeteners from high
lactose containing effluents of cheese manufacturing industries. Currently, the enzyme is mainly
obtained from Aspergillus niger and Kluyveromyces lactis, however, they are thermolabile with opti-
mum pH between 4.0 and 6.0 accompanied by low chemical resistance and high product inhibition
properties. Thermostable enzymes are suitable to perform the hydrolytic process at relatively high
temperatures, minimizing microbial contamination. Psychrophilic b-galactosidases are preferred for
the treatment of milk under refrigerated conditions (<158C), without maintenance of temperature
of milk. Lactic acid bacteria (LAB) are a potential source of food grade enzymes and other metabo-
lites. Although reports on the use of b-galactosidase of LAB in pure form is reported for hydrolysis
of lactose in milk and production of fermented milk products for reduced lactose content, methods
of immobilization for use in continuous bioreactors are less explored. Furthermore, recombinant
DNA technology has attained much significance to reveal molecular aspects of enzyme catalysis,
protein structure, gene organization for expression, and enhancement of enzyme activity of ther-
mophilic, psychrophilic, and mesophilic b-galactosidases. Heterologous expression of
extremozymes with b-galactosidase would help in overcoming the limitations faced within the bio-
process technology.
Practical applications
Lactic acid bacteria (LAB) producing extracellular cell bound b-galactosidase have been isolated
and enzyme production was carried out using modified MRS medium incorporating lactose as
enzyme inducer. Process for growing probiotic lactic acid bacteria for b-galactosidase from fer-
mented milk products has been patented (Indian Patent No. 247904). Thermostable
b-galactosidases were also extracted from Kluyveromyces lactis a yeast, isolated from curd samples
and Bacillus sp. MTCC 864. An Indian patent has been granted for a simple, cost effective perfo-
rated two compartment immobilized enzyme bioreactor was designed and fabricated for lactose
hydrolysis (Indian Patent No. 264609). The bioreactor can be operated continuously at tempera-
ture between 5.0 and 75.08C, employing different immobilized enzyme preparations for different
operational temperatures. b-galactosidase of cell confined LAB and yeast have been immobilized
in agarose and galactooligosaccharides (GOS) production was observed at 40% lactose concentra-
tion in 0.05 M phosphate buffer at pH 6.5. Immobilized bioreactor preparation could be used for
lactose hydrolysis of milk between 45 and 708C with 75% efficiency.
KEYWORDS
beta-galactosidase, food and industrial applications, lactose hydrolysis
wileyonlinelibrary.com/journal/jfbc V
C 2018 Wiley Periodicals, Inc. | 1 of 15
2 of 15 | XAVIER
1 | INTRODUCTION
Microbial enzymes are useful in a wide range of applications in food
processing industry. Enzymes derived from microorganisms are used as
important tools in basic research, synthesis of organic molecules, pro-
duction of pharmaceuticals, and in various biotransformation processes
for production of bulk chemicals. Glycoside hydrolases are capable of
hydrolyzing glycosidic bond between two or more carbohydrates or
between a carbohydrate and a noncarbohydrate moiety and are classi-
fied into 133 families (www.cazy.org/Glycoside-Hydrolases.html).b-
Galactosidase galacto hydrolysase, (EC 3.2.1.23) commonly referred as
lactase is one of the predominantly occurring hydrolytic enzymes in
higher plants, animals, and microorganisms. The worldwide production
of b-galactosidase is estimated at around 5.75 million metric tons per
annum. The enzyme has two main applications; the removal of lactose
from milk products and production of galactosylated products (Husain,
2010). In mammals the small intestine brush border cells produce
b-galactosidase or “lactase-phlorizin hydrolase” and its complete
absence or insufficient production in child hood is referred to as pri-
mary lactase deficiency. In adults it is known as adult type hypolactasia
or lactase nonpersistence. The absence or inadequate production of
lactase in human beings usually leads to an intestinal symptomatic
problem “lactose intolerance” with clinical symptom manifested in form
of abdominal pain, diarrhea, nausea, flatulence, and bloating after intake
of lactose containing foods. Estimates showed that 70% of the world
population, comprising various age groups, is lactose intolerant. How-
ever, dairy produce is the major source of calcium for individuals, and a
low intake of those products is associated with a higher risk of frac-
tures or even of osteoporosis. Lactose hydrolysis in milk prior to con-
sumption has been suggested to help in the absorption of
monosaccharide constituents of the disaccharide. New technologies to
produce lactose-reduced or lactose-free dairy products have therefore
been developed, including lactose hydrolysis or separation. Thus, appli-
cation of hydrolytic activity of b-galactosidase in food industry has
attracted worldwide importance over the last few decades, for reducing
the lactose content in milk transglycosylation reactions for the synthe-
sis of galactooligosaccharides (GOS) in recent years (Oliveira,
Guimar~aes, & Domingues, 2011; Park & Oh, 2010). The hydrolysis of
lactose acquires great significance from clinical, technological, and envi-
ronmental perspectives. Apart from providing relief to lactose intoler-
ant subjects, hydrolysis of disaccharide increases its solubility, and
sweetness of product due to monosaccharides glucose and galactose
thereby utilizing vast quantity of whey constitutes. Lactose a renew-
able carbohydrate and a disaccharide could also find application as a
starting material for the production of bulk chemicals, food, and feed
additives and in preparation of value added functional food products. It
is also added to several nondairy foods such as breads, beer, frozen
vegetables, salad dressings, soups, nutritive breakfast diets, and in pro-
duction of other low-calorie foods. In the background of these applica-
tions of lactose and its hydrolyzed products, that is, glucose and
galactose, the preeminence has been attributed to the b-galactosidase.
The aim of this review is to discuss the developments on
b-galactosidases in a more comprehensive form, amalgamating the
ET AL.
various pioneering works that took place in finding novel enzyme sour-
ces, novel means of enzyme preparations and production of GOS and
applications to enhance the efficiency and productivity, and bioprocess
developments that turned out to be more practical on industrial appli-
cations and also aimed at addressing some of the constraints currently
encountered in the enzyme production, utilization and reuse by
enhancing the half-life of various enzyme forms. In the present review,
various biotechnological aspects of b-galactosidases are also discussed
with emphasis on hydrolysis of lactose and production of oligosaccha-
rides using enzymes produced by lactic acid bacteria, yeast, molds, and
extremophiles. Biochemical properties of b-galactosidases with respect
to pH, temperature, and their food processing implications are also
reviewed based on different methods of permeabilization and enzyme
immobilization.
2 | MICROBIAL SOURCES OF
b- G A L A C T O S I D A S E
b-Galactosidases can be obtained from microbial sources such as bac-
teria, yeasts, and filamentous fungi (Oliveira et al., 2011). While the
previous reviews mainly focused on b-galactosidases obtained from
yeast and fungal genera, during the last few years several develop-
ments have took place in the area of enzyme production from psychro-
philes, mesophiles, and thermophiles and hyperthermophilic bacteria,
isolated from geographically distributed areas. Commercial production
of GOS using b-Galactosidases has been reported from Kluyveromyces
lactis, Bacillus circulans (Vivinal GOS), Bifidobacterium bifidum (Bimuno),
Aspergillus oryzae, and Streptococcus thermophilus (Oligomate55)
(Torres, Gonçalves, Teixeira, & Rodrigues, 2010). b-Galactosidase of
microorganisms display a great diversity in their functional properties
which could be further improved, by isolating suitable strains of micro-
organisms, producing enzymes amenable to immobilization, receptive
to chemical mutagenesis, enhanced enzyme secretion, and gene
expression by recombinant DNA techniques is constantly increasing
nowadays. Further, products obtained from certain microorganisms
such as Kluyveromyces lactis are generally recognized as safe (GRAS sta-
tus) for human consumption, which is significant for food processing
applications (Kosseva, Panesar, Kaur, & Kennedy, 2009). Currently,
recombinant DNA technology can be used to express and optimize the
production of interesting b-galactosidases from the most diverse sour-
ces, presenting tedious growth conditions and productivities including
microorganisms with no GRAS status, in heterologous microbial hosts
for highly efficient protein production. Furthermore, using state-of-the-
art protein engineering tools novel microbial b-galactosidases with
enhanced features such as higher product yield, reduced product inhi-
bition etc. could be produced (Gosling, Stevens, Barber, Kentish, &
Gras, 2010). The interest in exploiting recombinant enzymes as biocata-
lysts is constantly increasing nowadays due to plausible advantages
involved with their use including their (1) rigidity and permeability, (2)
hydrophobic/hydrophilic character, (3) ease of purification and large-
scale production, (4) immediate separation from the reaction mixture
after completion of reaction without using any chemicals or heating, (5)
regenerability as they impart stability to enzymes by protecting their
XAVIER ET AL. | 3 of 15

T AB LE 1 b-galacosidase from various sources for lactose hydrolysis

Sl. No. Organism Source Enzyme characteristics References

1. Pseudoalteromonas Antarctica soil Cold active b-galacosidase Singh & Ramana, 1998
sp. DFR1

2. Pseudoalteromonas Antarctica soil Cold active b-galacosidase Singh & Ramana, 1998
DFR6

3. Candida sp. Antarctica soil Active pH range 5.0–8.5 Active Ramana & Singh, 2000
temperature range 0.0–55.08C

4. Kluyveromyces lactis Curd Optimum pH 6.8 Pal et al. (2009)

Optimum temperature 408C
Resistant to organic solvents and
stable at 5–68C for 12–18 months
on immobilization using cellulose
acetate immobilization

5. Bacillus sp. MTCC 864 MTCC, Optimum pH 7.0 Patil et al., 2011
Chandigarh Optimum temperature 50.08C
Thermostable
b-galacosidase, resistant to organic
solvents and stable at 5–68C for
12–18 months on immobilization
using agar

6. Design of bioreactor for lactose hydrolysis in milk and other Ramakrishna et al., 2005
liquids that contain lactose as constituent sugar (Indian Patent No. 264609) Ramana et al., 2015

active sites from deactivation, and (6) convenience in tailoring within
utility limits (Ansari & Satar, 2012) (Table 1).
These prospects of modifying the b-galactosidase producing
microorganisms play a major role in economical and effective utilization
of industrial processes involving the enzyme. On the other hand,
growth of the microorganisms could be manipulated under semi-batch
or continuous fermentation conditions during bioprocess development.
Enzyme production can also be increased by incorporating suitable
media ingredients and extraction of the enzyme by suitable modifica-
tions of downstream processing protocols (Scheme 1).
pH 3.5–4.5 which is close to acid whey is used in treatment of acid
whey from cottage cheese. Lactose hydrolysis also permits more
whey to be fed to cattle and other domesticated animals (Holsinger
& Kligerman, 1991).
4. For the production of concentrated milk, hydrolysis of lactose is
essential to minimize lactose crystallization. Lactose is the largest
component in milk and whey which imparts sandy or gritty texture
to dairy products such as condensed milk, evaporated milk, frozen
milk, ice cream, and in confectionery products with high milk con-
tent (Zarate & Leiva, 1990).

5. Galacto-oligosaccharide production for prebiotic applications (Lu

3 | APPLICATIONS OF MICROBIAL et al., 2012; Vera, Cecilia, Rau s, 2012).
 l, & Andre
b- G A L A C T O S I D A S E S

1. Hydrolysis of lactose in milk for enhancing stability of milk and for
lactose intolerant population. A fungal lactase capable of reduction
of lactose content in fermented milk products and to enhance its
sweetness during distribution period of 3–5 days at 108C (Wilde,
Baumgartner, Fertsch, & Hinrichs, 2001).
2. Sweetener and functional protein production from hydrolyzed whey
lactose could be achieved in form of concentrated syrup containing
70–75% solids for sweetening ice creams, bakery, and confection-
ery products. Deproteinized, demineralized, and lactose hydrolyzed
whey concentrated to 60–65% solids can be used as sugar syrup as
replacement to corn syrup (Dziezak, 1991).
3. Recovery of proteins, enzymes, and lactose from whey is not eco-
nomical and the release whey, obtained as by-product of cheese
production is often banned in several countries due to the polluting
constituents contributing to high content of biochemical oxygen
demand (Zarate & Leiva, 1990). Aspergillus niger lactase with optimal
4 | b- G A L A C T O S I D A S E F R O M L A C T I C A C I D
BACTERIA
Lactic acid bacteria are highly preferred for the production of enzymes
in dairy and food processing application and have an important role in
production of fermented dairy products, owing to their technological
and health benefits. Optimal b-galactosidase production medium
required for lactic acid bacteria comprised of whey, skim milk, or whey
permeate supplemented with 1.0% whey protein or 0.2% yeast extract
or 1.2 de Man-Rogosa-Sharpe (MRS) broth. Skim milk alone was also
found to support b-galactosidase production of 5.49 U/mL (Vasiljevic
& Jelen, 2001). b-Galactosidases extracted from pure cultures of Bifido-
bacterium angulatum, B. bifidum BB-12, B. adolescentis ANB-7, B. infan-
tis DSM-20088, and B. pseudolongum DSM-20099 were investigated
for glycosyl transfer reactions for synthesis of GOS from lactose and
GOS was comparable to commercially available oligosaccharide Oligo-
mat 55 (Rabiu, Jay, Gibson, & Rastall, 2001). The induction of a- and
4 of 15 | XAVIER
SCHEME 1 Sources and applications of microbial b-galactosidase
b-galactosidases production by six strains of Lactobacillus reuteri using
six carbohydrate and four protein sources were studied. L. reuteri CF2–
7F grown on raffinose showed highest a-galactosidase activity (10.55
Gal U/mL), while lactose exhibited the highest b-galactosidase activity
(43.82 Gal U/mL) while L. reuteri CF2–7F grown on yeast extract exhib-
ited the highest a- and b-galactosidases activities (15.27 and 12.88 Gal
U/mL, respectively). Thus, L. reuteri strain CF2–7F could be a good pro-
biotic candidate with applications as beneficial food additive or food
supplement (Alazzeh, Ibrahim, Song, Shahbazi, & AbuGhazaleh, 2009).
Carnobacterium sp. isolated from meat and fish samples is a
recently reported genus of lactic acid bacteria. Psychrotrophic C. pisci-
cola strain BA with three b-galactosidase genes with homology to glu-
cosidases were detected in fragment cloned from the C. piscicola. The
a-galactosidase gene agaA was followed by two genes of
b-galactosidase genes in the same orientation. The bgaB was a 2 kb
b-galactosidase gene while bgaC encoded a structurally distinct
b-galactosidase protein with 1.76 kb base sequence. This gene arrange-
ment was found to be unique to this bacterium and not found in any
other lactic acid bacteria, including the Lactococcus lactis, for which the
entire genome sequence is known. The b-galactosidase bgaC showed
its optimum activity at 408C with a half-life of 15 min at 458C. Activity
of a- and b-galactosidase declined in the C. piscicola in the presence of
glucose or lactose media (Coombs & Brenchley, 1999). The
b-galactosidase from L. bulgaricus L3 synthesized GOS and through
beta galactosyl transfer to the OH-30 and OH-60 of lactose (Lu et al.,
2012).The b-galactosidase gene from L. bulgaricus L3 was cloned and
expressed in Escherichia coli using the pET-21b vector. The purified
recombinant enzyme could catalyze glycosyl transfer from lactose to
sucralose, one of the most popular sweeteners. The dominant product
reached a maximal yield of 41.0% at the lactose/sucralose ratio of
1.5:1 for 15 hr. In another study, Yi, Alli, Park, and Lee (2011) have
ET AL.
expressed a b-galactosidase gene of Bifidobacterium breve in E. coli and
found that the Km value for its natural substrate, that is, lactose is 52.5-
fold higher (95.58 mM) than the synthetic substrate ONPGC
(1.82 mM). The enzyme was optically active at pH 7.0 and 458C.The
enzyme resulted in 42.0% of GOS in the presence of 1 M lactose.
Another merit of the enzyme was nearly 97.0% lactose was hydrolyzed
in milk at 458C a desirable property for its industrial scale applications.
5 | FUNGAL AND YEAST ENZYME SOURCES
O F b- G A L A C T O S I D A S E
Although, several organisms are known to produce b-galactosidase,
currently it is mainly obtained from a few Generally Recommended As
Safe (GRAS) status microorganisms such as, Aspergillus niger, A. oryzae,
and Kluyveromyces lactis. Lactase from K. lactis shows an optimum pH
6.8–7.0 and optimally active at 358C whereas a lactase from K. fragilis
displays optimum activity at pH of 4.8 and at 508C. K. lactis lactase is
suitable for treating milk (pH 6.8) and sweet whey (pH 6.2). Lack of sta-
bility below pH 6.0 precludes its use for treating acid whey at pH 4.6.
K. fragilis lactase and A. niger lactase with pH optimum 4.0–4.5 and
good stability at pH 3.0–7.0 and a temperature optimum at 558C are
suitable for hydrolysis of lactose in acid whey (Holsinger & Kligerman,
1991). b-Galactosidase production and ethanol fermentation from
whey was reported using K. marxianus NCIM 3551 at laboratory scale.
Optimum b-galactosidase production and ethanol fermentation were
obtained with 16 hr old culture at an inoculum size of 10% for 20 hr at
pH 5.0 and at 258C (Gupte & Nair, 2010). Lactose-free, low cost
medium containing wheat bran 0.5% (w/v), corn steep liquor 1% (w/v),
and NaCl 5% (w/v) was optimized using response surface methodology
for production of b-galactosidase by a halo tolerant A. tubengensis GR1
(Raol, Vimal, & Raol, 2014). The enzyme production was maximum
XAVIER ET AL.
(352.0 6 2.0 U/mL) after 96 hr of incubation and feasible for large scale
production on an industrial scale. b-Galactosidase obtained had a
molecular weight of 93 kDa and was also found to be stable over wide
pH range and thermally stable at 60–658C up to 60 min of incubation
while maximum activity was exhibited at 658C with pH 3.0. However,
the enzyme activity was found to be significantly activated in the pres-
ence of 15–30% NaCl concentration. In another study, b-galactosidase
exhibiting high activity in an extremely acidic pH region to neutral pH
region was efficiently purified by Isobe, Naomi, Serina, Miho, and Taka-
humi (2013) from an acidophilic fungus, Teratosphaeria acidotherma
AIU BGA-1isolated from an acidic and high temperature hot spring.
Four intracellular b-D-galactosidases were produced with different pH
activity profiles, in which three were acidophilic and one was alkalo-
philic in nature. The enzyme was stable in the pH range from 1.5 to
7.0, and exhibited optimal activity at pH 2.5–4.0 and 708C. 2-Nitro-
phenyl-b-D-galactopyranoside, 4-nitrophenyl-b-D-galactopyranoside,
and lactose were rapidly hydrolyzed, and apparent Km values of the
enzyme were found to be 0.19, 1.2, and 170 mM, respectively. The
molecular mass of the enzyme was estimated to be 140 kDa with two
hetero subunits of 86 and 50 kDa. The enzymes were remarkably dif-
ferent from those of previously known fungal b-galactosidases and
could be used in a wide pH range for hydrolysis of lactose.
Recombinant Aspergillus b-galactosidases are robust glycomic and
biotechnological tool, the recombinant expression and purification of
two previously uncharacterized b-galactosidases from A. nidulans as
well as one from A. niger suitable for various glycobiological and bio-
technological applications was reported by Dragosits et al. (2014). The
recombinant enzymes were active toward p-nitrophenyl-b-D-
galactopyranoside as substrate and digested various complex sub-
strates containing terminal galactose b-1,4 linked to either N-
acetylglucosamine or fucose, such as N-glycans derived from bovine
fibrin and Caenorhabditis elegans. Comparative study of the recombi-
nant galactosidases with the commercially available galactosidase from
Aspergillus oryzae, displayed various degrees of activity toward complex
oligosaccharides containing b-1,3-linked terminal galactose residues.
The enzymes were found to be robust in the presence of various
organic solvents, temperature variations, and freeze/thaw cycles and
were able to synthesize GOS. Furthermore, the use of fermenters con-
siderably increased the yield of recombinant galactosidases. Moreover,
fungal b-galactosidases have also been successfully engineered in order
to achieve decreased product inhibition by galactose (Hu, Robin, Con-
nell, Walsh, & Wall, 2010; Kim & Rajagopal, 2000). In this regard, A.
nidulans rlacA with high Ki for galactose, is a very interesting and suita-
ble candidate for future protein engineering studies. The availability of
crystal structures of closely related enzymes, including the recently
reported structure of A. oryzae b-galactosidase (Maksimainen, Lampio,
Mertanen, Turunen, & Rouvinen, 2013) could facilitate the directed
engineering of fungal galactosidases toward decreased product inhibi-
tion and engineering toward other relevant improved activities such as
fucosidase (Zhang, Dawes, & Stemmer, 1997), glucuronidase (Xiong
et al., 2007), or glycosynthase activity (Shim, Chen, Rich, Goddard-
Borger, & Withers, 2012).
| 5 of 15
6 | PERMEABILIZATION OF BACTERIA AND
YEAST FOR ENZYME EXTRACTION
Several chemicals including solvents, surfactants and emulsifying
agents have been recognized to permeabilize yeast, fungi, and bacteria
for utilizing their intracellular b-galactosidase for lactose hydrolysis.
Among them, sodium dodecyl sulfate, triton X-100, sodium deoxycho-
late, ox gall, ethanol, chloroform, toluene, acetone, and cetyl trimethyl
ammonium bromide were found to preserve the enzyme activity and
to limit the cell viability. In addition to the ease of preparation, perme-
abilized cells provide stability to enzymes in intracellular environment
against several chemical and physical inhibitors in complex milieu of
process. L. reuteri was permeablized using 0.15% (v/v) ox gall and
enzyme activity of whole cell preparation was noticed to increase sev-
eral fold after lyophilization of bacterial biomass (Valdez et al., 1997).
Once the enzyme is produced, down-stream processing becomes a
potential and significant factor depending on the location of enzyme,
as they are sometimes found to be cell wall bound only for a certain
period during growth cycle and after a particular growth phase is
released into the culture medium (Rapp, 1995). L. delbrueckii subsp bul-
garicus 11842 produced intracellular b-galactosidase, hence lysis of
cells for extraction of intracellular pool of enzyme is the primary step
for extraction. Sonication or homogenization at high pressure and use
of bead mill for disruption of cell biomass were found suitable for intra-
cellular enzyme extraction. Three passages through a homogenizer at
135 MPa or a onetime 200 MPa pressure resulted in maximum enzyme
extraction (Bury, Jelen, & Kalab, 2001). In another study, production of
b-galactosidase by an autolytic strain of Streptococcus salivarius sub. sp.
thermophilus 11F was noticed on cell lysis immediately after cessation
of growth of organism. The lactic acid bacteria were found suitable for
lactose hydrolysis as they are known to produce thermostable
b-galactosidases and methods exist for their permeabilization and for
hydrolysis of lactose (Vasiljevic & Jelen, 2001).
7 | T H E R M O S T A B L E b- G A L A C T O S I D A S E S
Extremophile organisms are a valuable source for production of
enzymes and development of various biotechnological processes (Agui-
lar, 1996). The glycoside hydrolase family 42 (GH42) consists of ther-
mostable b-galactosidases that display significant variations in their
temperature optima and stabilities. b-Galactosidase is one of the rela-
tively few enzymes that have been used in large-scale processes to
perform lactose hydrolysis and galacto-oligosaccharide production.
Thus, the b-galactosidases obtained from various mesophilic, psychro-
philic, and thermophilic sources and their potential applications in bio-
technology industry are discussed. Thermostable enzymes are wide
spread among plants, animals, and microorganisms. They are optimally
active between 60 and 1258C. These enzymes are unique in terms of
their protein structure and stability toward temperature, pH and chemi-
cals. Although thermozymes are predominantly found in thermophilic
microorganisms their growth temperature is in the range of 60–808C,
for instance some of the food spoilage psychotrophic bacteria are
6 of 15 | XAVIER
found with thermostable proteases. Novel thermostable enzymes are
being investigated to make bioprocesses more efficient. In general ther-
mostable enzymes are resistant to various chemicals and solvents in
addition to a higher optimum temperature, indicating their rigid protein
conformation attributed to lower hydrogen exchange rate and lower
susceptibility to proteolytic degradation. Molecular cloning has made it
possible to express thermophilic and hyper thermophilic genes in meso-
philic bacteria such as E. coli. Novel b-galactosidases derived from
recombinant vectors and by protein engineering approach are impor-
tant for efficient recombinant microbial production systems and
thereby applications of recombinant enzymes as a relevant synthetic
tool in biotechnology. The union of specific physical and chemical prop-
erties of recombinant proteins with specific recognition of catalytic
properties of biomolecules has led to their appearance in myriad novel
biotechnological applications.
b-Galactosidases from Aspergillus oryzae was used as a model
enzyme to study the effect of amino-epoxy (amino-epoxy-sepabeads)
supports for enzyme immobilization and compared with conventional
epoxy supports. The layer of high density epoxy groups are embedded
over a layer of ethylene diamine on the substrate with great anionic
exchanger strength. Rapid immobilization of enzymes at moderately
low ionic strength with improved enzyme stability over 12-fold was
also noticed which could be attributed to some multipoint covalent
attachment between the enzyme and the support (Mateo et al., 2003;
Torres et al., 2003).The immobilization of a thermophilic
b-galactosidase on Sepabeads support to decrease product inhibition,
immobilization conditions for strong reversible immobilization of large
beta-galactosidases from E. coli and Thermus sp. proteins, on tailor-
made anionic exchangers by controlling the nature of support were
studied by Pessela et al. (Pessela et al., 2003; Pessela et al., 2006). Pes-
sela, Fernandez-Lafuente, et al. (2007) also cloned and overexpressed
aglA gene from Thermus sp. strain T2 of a thermostable alpha-
galactosidase, in Escherichia coli strain MC 2508 and the purified
enzyme proved to be thermostable, retaining high levels of activity
even after incubation at 708C. The b-galactosidases from hyperthermo-
philes such as Thermotoga maritima (BgalC), Pyrococcus woesei, and
Thermus sp. IB-21 (BgaA) were reported to be highly thermostable and
typically exhibit optimal activity between 80 and 958C and from
pH5.5–6.5 using o-NPG or p-NPG as a substrate. Recombinant
b-galactosidase from T. naphthophila RUK-10 of GH family 42 has
been cloned and expressed in E. coli. The enzyme showed maximum o-
NPG hydrolysis activity at 908C and pH 6.8, and showed optimum lac-
tose hydrolysis activity at 708C and pH 5.8. The excellent thermostabil-
ity and promising transglycosylation activity of this enzyme suggests its
usefulness for industrial applications, especially for the synthesis of
alkyl galactopyranosides (Katrolia et al., 2011). A gene encoding an
extremely thermostable b-galactosidase from Pyrococcus furiosus (Pflac-
tase) cloned and expressed in E. coli BL21. The enzyme exhibited both
b-galactosidase and b-glucosidase activity and displayed optimal activ-
ity at 908C and pH 7.0 in phosphate buffer (Dong, Xufan, Minhui, &
Ziwen, 2014).
A metagenomic investigation was carried out by Zhang et al.
(2013) for the production of b-galactosidase with high thermostability
ET AL.
and to the lactose hydrolyzed products such as galactose and glucose.
The study also explained the importance of metagenomic approach for
microorganisms containing 99% of the natural microflora. The investi-
gators produced b-Galactosidase optimally active at pH 6.8 and 788C;
however, displaying considerable activity between 40 and 708C. Sub-
strate binding modes of 2 GH42 b-galactosidases, Bga B from Geobacil-
lus stearothermophilus, and A4-b-Gal from Thermus thermophilusA4
were compared with know the basis of the catalytic efficiency of the
enzyme. The A4-b-Gal has a catalytic triad (Glu312-Arg32-Glu35) with
an extended hydrogen bond network that has not been observed in
BgaB (Dong, Hai-Qin, Yan-Hui, Hao, & Wei, 2015).
Biochemical studies were carried out in our laboratory on thermo-
stable b-galactosidase extracted from yeast, Kluyveromyces lactis iso-
lated from curd. The enzyme was optimally active at pH 6.8 and
temperature 408C. The divalent cations (Mn12, Mg12) increased the
enzyme activity maximally followed by monovalent cations (Na11, K11,
Li11) Activity of the enzyme could be enhanced by providing reducing
environment to the assay mixture (Pal, Pal, Ramana, & Bawa, 2009).
b-Galactosidase from Bacillus sp. MTCC 864 was extracted, partially
purified and characterized in our laboratory revealed intracellular
b-galactosidase was a metalloenzyme with strict divalent metal ions
such as Mg11 and Mn11 to enhance its activity. The optimum enzyme
activity was found at 50.08C and pH 7.0 and was found to be thermo-
stable and retained 70% of its original activity after 30 min of incuba-
tion at 60.08C. The properties of this enzyme is suitable for hydrolysis
of lactose in milk around neutral pH and other dairy operations (Patil,
Ramana, & Bawa, 2011).
8 | C O L D - A C T I V E b- G A L A C T O S I D A S E S
FROM PSYCHROPHILIC AND
PSYCHROTROPHIC MICROORGANISMS
Cold active enzymes are widely produced by marine bacteria that are
naturally either psychrophiles or psychrotrophs. Dalmaso, Davis, and
Alane (2015) reported that cold-adapted enzymes unfold at moderate
temperatures due to the lower intramolecular stabilizing interactions
necessary for flexibility at low temperatures. Cold-adapted b-galactosi-
dase obtained from marine bacteria have diverse biofunctional charac-
teristics and could hydrolyze lactose producing low-lactose or lactose-
free dairy products at low temperatures (48C). Bacteria which grow at
0–58C and even at temperatures exceeding 258C are considered psy-
chrotrophic/cold-active. Cold-active microorganisms profusely grow at
low-temperatures in comparison to mesophiles. These bacteria could
be isolated from sub-zero temperature habitats of Antarctica and Arctic
pole as well as from marine and fresh water lake sediments, which are
permanently or temporarily exposed to extreme cold-habitats (Singh &
Ramana, 1998; Ramana, Singh, & Dhaked, 2000; Ramana & Singh,
2000; Ramana, Sulekha, & Bawa, 2003). Initially these microorganisms
have been isolated for elucidating their basic mechanism of cold-
tolerance.
An ideal b-galacosidase should be able to hydrolyze the substrate
at pH 6.7–6.8 with substantial activity at lower temperature (4- 88C) to
achieve enzymatic hydrolysis of lactose during shipping and storage of
XAVIER ET AL.
milk. In principle b-galactosidases produced by yeast and fungi are
active in acidic range of pH 5.0–6.5, whereas the b-galactosidase pro-
duced by cold-active Candida sp. had displayed activity between pH
5.0 and 8.5 and was active over a wide range of temperature from 0.0
to 55.08C. The ability of the cold-active bacteria and yeast such as Can-
dida sp., Pseudoalteromonas sp. DFR1, and Pseudoalteromonas DFR6 to
grow in nutrient broth supplemented with 1.0% (w/v) lactose shows
that the enzyme containing biomass can be readily obtained for its
large scale application. The hydrolytic activity of these enzymes in very
dilute buffer systems or even distilled water indicates that these
enzymes can be functional in the absence of buffer. We suggest that it
is useful property for this enzyme application in food processing, where
buffer addition is impractical. Psychrophilic bacteria can grow on lac-
tose as a sole carbon source at less than 58C. b-Galactosidase of
Arthrobacter psychrophilus was active even at 08C, while some strains
were capable of producing isozymes (Ramana & Singh, 2000).
A cold-active b-galactosidase was isolated from Paracoccus sp. 32d
by Wierzbicka et al. (2011). The enzyme was found suitable for the
hydrolyzing lactose at refrigerated temperatures in milk. Hydrolysis of
lactose is also needed to reduce its hygroscopicity in addition to
increasing sweetness and reducing lactose intolerance. The cold active
b-galactosidase is found to reduce mesophile contamination during the
hydrolysis of lactose in milk and to save energy in industrial scale proc-
esses. A novel bgaL gene from Paracoccus sp. 32d encoding a novel
cold-active b-D-galactosidase and expressed in E. coli and enabled effi-
cient production of soluble form of BgaL Paracoccus sp. 32d, a new
industrial biocatalyst for efficient lactose hydrolysis in milk. A novel
gene encoding a cold-adapted b-galactosidase (R-b-Gal) belonging to
GH 42 from a psychrophilic gram-negative bacterium Rahnella sp. R3
isolated and expressed in E. coli BL21 (DE3) by Fan et al. (2015). In
solution, the enzyme was found to be a homo-trimer and was active at
temperatures as low as 48C while the purified enzyme displayed Km val-
ues of 6.5 mM for ONPG and 2.2 mM for lactose at 48C. Another cold
active b-galactosidase from Planococcus sp-L4 (bgal) was optimized for
expression of recombinant “BGalP” in Pichia pastoris (Mahdian et al.,
2016). It was active at temperatures ranging from 0.0 to 558C and
denatured at or above 608C with an optimal activity at pH 6.5. The
enzyme aids in breakdown of lactose in chilled milk and gets deacti-
vated on pasteurization which would minimize energy consumption
and also found to be useful in preservation of nutritional elements of
milk. Cold active b-galactosidase had a strong preference toward tiny
and small amino acids with more of a -helix region attributed for tem-
perature tolerance, whereas high temperature inhabitants had higher
content of basic and aromatic amino acids with higher percentage of
sheet and coil region. Rao, Rao, Vimal, and Kamlesh (2015) reported
preference or prevalence of certain amino acids in primary and second-
ary structure of psychrophilic, mesophilic, and thermophilic b–galacto-
sidases. Cold-adapted b-galactosidase capable of producing low-
lactose or lactose-free dairy products at low temperatures (48C) was
extracted from Rahnella sp. R3. The enzyme was encapsulated using
gellan gum using calcium and magnesium based hydrogel beads to
increase stability and usage. b-Galactosidase was found to release
more rapidly from the Ca21 hydrogel beads than the Mg21 hydrogels
| 7 of 15
during storage in aqueous solution which could be attributed to the
larger inner/surface pores of the calcium matrix (Fan, Jiang, Xiao,
Yuzhu, & Ruijin, 2017).
b-Galactosidase from the Antarctic marine bacterium P. haloplank-
tis was studied for hydrolysis of lactose in whey permeate. Whey per-
meate contains a valuable amount of lactose, which is an interesting
source for d-galactose production, which in turn can be converted into
the alternative low-calorie bulk sweetener d-tagatose. Optimal hydroly-
sis efficiencies were reached at an incubation temperature of 238Cand
at a pH of 7.0 and 8.0, although a higher production of GOS was
observed in whey permeate at 238C compared with 158C, indicating
the promotion of GOS formation at a higher temperature in whey per-
meate. These results indicated lactose hydrolysis of whey permeate at
refrigeration temperatures reducing the risk of microbial contamination,
an important factor during dairy products processing and for produc-
tion of GOS at higher temperatures (Voorde, Goiris, Syryn, Van den, &
Aerts, 2014).
A gene encoding a novel b-d-galactosidase from the psychrotoler-
ant Antarctic bacterium Arthrobacter sp. 32cB was cloned and
expressed in E. coli. Two units of the enzyme hydrolyzed 90% of the
lactose in 1 mL of milk at 108C in 24 hr. GOS were also synthesized in
temperature range from 10 to 308C along with heterooligosaccharides
such as lactulose, galactosyl-xylose and galactosyl-arabinose, alkyl gly-
cosides, and glycosylated salicin from lactose and the appropriate
acceptors at 308C. b-d-Galactosidase from Arthrobacter sp. 32cB is an
interesting candidate for industrial removal of lactose from milk and a
promising tool for the glycosylation of various thermosensitive accept-
ors (Anna et al., 2014).
9 | CELL AND ENZYME IMMOBILIZATION
AND PROCESS OPTIMIZATION
Application of soluble enzymes for industrial scale production of lac-
tose hydrolyzed milk is limited in view of high enzyme costs. Logistics
of large scale preparation of lactose hydrolyzed milk could be improved
using immobilized enzyme preparations in continuous reactors. Enzyme
immobilization methodology offers various advantages such as efficient
reuse of the enzyme, continuous operation, enhanced stability, under
storage and operational conditions, trouble-free separation from the
reaction medium, minimizing, or eliminating protein contamination of
the product, low or no allergenicity, due to the inability of since an
immobilized enzyme to penetrate the skin easily (Sheldon & Van Pelt,
2013). Enzymes immobilized in various matrices facilitate enzyme reus-
ability, continuous bioprocessing, better control over the biochemical
process, and product formation along with automation. Immobilized
enzymes are preferred over free enzymes owing to their stability at
over a wide range of pH and temperatures. The immobilized enzymes
are easy to handle and are essential to avoid product contamination
with enzymes when the products are intended for application as foods
and pharmaceuticals. It is widely reported that immobilized enzymes
are robust and can be functional in organic solvents owing to protein
stabilization in the process of immobilization. Broadly enzyme immobili-
zation is achieved by adsorption, covalent bonding, entrapment, or
8 of 15 | XAVIER
through cross-linking of enzyme molecules. All the above process has
been employed for immobilization of b-galactosidase for application in
process of lactose hydrolysis in milk and whey products as well as for
the preparation of oligosaccharides (Razi & Sardar, 2016). The moder-
ate stability of enzyme b-galactosidase is a major limitation for its appli-
cations in food and dairy industry which could be stabilized by using
strategies such as enzyme immobilization. The increase in the enzyme
stability in order to facilitate repetitive usage in diverse processing con-
ditions including continuous reactors is the major advantage of immobi-
lized enzyme technology. Subunit-subunit interactions of multimeric
enzymes could be stabilized or enhanced by covalent immobilization
using various supports (Fernandez-Lafuente, 2009; Mateo, Urrutia, Gui-
san, Wilson, & Illanes, 2013). The covalent immobilization of enzyme
also protects it from high temperature, microbial contamination, and
oxidative inactivation. Intermittent rinsing and sterilization by method
of pasteurization using acetic acid solution or quaternary ammonium
salt such as benzalkonium chloride along with changing in direction of
medium feeding would be helpful in reducing the problems of microbial
contamination, protein adherence and channeling (Honda et al., 1987;
Honda et al., 1993; Honda & Takafuji,1987). Increased heat stability of
lactases through immobilization methods were reported (Zhou & Chen,
2001). Moderate stability of b-galactosidase is one of the limitations
that hamper its implementation for industrial scale applications. For lac-
tose hydrolysis, addition of immobilized b-galactosidase directly to
whole milk during large scale industrial applications is cost-effective
due to the possibility of reuse of the enzyme after complete lactose
hydrolysis. Centrale del Latte of Milan, Italy, utilized the SNAM Progetti
technology for cellulose triacetate fiber entrapped Kluyveromyces lactis
lactase.
Microenvironment of enzymes tend to get altered due to varied
interactions of matrices and substrates/products based on physical and
chemical properties and pH is one of the important factors which gets
altered due to immobilization thus changing the optimum pH for
enzyme activity. The change in acidic and basic amino acid side chain
ionization around its active site and surrounding microenvironment
results in shift in optimum pH, that is, broadening of pH range or shift-
ing to acidic or basic range. The charge and surface area of the matrix
plays a crucial role along with the type of matrix used for immobiliza-
tion. A positively charged matrix leads to repulsion of protons and
attracts electrons thus shifting the optimum pH toward the basic side.
In the same way, negatively charged matrix shifts the optimum pH to
the acidic side. The surface charge density of the matrix is important
for the shift in pH. The optimum pH range is broadened when a large
quantity of enzyme activity is available per unit of matrix resulting in
increased concentration of products, due to high intrinsic specific activ-
ity termed as Zulu effect. The substrate molecules would be hindered
from reacting with active site due to increased enzyme concentration
at surface or inside of the matrix used for immobilization. This mecha-
nism leads to constant reaction rates for longer duration despite varia-
tions in pH of the reaction mixture (Dwevedi, 2016).
For food applications, polymers of biological origin are favored
while simple methods and inexpensive supports are important for
industrial applications. The enzyme immobilization methods using
ET AL.
substrates such as micro porous sheets, glass beads, and carbon for
packed bed reactors and alginate, cellulose acetate, and titanium diox-
ide for process applications may result in increased activity by provid-
ing a suitable microenvironment and better thermostability, and pH
tolerance for the enzyme. Covalent attachment of enzymes to chitosan,
polyurethane foam, alginate, and gelatin have been investigated to a

large extent (Garcia-Galan, Angel, Roberto, & Rafael, 2011). An immobi-
lized Kluyveromyces lactis lactase entrapped in cellulose triacetate fibers
was the first commercial application of immobilized enzyme by the firm
Centrale del Latte of Milan, Italy. Sumitomo Chemical, Japan, also
immobilized b-galactosidase on a amphoteric ion exchange resin of
phenol formaldehyde polymer and is used for producing lactose free
milk and hydrolyzed whey (Parmjit, Panesar, Shweta, & Reeba, 2010).
Pessela et al. (2008) have used a battery of new heterofunctional
epoxy supports to immobilize beta-galactosidase. The capability of a
standard Sepabeads-epoxy support to immobilize beta-galactosidase
from Thermus sp. strain T2 can be equal with other Sepabeads-epoxy
supports partially modified with boronate, iminodiacetic, metal che-
lates, and ethylenediamine. Immobilization yields depended on the sup-
port, ranging from 95% using Sepabeadsepoxy-chelate, Sepabeads-
epoxy-amino, or Sepabeads-epoxy-boronic to 5% using Sepabeads-
epoxy-IDA. Modulation of catalytic properties of multimeric enzymes
using various immobilization strategies were studied using
b-galactosidases obtained from E. coli by using substrates such as
glyoxyl, epoxy, or BrCN groups or by glutaraldehyde crosslinking on
matrices containing primary amino groups were studied.
b-Galactosidase was used as a model multimeric enzyme, whose active
center may be determined by the exact assembly of various enzyme
subunits, which causes alterations in catalytic properties by slight dis-
tortion in the enzyme subunit. Although, yield and activity recoveries
were 100% for immobilized enzyme the specificity was altered. Euper-
git C immobilized b-galactosidases indicated the possibility of obtaining
enzymes with different properties using various immobilization meth-
ods (Pessela et al., 2007). Lactose hydrolysis by covalently immobilized
b-galactosidase into carrageenan coated with chitosan was accom-
plished by Elnashar and Yassin (2009). Immobilized cold-active
recombinant b-galactosidase of Antarctic marine bacterium Pseudoal-
teromonas sp. 22b on glutaraldehyde-treated chitosan beads was
reported by Klein et al. (2013). The immobilized preparation was not
inhibited by glucose and optimum temperature for activity was 508C,
optimum pH range was pH 6–9 and was stable in a broad range of
NaCl concentrations (up to 3 M). The immobilized b-galactosidase was
active for 40 days and continuously hydrolyzed lactose at 158C and
was shelf stable at 48C up to12 months. Immobilization of the yeast K.
lactis NRRL Y-1104 in agar was also found to impart remarkable and
prolonged enzyme stability over a period of 12–18 months. The K. lac-
tis b-galactosidase covalently immobilized by Ansari, Rukhsana, San-
desh, and Mohd (2013) on multiwalled carbon nanotubes (MWCNTs)
functionalized by glutaraldehyde increased binding of enzyme and was
found to be an ideal matrix for enzyme immobilization. The optimal pH
for soluble and immobilized b-galactosidase was observed at pH 7.0
while the optimal operating temperatures were 40.0 and 50.08C,
respectively. Moreover, the immobilized b-galactosidase retained
XAVIER ET AL.
greater biocatalytic activity at higher galactose concentration and upon
repeated uses as compared with enzyme in solution. b-Galactosidase
from K. fragilis immobilized on chitosan coagulated with KOH and acti-
vated with glutaraldehyde, showed immobilization yield and recovered
activity up to 100%. Immobilized b-galactosidase was 3- to 5-fold
more stable than the soluble enzyme at 40 and 208C, respectively. The
immobilized biocatalyst was capable of efficiently hydrolyzing lactose
into the whole milk at 258C, and showed similar performance even
after four consecutive batches (Vieira et al., 2013).
In recent times, even nanoparticles are investigated as efficient
enzyme immobilization matrices. Furthermore, magnetic nanoparticles
have attracted much attention for enzyme immobilization as they are
readily recoverable from reaction milieu. Biospecific adsorption of A.
oryzae b-galactosidase on glutaraldehyde functionalized nanodiamonds
(NDs) of 20 nm size with enzyme activity of 7,420 U/g was reported.
The optimal pH and temperature for soluble and immobilized
b-galactosidase was observed at pH 4.5 and at 508C, respectively.
Modified NDs bound b galactosidase showed improved hydrolysis of
lactose from solution in batch processes at various temperatures even
after 10 hr even after 2 months of storage, thereby suggesting its use
for hydrolyzing lactose in dairy products. The use of packed-bed reac-
tor (PBRs) for lactose hydrolysis and GOS synthesis may replace batch
reactors, with several cost reductions and operational advantages,
including reduced reaction inhibition by substrate and products (Ansari
et al., 2015). Immobilization of b-galactosidase from Kluyveromyces lac-
tis on glutaraldehyde-activated chitosan and used in a PBR for continu-
ous hydrolysis of lactose and synthesis of GOS was studied.
Immobilization increased the range of operational pH and temperature.
Almost complete lactose hydrolysis was achieved for both milk whey
and lactose solution at 378C at flow rates up to 2.6 mL/min. Maximal
GOS concentration of 26 g/L was obtained at a flow rate of 3.1 mL/
min, with a productivity of 186 g/L/h. Steady-state operation for 15
days showed the reactor stability concerning lactose hydrolysis (Klein
et al., 2013). In a recent study, b-galactosidase was immobilized on chi-
tosan coated Fe3O4 nanoparticles with or without tris(hydroxymethyl)
phospine, the immobilized enzyme was found with high activity in a
wide range of temperatures and pH of in the case of immobilized
enzyme preparations. The enzyme was used for the production of GOS
using 36% (w/v) lactose resulting in 50.5% GOS on dry weight basis.
Immobilization of b-galactosidase on magnetic nanoparticles are mainly
for feasible recovery of the enzyme and reuse for economic considera-
tions (Chen, Qiaojuan, Zhengqiang, Yu, & Yuchen, 2015). Peirce et al.
(2016) reported coimmobilization of b-galactosidase from Aspergillus
oryzae and lipase B from Candida antarctica, where the combi-catalyst
provided optimum conditions for lipase immobilization on octyl-
agarose. The reduced stability of b-galactosidase even after multipoint
covalent attachment permitted the reuse of the most stable enzyme
after inactivation of the least stable b-galactosidase used for coimmobi-
lization. The combi-catalyst could be inactivated using 300 mM NaCl at
608C and pH 7, to rapidly inactivate b-gal without altering the activity
of coimmobilized lipase. Klein et al. (2016) reported immobilization of
b-d-galactosidase obtained from Aspergillus oryzae on a chitosan sup-
port with genipin, a naturally occurring cross linking reagent for food
| 9 of 15
processing applications. The food grade chitosan–genipin particles
showed increased operational stability maintaining 100% of the initial
activity of enzyme after 25 batches of lactose hydrolysis in comparison
to the traditional methodology of immobilization using glutaraldehyde
which is not food grade. Pectinex Ultra SP-L,a commercial preparation
of b-galactosidase extracted from Aspergillus aculeatus was covalently
immobilized using glyoxyl-modified mesoporous silica supports and
assayed for production of GOS. The yield of GOS was 20.2% by trans-
galactosylation activity of enzyme within the porous support frame-
work whereas yield was only 11.2% in free conditions. In addition,
these silica-based biocatalysts were reusable for three times even at
508C the thermal deactivation temperature of the enzyme (Gonzalez-
s (2017) also
Delgado et al., 2018). Guerrero, Carlos, and Andre
reported highest yields of lactulose using Aspergillus sp. b-galactosidase
immobilized in glyoxyl-agarose (GA-bG), amino-glyoxyl-agarose (Am-
GA-bG), and chelate-glyoxyl-agarose (Che-GA-bG) in comparison to
free enzyme. Immobilized b-galactosidase on macrospheres of chitosan
synthesized products such as lactosucrose (79 g/L), GOS (37 g/L), and
250 g/L of total oligosaccharides at pH 7.0 and 308C. The immobiliza-
tion also increased the thermal stability up to 260-fold. The maximal
lactosucrose synthesis occurred for 30 cycles on substrate composition
of 300 g/L of sucrose, 300 g/L of lactose, and 8.5 mg of chitosan per
mL which fulfills the important criteria of enzyme reuse through immo-
bilization (Duarte et al., 2017).
b-Galactosidases produced in our laboratory using Kluyveromyces
lactis NRRL Y-1104, Pseudoalteromonas sp. DFR1, and Pseudoalteromo-
nas DFR6, were found to be suitable for lactose hydrolysis at sub-
ambient temperatures. Whereas another K. lactis sp. isolated from curd
sample was found to be thermostable but inactivated in the presence
of acetone. The K. lactis N-1104 was particularly suitable for immobili-
zation as it could with stand treatment with ethanol and acetone (Pal,
Pal, & Ramana, 2007). The enzyme from B. coagulans was also found to
be thermostable as well as resistant to treatment with organic solvents
such as acetone and ethanol (Patil et al., 2011). We have been working
with lactic acid bacteria, Antarctic bacteria, Bacillus sp. and K. lactis and
Kluyveromyces sp. to produce the cell bound (intracellular and extracel-
lular) b-galactosidase resulting in the development of processes for
their utilization to lactose hydrolysis. Because these microorganisms
could produce cell bound b-galactosidase various permeabilization
methods have been developed using ethanol/CTAB in buffer, toluene–
chloroform mixture, and acetone in phosphate buffer. The permeabl-
ized lactic acid bacterial biomass was utilized for the preparation low-
lactose containing milk products. Various immobilization methods such
as agar, cellulose acetate, and cellulose sheet have been developed
which are the prominent for their real time applications for the cell
bound enzyme. The enzyme activity in permeablized whole cells dis-
played remarkable stability in agar entrapped yeast cells over a period
of 12–18 months when stored at 5–68C. The yeast biomass immobili-
zation in cellulose acetate demonstrated remarkable stability wherein
the entrapped enzyme was found to be highly suitable for multiple
uses (as high as 60 times on repeated washing buffer and incubation
with substrate mixture) over a period of 9–10 days (Pal et al., 2009).
10 of 15 | XAVIER
10 | LACTOSE HYDROLYSIS USING
BIOREACTORS
Application of soluble b-galactosidases on industrial scale is limited
mainly due to the logistics involved in use of enzymes. Immobilized
enzymes and cells enable the optimum utilization of their activity in a
bioreactor resulting in increased overall process efficiency. GOS pro-
duction in a continuous packed bed reactor (PBR) using immobilized b-
galactosidase could be an alternative for GOS production. Fed-batch
fermentation (3,694.6 U/L) and semi-continuous fermentation (5,551.9
U/L) strategies further enhanced b-galactosidase production by 1.8
and 2.8 fold, respectively (Choonia & Lele, 2013). A highly active and
stable derivate of immobilized B. circulans b-galactosidase was
prepared for the synthesis of GOS under repeated-batch operation.
B. circulans b-galactosidase was immobilized on monofunctional glyoxyl
agarose and three hetero functional supports: amino-carboxy-, and
chelate-glyoxyl agarose. Glyoxyl agarose was the support with highest
immobilization yield and stability being selected for central composite
rotatable design for optimization of immobilization conditions and
application in GOS synthesis. Optimal conditions of immobilization
were 28.9 mg/g and 36.4 hr of contact, resulting in a biocatalyst with
595 IU/g and a half-life 89-fold higher than soluble enzyme. Immobili-
zation process was found not to alter the synthetic capacity of
b-galactosidase, obtaining the same GOS yield and product profile than
the free enzyme. GOS yield and productivity also remained unchanged
along ten repeated batches, with values of 39% (w/w) and 5.7 g GOS/
g of biocatalyst per batch. Total product obtained after 10 batches of
reaction was 56.5 g GOS/g of biocatalyst (1956 g GOS/g protein).
Cumulative productivity in terms of mass of contacted protein was
higher for the immobilized enzyme than for its soluble counterpart
from the second batch of synthesis onward (Mateo et al., 2013). War-
merdam et al. (2014) reported that the product composition of GOS
obtained on using immobilized enzyme in a PBR was on par with GOS
composition obtained using a batch reactor with free enzyme, and the
productivity of GOS was six times higher. The immobilized enzyme
was stable at a lactose concentration of 38% (w/v) and at 508C while
the half-life time of the immobilized enzyme was around 90 days.
Carević et al. (2016) reported immobilization protocol
b-galactosidase extracted from Aspergillus oryzae using Purolite® A-
109, a polystyrenic macroporous resin for GOS synthesis in a bioreac-
tor. The immobilized enzyme retained 73% activity after 10 cycles of
usage and yielded GOS of 100 g/L in a fluidized bed reactor in a
medium containing 400 g/L lactose, pH 4.5, 508C. The immobilized
enzyme was found to have higher affinity toward catalyzing transgalac-
tosylation than hydrolysis and shift of pH optimum toward more acidic
conditions. Vasileva, Yavor, Stanka, Iliana, and Tzonka (2016) studied
the covalently immobilization of b-galactosidase using a polypropylene
membrane modified using glutaraldehyde. The lactose hydrolysis and
stability for the immobilized enzyme was found to be 1.6- and 2.0-fold,
respectively. Glucose–galactose syrup from waste whey was obtained
by lactose hydrolysis in a bioreactor using the immobilized enzyme on
a spirally wound membrane. After 10 hr, the degree of lactose hydroly-
sis increased to 91% and even after 20th cycle the yield of bioreactor
ET AL.
was 69.7%. Al-Qodah, Al-Shannag, Al-Busoul, Penchev, and Orfali
(2017) recently reviewed the usage of immobilized enzyme bioreactors
utilizing magnetic field where the magnetic supports or beads with
immobilized enzymes as the solid phase. The magnetic field was found
to be employed in bioreactors for purposes such as mixing, attracting
the particles to prevent their washout from the column and to operate
with higher substrate velocities to enhance mass transfer processes.
The magnetic field enhanced the activity and conversion rates of the
immobilized enzyme which could be due to the intensive mixing or the
operation with higher medium flow rates leading to improved mass
transfer between the medium and enzyme.
We have designed and developed a bioreactor for lactose hydroly-
sis in milk and other liquids that contain lactose as constituent sugar.
Bioreactor technology for the hydrolysis of lactose in milk through an
immobilized enzyme was developed in our laboratory (Ramakrishna,
Ramana, & Bawa, 2005). An Indian patent titled has been granted for
the development of temperature controlled immobilized enzyme bio-
reactor (Ramana, Ramakrishna, & Bawa, 2015). The bioreactor has
been developed for the retention of immobilized enzyme preparation
for continuous operation at selected temperature between 5.0 and
75.08C, employing different immobilized enzyme preparations for dif-
ferent operational temperatures. The bioreactor temperature is adjusta-
ble by continuous circulation of cold or hot water to maintain the
temperature. The bioreactor is intended for use at lower, ambient and
high temperatures and comprises of three concentric cylinders of dif-
ferent diameters. The lower and high temperatures are preferred for
reasons of minimizing the microbial contamination. Most of the bio-
reactors reported previously by various workers are simple columns for
use as packed bed columns without much concern for temperature
control of the reactor.
11 | PREBIOTIC OLIGOSACCHARIDE
SYNTHESIS
Transgalactosylation is inherent property associated with
b-galactosidase and oligosaccharide formation occur during lactose
hydrolysis. The transfer of galactosyl moiety of lactose into acceptor
for molecules containing hydroxyl groups other than water yields, prebiotic
GOS with varied degree of polymerization and different glycosidic link-
ages (Park & Oh, 2010). b-linked oligosaccharides with a degree of
polymerization (DP) of 2–9 that are composed of galactose and may
contain one glucose unit, typically at the reducing end are termed as
GOS (Van Leeuwen, Kuipers, Dijkhuizen, & Kamerling, 2016). Depend-
ing upon the substrate, transgalactosylation of lactose by
b-galactosidase yields a mixture of glucose, galactose, lactose, and
GOS. Production of delactosed milk and dairies has been traditionally
carried out by the hydrolytic activity of b-galactosidases, while transga-
lactosylation activity is used for synthesis of lactose-derived oligosac-
charides. Transgalactosylation takes place at elevated temperatures
from concentrated lactose solutions so b-galactosidases acting at ele-
vated temperatures would be helpful in achieving high reaction rates
and higher GOS yields (Hsu, Yu, & Chou, 2005; Park & Oh, 2010).The
prebiotic properties and increasing the yield of GOS through process
XAVIER ET AL.
engineering were reviewed by Gänzle (2012) and Macfarlane et al.
(2008) while the structure of GOS has been elucidated by Van Leeu-
wen et al. (2016). Chen, Hsu, and Chiang (2002) reported that the
amount of GOS formed in milk is much less in comparison to the con-
centration of lactose in buffer solutions. Two protocols were used for
making low-lactose and high-oligosaccharide milk. In the first method
b-galactosidase was used to transform lactose in evaporated milk into
oligosaccharides, while in the second ultra-filtration was used to sepa-
rate the lactose from milk and the permeate devoid of milk proteins
was concentrated by evaporation. Furthermore, GOS production was
investigated by an immobilized enzyme preparation by Neri et al.
(2009). The enzyme immobilization was achieved using magnetic poly-
siloxane–polyaniline particles.
b-Galactosidases from A. oryzae and B. circulans exhibited high
transgalactosylation activity, while those from Kluyveromyces exhibited
high hydrolytic activity but quite low transgalactosylation activity. The
effect of enzyme to substrate ratio, initial lactose concentration and
temperature has been studied for the kinetically controlled reaction of
lactose transgalactosylation with A. oryzae b-galactosidase, to produce
prebiotic galacto-oligosaccharides (GOS) was reported. Highest yield of
29 g GOS/100 g lactose added was obtained at a lactose monohydrate
initial concentration of 50% (w/w) and 47.58C. Highest specific produc-
tivity of 0.38 g GOS hr21 mg enzyme21 was obtained at lactose mono-
hydrate initial concentration of 40% (w/w) and 558C, where a
maximum yield of 27 g GOS/100 g lactose added was reached. When
lactose precipitation occurred, values of yields and specific productiv-
ities lower than 22 g GOS/100 g lactose added and 0.03 g GOS hr21
mg enzyme21 were obtained, respectively (Vera et al., 2012).
Galactosidase from A. oryzae was immobilized in glyoxyl-agarose
for the synthesis of GOS from lactose at high concentrations. In the
sequential batch production of GOS, biocatalyst efficiency was
increased by 190% with respect to the free enzyme in solution, and
8,500 g of GOS per gram of enzyme preparation were produced after
ten batches (Huerta, Carlos, Cecilia, Lorena, & Andres, 2011). Enzy-
matic transgalactosylation of lactose in presence of fructose using
b-galactosidase from K. lactis leading to the formation of oligosaccha-
rides was investigated in detail by Shen et al. (2012). The reaction mix-
ture discovered two main transgalactosylation products such as allo-
lactulose as the major transgalactosylation product and lactulose the
minor one. The maximum yields of allolactulose and lactulose were
47.5 and 15.4 g/L, respectively, at 66.5% lactose conversion.
Palai et al. (2014) immobilized b-galactosidase enzyme on a hydro-
phobic PVDF membrane by cross-linking via glutaraldehyde and inves-
tigated GOS formation feed under recirculation loop. Maximum GOS
formation was observed to be 30% at 50 g/L ILC, a 0.5 mL/min feed
flow rate (1.5 mL/min recirculation rate), and 408C. The immobilized
enzyme could be stored for longer periods of time without losing much
initial activity as compared with the native enzyme. The usage of
immobilized enzyme in a continuous packed bed reactor (PBR) is a
good alternative for GOS production instead of the traditional use of
free enzyme in batch reactor. The stability of the immobilized enzyme
at a lactose concentration of 38% (w/v) and at 508C was very high
with a half-life time of approximately 90 days. The enzymatic
| 11 of 15
productivity of GOS production using immobilized enzyme in a PBR
could be more than six times higher than that of GOS production with
free enzyme in a batch reactor as reported by Warmerdam et al.
(2014).
GOS were synthesized by enzymatic transgalactosylation in
skimmed milk permeate fortified with lactose 40% (w/w) using
b-galactosidases derived from A. aculeatus, A. oryzae, K. lactis, and B.
circulans. The GOS yields expressed as a percentage of the initial lac-
tose content were 41.0%, 21.0%, 13.0%, and 11.0% under optimal con-
ditions. B. circulans enzyme also revealed highest affinity to
transgalactosylation relative to hydrolysis. HPAEC-PAD/MS results
showed similarities in GOS are mainly di-, tri-, and a few tetra-
saccharides, especially with b-galactosidases from A. oryzae, A. aculea-
tus, and K. lactis with a preference toward b-galactosyl residues b(1–6)
(Frenzel, Katja, Clawin-Rädecker, & Peter, 2015). Some of the enzyme
properties play a crucial role for development of optimal bioprocesses
based on b-galactosidases for lactose hydrolysis and GOS formation
are listed below:
(a) Extensive screening to find suitable microorganisms with superior
enzyme traits.
(b) Identification of renewable and readily available media
ingredients.
(c) Availability of thermostable and cold active enzymes for reducing
the chances of contamination during the process.
(d) b-galactosidase preparations free from contaminating proteases
and lipases to ameliorate the quality of the product.
(e) Immobilized b-galactosidase preparation for continuous
operation.
(f) Constitutive production of b-galactosidase in microorganisms
without the need for incorporating inducers and to overcome
complex regulatory mechanisms.
(g) Protein engineering and heterologous expression of enzyme for
enhanced stability and to scale-up of production.
(h) Screening for enzymes with low feedback-inhibition in the pres-
ence of high concentrations of glucose and galactose.
(i) Solvent tolerant version of b-galactosidase for organic synthesis
in organic solvents.
(j) Effective use of aqueous-two phase systems for enzyme purifica-
tion or single step methods such as expanded bed chromatogra-
phy for enzyme purification which may lead to cost effective
production of enzymes thereby leading to their large scale appli-
cations in food processing.
12 | CONCLUSIONS
b-Galactosidase is a widely occurring enzyme in plants, animals, yeast,
and molds, and bacteria in the nature with varied biochemical proper-
ties and production pattern in each genus. Although the purified
enzyme from yeast and molds is already in use, the lactic acid bacteria
12 of 15 | XAVIER
are equally promising for lactose hydrolysis due to their GRAS status.
Much information is available on the Streptococcus thermophilus and its
b-galactosidase for milk processing applications. The advantages
offered by b-galactosidases of thermophiles and psychrophilic microor-
ganisms are very novel and their application needs further in-depth
study through production of these enzymes by expressing in well
described mesophilic hosts. Galacto-oligosaccharides production by
microbial b-galactosidases has been studied at various temperatures by
several groups. Furthermore, protein engineering along with immobili-
zation of the enzyme or the whole permeabilized cell biomass can
make the process economically viable through improved yields and pro-
ductivity. At the same time, the importance of investigations aimed at
revealing the active site residues and protein conformation as well as
subunit structure under the effect various physico-chemical parameters
is also of major importance. The b-galactosidase appears to be more
promising in the preparation of fruit juices and hydrolysis of plant poly-
saccharides comprising of galactose and other hexoses, the enzyme
important for the synthesis of various glycosides of biomedical impor-
tance. In addition several novel applications for these enzymes can be
expected in the near future.
OR CID
Janifer Raj Xavier http://orcid.org/0000-0001-9848-7539
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How to cite this article: Xavier JR, Ramana KV, Sharma RK.
b-Galactosidase: Biotechnological applications in food process-
ing. J Food Biochem. 2018;e12564. https://doi.org/10.1111/
jfbc.12564