Lactose Crystallization, Hydrolysis and Value-Added Derivatives SUPER INTERESANTE
Lactose Crystallization, Hydrolysis and Value-Added Derivatives SUPER INTERESANTE
Review
abstract
Lactose, the most abundant component of milk of most mammals, has been thoroughly studied for its
physico-chemical properties, crystallization behavior and importance as a fermentation medium.
Studies of various approaches to lactose modifications to increase its value as a food ingredient or
nutraceutical component are more recent and presently predominate the research interest concerning
lactose. This review, while summarizing briefly some physico-chemical properties and older studies
concerning crystallization behavior (mutarotatory equilibrium, solubility, crystalline habit and form)
focuses also on the modification alternatives to increase the utilization of lactose through value-added
products. Various approaches to lactose hydrolysis leading to increased solubility, higher sweetness and
expanded availability of milk and dairy products for lactose intolerant consumers are compared with an
emphasis on crude enzyme extracts. Principles and processes for conversion of lactose to lactitol,
lactobionic acid, lactulose, lactosucrose, and galacto-oligosaccharides are highlighted.
& 2008 Elsevier Ltd. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 686
2. Characteristics of lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 686
2.1. Occurrence and properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 686
2.2. Mutarotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 686
2.3. Solubility and sweetness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 686
3. Lactose crystallization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 686
3.1. Crystalline habits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 686
3.2. Shapes and crystallization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 687
3.3. Effects of acidity, alkalinity, and salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 687
3.4. Other milk components, chemicals and food ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 687
3.5. Industrial process conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 687
3.6. Amorphous noncrystalline lactose (lactose glass) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 688
3.7. Lactose in food products—quality and industrial applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 688
4. Lactose hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 688
4.1. Principles and theoretical considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 688
4.2. Industrially applicable alternatives for lactose hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 688
4.3. Lactose hydrolysis by mechanically disrupted bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689
4.4. Future prospects for industrial applications of lactose hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689
5. Conversion of lactose to value added materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689
5.1. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689
5.2. Lactulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 690
5.3. Lactitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 690
5.4. Lactobionic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691
5.5. Lactosucrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691
5.6. Galacto-oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691
6. Conclusions and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 692
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 693
Corresponding author. Tel.: +1780 492 0774; fax: +1780 492 4265.
E-mail address: [email protected] (M.G. Gänzle).
0958-6946/$ - see front matter & 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.idairyj.2008.03.003
ARTICLE IN PRESS
OH OH 3. Lactose crystallization
α−lactose
(Nickerson, 1974). In general, the rate of crystal growth increases (Jelen & Coulter, 1973b; Smart, 1988). Some salts led to significantly
rapidly as supersaturation (or precipitation pressure, expressed as higher crystal growth rates, and their effect was concentration
ratio of actual concentration to solubility) is increased (Twieg & dependent. Thus, salt concentrations per se appear to be important
Nickerson, 1968; van Kreveld & Michaels, 1965). The rate is factors influencing growth rates, with each salt exerting a different
different for the different faces of the crystals, an effect that alters effect. In addition, some salts seem to change the shape of crystals
the shape of the crystals during growth. Crystal surface integra- leading to elongations, needle-like shapes or triangular flake forms
tion is a critical factor in the formation of crystals, while the (Bhargava & Jelen, 1996; Jelen & Coulter, 1973b).
diffusion rate of a-lactose to the surface is not rate-limiting
(Thurlby, 1976). The rate of lactose crystal growth is not much
3.4. Other milk components, chemicals and food ingredients
different from that of sucrose, when considering that the growth
of lactose crystals occurs predominantly or solely on only one
Methanol and ethanol accelerate crystallization by as much as
face—the bottom—of the pyramidal crystal (Bhargava & Jelen
30–60% even at low (1%) concentrations (Nickerson, 1974; van
1996; Jelen & Coulter, 1973a, 1973b). Several older investigations,
Kreveld & Michaels, 1965). Alcohols reduce the solubility of lactose
especially in the Netherlands at the Cooperative Condensfabriek
and support spontaneous nucleation. The latter effect, accelerating
Friesland, focussed on the mechanisms and kinetics of the lactose
the formation of crystals, may support the step theory (step
crystal formation and growth in great detail (e.g. van Kreveld,
generation and rapid growth) in lactose crystallization (Michaels &
1969; van Kreveld & Michaels, 1965; Visser, 1983).
van Kreveld, 1966; Nickerson & Moore, 1974a, b).
In dairy products, the presence of ‘‘impurities’’ or chemicals
3.2. Shapes and crystallization used as food additives may inhibit or accelerate the growth of
lactose crystals, alter crystal form (Jelen & Coulter, 1973b) or have
The pyramid, tomahawk and prism shapes are the most no effect. In some instances, impurities may inhibit the formation
common forms but lactose crystals can be observed in a variety of the nuclei. The effects on crystal growth are a function of
of other shapes, depending on the conditions of crystallization. intricate physico-chemical interactions of available energies,
The principal factor governing the form of lactose crystals is the adsorption rate to the various crystal faces, steric hindrance
supersaturation of the solution (Herrington, 1934). High super- caused by proteins and polysaccharides, tendency to spontaneous
saturation forces rapid crystallization and only prisms form. As nucleation, and the concentration of substances in question
supersaturation decreases, the dominant crystal form changes to (Nasirpour, Scher, Lindner, & Desobry, 2006; Nickerson, 1974). In
diamond-shaped plates, then to pyramids and tomahawks, and the presence of impurities, lactose crystals tend to be irregularly
finally to fully developed crystal showing a multitude of faces shaped and clumped, instead of yielding the characteristic crystals
(Holsinger, 1988; Jelen & Coulter, 1973a; Nickerson, 1979; van obtained from simple lactose solutions (Nickerson, 1962).
Kreveld & Michaels, 1965). More recent studies demonstrated the Gelatine is an example of a crystallization inhibitor that
physico-chemical interrelationships of aqueous lactose solutions reduces the rate by 25–60%. However, gelatine cannot suppress
at different degrees of supersaturation, temperatures, different nucleation in highly supersaturated lactose solutions, which
stages of crystallization and in the presence or absence of explains its ineffectiveness in preventing sandiness in ice cream
different water-miscible organic solvents (Zeng, Martin, Marriott, (Nickerson, 1962). On the other hand, various marine and
& Pritchard, 2000b). Although the majority of lactose crystals vegetable gums are now widely used in ice-cream formulations,
were found to be either tomahawk-shaped or pyramidal, at higher because they inhibit the formation of lactose crystal nuclei.
concentrations elongated cuboidal crystals were observed, with Carrageenan-containing systems have been shown to inhibit
higher initial lactose concentrations resulting in more elongated crystallization (Kouassi, Jouppila, & Roos, 2002). Gels can provide
particles. a ‘‘protective environment’’, thus presenting a ‘‘control’’ mechan-
ism for the rate of crystallization as well as the uniformity of the
crystals (Zeng, Martin, Marriott, & Pritchard, 2000a).
3.3. Effects of acidity, alkalinity, and salts
Valio, one of the principal industrial pioneers in the field of lactose Enhancement of starter culture production
hydrolysis. The patented process is based on removing most of the Development of new flavors
lactose from regular milk by a chromatographic process developed New product ideas combining lactose hydrolysis and flavor developments
by Harju (1987b), followed by hydrolysis of the remaining lactose New/modified products (whey cheeses, spreads, drinks, ‘‘milk honey’’, dairy-
based confectionery)
by a soluble enzyme. Now marketed successfully in several
Scandinavian and other European countries, the Finnish process
results in comparable taste to the regular cows milk and avoids
the uncharacteristic excess sweetness caused by liberated glucose cells and production of ‘‘enzyme cocktails’’ with high b-galacto-
and galactose associated with traditional in situ lactose hydrolysis sidase activities. The application of these ‘‘in-house’’ produced
methods (Jelen & Tossavainen, 2004). cocktails for lactose hydrolysis under carefully selected conditions
The requirement for enzyme preparations in the free enzyme could result in optimal hydrolysis rates, without significant
approach is reduced by membrane reactors in which the soluble detrimental effects of other enzymatic processes. Our research,
enzymes are being recovered for repeated use (e.g., Mehaia, using a high b-galactosidase producer Lactobacillus delbrueckii
Alvarez, & Cheryan, 1993). However, the practicality and econom- subsp. bulgaricus strain ATCC 11842, focussed on the economic
ical feasibility of this technology in industrial applications seems (Bury & Jelen, 2000) and technical (Kreft, Roth, & Jelen, 2001)
doubtful. Effectiveness of membrane reactors for lactose hydro- feasibility of the process; optimization of the growth media
lysis in milk or whey is affected by several of the typical problems (Vasiljevic & Jelen, 2001) and of the mechanical disruption process
of the membrane processes (membrane fouling, membrane (Bury, Jelen, & Kalab, 2001; Geciova, Giesova, & Jelen, 2002);
selectivity, effects of some solutes on flux) as well as by the suppression of competing enzymatic reactions (Vasiljevic & Jelen,
gradual loss of enzyme activity. 2002); stability of the enzyme during the hydrolysis process (Kreft &
At the height of the ‘‘lactose hydrolysis euphoria’’ in the mid- Jelen, 2000); and sensory aspects of the final products (Vasiljevic,
1980s, developments of the immobilized enzyme technology Wismer, & Jelen, 2003). Clearly, before this approach could become
appeared to offer the ultimate solution. The much publicized successful, additional research would be needed, including a search
‘‘Corning process’’, involving enzyme immobilization on sintered for a more effective microbial producer of the enzyme and/or
glass beads, seemed to be ‘‘winning the race’’ of the day involving genetic engineering leading to significant overproduction
(Anonymous, 1981, 1984). Unfortunately, even in this very of the b-galactosidase and suppression of some of the other enzymes
promising case, the unfavorable economic realities and technical catalyzing potentially undesirable reactions.
difficulties resulted its final demise. Several other commercial
processes using the immobilized enzyme approach were tabu-
4.4. Future prospects for industrial applications of lactose hydrolysis
lated by Harju (1987a) and the most prominent ones described
briefly by Sienkiewicz and Riedel (1990). The Finnish develop-
ment by the Valio company, based on the b-galactosidase Lactose hydrolysis as a major industrial process appears to be
immobilization on a special resin, is still being used for grossly underutilized today. The amount of lactose available just
production of a successful whey drink (Jelen, personal observa- in the dried whey produced in Europe and North America can be
tion, 2006) as well as for production of some of the many lactose- estimated as at least 1.4 MT annually (IDF, 2005). In addition to
hydrolyzed dairy products marketed under the HYLA (HYdro- one of the main uses of lactose hydrolysis for alleviation of the
lyzed-LActose) brand (Jelen & Tossavainen, 2004). lactose malabsorption problem, other prospective applications
both in mainstream processing and as a route for by-products
valorization are listed in Table 2. In this regard, lactose hydrolysis
4.3. Lactose hydrolysis by mechanically disrupted bacteria can be considered as belonging to the family of enzymatically
catalyzed or chemical processes leading to added-value conver-
The unfavorable economies of either free or immobilized sion of lactose to other industrially attractive products.
enzyme routes limit the large-scale applications of lactose
hydrolysis for many potential uses. In an attempt to find an
inexpensive solution to this problem, a recent research project has 5. Conversion of lactose to value added materials
been carried out within the framework of an NSERC-supported
Canadian Research Network centered at the Université Laval. The 5.1. Overview
project aimed at using traditional dairy bacteria, known to be high
producers of the b-galactosidase, as an ‘‘in-house’’ source of the An overview on commercially produced derivatives of lactose
enzyme that could be produced by the potential users themselves, is shown in Fig. 2 and their properties and applications are
similarly to the traditional production of bulk cheese starters in outlined below. Currently, only a small proportion of the lactose is
the cheese factories. However, in contrast to starter cultures for used as feedstock for the chemical, enzymatic or microbiological
use in cheese, the fermentative activities of b-galactosidase conversion to lactose derivatives. However, because the market
producing bacteria in the subsequent lactose hydrolysis process value is substantially higher than lactose, these compounds
would be detrimental to many such applications. Thus, the second provide significant opportunities for value-added conversion of
important aspect of this approach is the mechanical disruption of lactose to functional food ingredients. In recent years, attention
the bacterial cultures, resulting in the inactivation of the bacterial was focussed on prebiotic lactose derivatives. Prebiotics were
ARTICLE IN PRESS
O OH
HO O OH
O O OH HO OH
HO
OH O O OH
HO
HO OH
OH Lactobionic acid HO OH
OH Lactulose
isomerisation
oxidation
OH O OH
HO transgalactosylation O OH
HO HO
O O OH O O OH OH
HO O O
HO OH O O
OH OH
HO
HO OH reduction HO OH
HO OH
OH Lactitol OH Lactose HO OH OH
OH
hydrolysis Galacto-oligosaccharides
fructosyl transfer (β-(1-4) or β− (1-6) linkage)
HO HO
HO
O O O
HO OH HO OH OH
O O
HO
OH HO OH HO OH
OH O O OH
HO
Galactose + Glucose OH
HO OH
OH Lactosucrose
defined in 1995 as ‘‘non digestible food ingredients that to act as soluble receptors for pathogens (Kunz et al., 2000). While
beneficially affect the host by selectively stimulating the growth functions other than the prebiotic effects cannot be mimicked by
and/or activity of one or a limited number of bacteria in the colon, lactose derivatives, the addition of prebiotic oligosaccharides to
and thus improve host health’’ (Gibson & Roberfroid, 1995). infant formulae resulted in levels of bifidobacteria in the intestine
Prebiotics are frequently referred to as ‘‘bifidus factor’’ in the older that was comparable to the one found in breast-fed infants
literature. Prebiotic compounds are not digested and adsorbed in (Veereman-Wauters, 2005).
the small intestine, therefore, they are classified as dietary fibre
and contribute to a fibre-rich diet recognized as beneficial to
5.2. Lactulose
human health (Englyst & Englyst, 2005). The end products of the
metabolism of prebiotics by intestinal bacteria are lactate and
As small quantities of lactulose are formed during heating of
short chain fatty acids (SCFA) which contribute to the beneficial
milk, it has been used as an indicator for milk heat treatment
effects of prebiotics (Topping & Clifton, 2001). Moreover, pre-
(Elliott, Datta, Amenu, & Deeth, 2005). Lactulose is produced by
biotics are generally applied to stimulate growth and metabolism
isomerization of lactose in alkaline solution; boric acid shifts the
of bifidobacteria and the consumption of prebiotics results in an
isomerization equilibrium in favor of lactulose and prevents side
increase in both the occurrence and number of bifidobacteria
reactions. Lactulose, the first lactose derivative that was commer-
isolated from fecal material (Böhm et al., 2004; Cummings,
cialized (Kozempel, Kurantz, Craig, & Hicks, 1995; Timmermans,
Macfarlane, & Englyst, 2001). The establishment of metabolically
1997), can be produced enzymatically with b-galactosidases and
active bifidobacteria is considered to be beneficial for the host.
fructose as galactosyl acceptor (Lee, Kim, & Oh, 2004; Mayer et al.,
Beneficial effects may include the regulation of bowel habit,
2004). The relative sweetness of lactulose is 0.6 in comparison to
stabilization of the gut mucosal barrier and the prevention of
sucrose. Lactulose is a non-digestible carbohydrate which is
diarrhea, and increased mineral absorption (Cummings et al.,
fermented in the human colon (Nilsson & Nyman, 2005) but its
2001; Hopkins & Macfarlane, 2003; Lee, 1999). The application of
specific bifidogenic effects is less pronounced when compared with
prebiotics in infant foods is especially relevant to substitute the
fructo-oligosaccharides or galacto-oligosaccharides (Bouhnik et al.,
bifidogenic effect of oligosaccharides that are present in human
2004). Lactulose is widely applied as laxative and administration of
milk at a level of 1–1.3%, whereas the milk of ruminants has much
lactulose or lactitol is a standard treatment for chronic hepatic
lower concentrations of oligosaccharides (Kunz, Rudloff, Baier,
encephalopathy, although the scientific basis for the latter therapy
Klein, & Strobel, 2000). More than 130 different human milk
is questionable (Als-Nielsen, Gluud, & Gluud, 2004).
oligosaccharides were identified. Most compounds carry lactose at
the reducing end and are substituted with N-acetylglucosamine, L-
fucose, and/or N-acetylneuraminic acid (sialic acid). In addition to 5.3. Lactitol
the modulation of the microbial colonization of infants, possible
functions of these compounds are the stimulation of the immune Lactitol is produced by chemical hydrogenation of lactose; the
system, anti-microbial and/or anti-inflammatory protection, and relative sweetness of lactitol is 0.3–0.4 compared with sucrose.
ARTICLE IN PRESS
Lactitol hydrolysis to galactose and sorbitol in the human hydrolyze lactose to glucose and galactose and alternatively
intestine is dependent on the intestinal microflora; most of the catalyze the transgalactosylation of lactose to produce galacto-
lactitol is metabolized to short chain fatty acids by the colonic oligosaccharides (Matthews, 2005, Fig. 3). Depending on the
microflora while a part of the galactose is resorbed (for review, see source of the enzymes, the oligosaccharides have predominantly
Dills, 1989). Lactitol is a strong laxative and its metabolism by b(1-4) and/or b(1-6) linkages. For example, the b-galactosidase of
humans is insulin-independent. Polyols such as mannitol, xylitol Kluyveromyces lactis produced predominantly b-(1-6) oligosac-
or arabitol, lactitol are applied as non-caloric sweeteners in charides (60 -galactosyl-lactose and b-D-Gal(1-6)D-Gluc), a b-
calorie-reduced and diabetic foods. Moreover, lactitol is used as an galactosidase of Sterigmatomyces elviae produced predominantly
alternative to lactulose in the treatment of hepatic encephalo- 40 -galactosyl-lactose whereas Bacillus circulans b-galactosidase
pathy (Als-Nielsen et al., 2004). forms b-(1-2), b-(1-3), b-(1-4) or b-(1-6) linkages to produce a
large variety of oligosaccharides (Asp, Burvall, Dahlqvist, Hallgren,
5.4. Lactobionic acid & Lundblad, 1980; Onishi, Yamashiro, & Yokozeki, 1995; Yanahira
et al., 1995). Glucose, galactose, mannose, fructose, maltodextrins,
Lactobionic acid is commercially produced by chemical oxida- N-acetylneuraminic acid, glucuronic acid and a number of
tion of lactose (Gerling, 1997). Alternative methods for the aromatic compounds have been shown to act as galactose-
production of lactobionic acid employing a glucose-fructose acceptor for b-galactosidases, providing a virtually unlimited
oxidoreductase from Zymomonas mobilis were described (Satory variety of oligosaccharides (Bridiau, Taboubi, Marzouki, Legoy, &
et al., 1997). Lactobionate is a strong chelator of calcium and is used Maugard, 2006; Lee et al., 2004; Miyasato & Ajisaka, 2004;
in calcium supplements in pharmaceuticals. The chelating proper- Takada, Ogawa, Saito, Murata, & Usui, 1998; Yanahira et al., 1998,
ties of lactobionate also enable applications as ion sequestrant in Fig. 3). Both 40 -galactosyl lactose and 60 -galactosyl lactose are
detergent solutions (Gerling, 1997). Moreover, lactobionate at a considered to have prebiotic properties (for review, see van Loo et
concentration of 100 mmol L1 is a key component of solutions al., 1999). However, different oligosaccharide preparations may
used for the cold storage of transplant organs. The cell impermeant vary with respect to technological benefits such as flavor
lactobionate prevents hypothermically induced cell swelling; in enhancing properties, sweetness, hygroscopicity, and solubility.
addition, iron chelation by lactobionate is thought to reduce The transgalactosylation reaction of b-galactosidases is favored
oxidative injury during storage (Southard & Belzer, 1995). at high lactose concentrations (Huber, Kurz, & Wallenfels, 1976)
and typical oligosaccharide yields range from 10% to 40% (GOS/
initial lactose). The effect of lactose concentration on lactose
5.5. Lactosucrose
turnover by a b-galactosidase from L. delbrueckii spp. bulgaricus is
depicted in Fig. 4. High temperatures enable increased initial
The trisaccharide lactosucrose, derived from transfructosylation lactose concentration (Boon, Janssen, & van’t Riet, 2000; Bruins,
of lactose, is commercially produced in Japan. Its sweetness relative Strubel, van Lieshout, Janssen, & Boom, 2003). Moreover, high
to sucrose is 0.3–0.6. Lactosucrose is not resorbed in the upper incubation temperatures strongly favored oligosaccharide forma-
intestine and is, thus, available for hydrolysis and metabolism by tion over lactose hydrolysis by b-galactosidase from L. delbrueckii
the colonic microflora. Lactosucrose has a bifidogenic effect and its spp. bulgaricus and the optimum yield of GOS was achieved under
consumption was reported to decrease fecal pH and to inhibit denaturing conditions at 50 1C (Fig. 4, Vasiljevic & Jelen, 2003).
growth of colonic clostridia (Ogata et al., 1993). However, the Other approaches to optimize oligosaccharide formation by b-
prebiotic effect of lactosucrose is not as well documented as is the galactosidases include screening for enzymes with high prefer-
case for fructo-oligosaccharides or galacto-oligosaccharides. ence for trangalactosylation (Boon et al., 2000; Cheng et al., 2006;
Transfructosylation of lactose is carried out by bacterial or fungal Vasiljevic & Jelen, 2003), enzyme immobilization to achieve an
fructosyltransferases using sucrose or raffinose as fructosyldonor. improved enzyme stability at high temperatures (Albayrak &
Fructosyltransferases catalyze the hydrolysis of sucrose as well as Yang, 2002), and use of high hydrostatic pressure to improve the
the transfructosylation to acceptor carbohydrates. Fructosyltrans- yield of oligosaccharides (Kawade, Sakakibara, Nomura, Suzuki, &
ferases catalyze the formation of high molecular weight fructan Kunugi, 1999). Moreover, protein engineering was successfully
polymers and the formation of oligosaccharides in addition to employed to obtain truncated or modified b-galactosidases with
sucrose hydrolysis. Depending on the type of polymer formed, high preference for production of oligosaccharides (Jørgensen,
fructosyltransferases are referred to as levansucrases or inulosu- Hansen, & Sougaard, 2001).
crases (Tieking & Gänzle, 2005; van Hijum, Kralj, Ozimek, The use of lactic acid bacteria (LAB) as producers of b-
Kijkhuizen, & van Geel Schutten, 2006). The formation of lactosu- galactosidase enzymes offers substantial potential for the produc-
crose by levansucrase was initially described using an enzyme from tion of GOS. First, LAB are known to be good producers of
Rhanella aquatilis (Hestrin & Avigad, 1958; Ohtsuka et al., 1992). extracellular b-galactosidases that enable GOS production from
Because the spectrum of acceptor-carbohydrates is essentially lactose (Garman, Coolbear, & Smart, 1996; Hung, Xia, Hu, & Lee,
comparable in all bacterial levansucrases characterized so far (Cote 2001; Smart, 1991). Second, LAB have a safe tradition in food
& Ahlgren, 1993; Hestrin & Avigad, 1958; Tieking, Kühnl, & Gänzle, fermentations and exhibit rapid anaerobic growth on agricultural
2005), it can be assumed that the synthesis of lactosucrose from substrates including waste products such as whey. Therefore, GOS
sucrose and lactose is a general property of bacterial levansucrases. may be produced from crude cellular extracts without costly
Levansucrase activity is frequently found in food fermenting lactic downstream processing (Vasiljevic & Jelen, 2001, 2003). Moreover,
acid bacteria, particularly L. reuteri, L. pontis and L. acidophilus. These GOS may be produced in situ during food fermentations or by
organisms have been successfully used for the generation of high using whey to produce food-grade GOS preparations. Bifidobacter-
levels of oligosaccharides in food fermentations (Tieking, Ehrmann, ium spp. deserve special attention as a relevant source of enzymes
Vogel, & Gänzle, 2005; Tieking et al., 2005). suitable for production of galacto-oligosaccharides. The competi-
tiveness of bifidobacteria in the intestinal tract of mammals is
5.6. Galacto-oligosaccharides linked to their ability to metabolize a large variety of oligo- and
polysaccharides. It was estimated that 8% of the genomic
Galacto-oligosaccharides (GOS) are commercially produced information in Bifidobacterium longum is dedicated to the
from lactose using fungal b-galactosidases. The b-galactosidases metabolism of complex carbohydrates (Ventura, van Sinderen,
ARTICLE IN PRESS
O
O O
Lactose O OH
O
O OH HO
O
O
O OH
O
Glucose
O H2N
OH
OH
O O
O O
O O HO
O O OH
E537 O OH
β (1-4) O O
H2O
O OH
O OH
O O OH
O O O Mannose, fructose
O
OH Galactose
Fig. 3. Schematic overview of the hydrolysis- and acceptor-reaction catalyzed by b-galactosidases. Carbohydrate moieties are drawn to indicate the carbon atoms and the
oxygen atoms of interest; galactose moieties are shaded gray. Lactose hydrolysis involved intermediate linkage of the galactosyl moiety to the glutamine residue in the
active site of the enzyme (E537 in the b-galactosidase of Escherichia coli) and subsequent transfer to H2O acting as galactosyl acceptor. Transgalactosylation to lactose or
galacto-oligosaccharides yields b(1-2), b(1-3), b(1-4) or b(1-6) linked galacto-oligosaccharides (the latter two predominating in most enzymes); other monosaccharides as
well as phenolic compounds have additionally been shown to act as galactosyl acceptor.
3.2 3.2
[Products] (%)
[Products] (%)
2.4 2.4
1.6 1.6
0.8 0.8
0.0 0.0
0 5 10 15 20 25 30 30 40 50 60
[Lactose ] (%) Temperature (°C)
Fig. 4. Lactose turnover by b-galactosidase in crude cellular extract from Lactobacillus delbrueckii spp. bulgaricus. Panel A: influence of lactose concentration on product
composition and Panel B: influence of temperature; K, glucose; ’, galactose; J, disaccharides; &, trisaccharides; D, tetrasaccharides. Replotted from Vasiljevic and Jelen
(2003).
Fitzgerald, & Zink, 2004). Several enzymes with activity on lactose biological as well as physico-chemical treatments are becoming
or galacto-oligosaccharides were characterized at a biochemical more complex, and (iv) available waste treatment scenarios are
level. The enzyme b-Gal-II from Bifidobacterium adolescentis becoming more expensive. Therefore, the desire to convert lactose
DSM20083 has a low transgalactosylation activity and a remark- and lactose-containing components into commercially viable
able specificity for hydrolysis of lactose, 40 galactosyllactose and products is increasing, and additional innovative applications in
higher b-(1-4) linked galacto-oligosaccharides (Hinz, van den the pharmaceutical and chemical industries are needed.
Broek, Beldman, Vincken, & Voragen, 2004). Bifidobacterium The traditional view of lactose as a commodity, produced by
infantis HL96 harbors two b-galactosidase genes, b-Gal-I and b- traditional crystallization or possibly other processes such as
GaI-III. Both enzymes are active towards lactose but the spray drying, has not been economically advantageous in the past.
oligosaccharide yield of b-Gal-I exceeded that of b-gal-III more Although there are several industrial lactose manufacturers
than threefold (Hung et al., 2001). offering a wide array of lactose products for diverse uses (from
infant foods to confectionery to pharma lactose), the overall
market for the traditional lactose products is relatively static and
6. Conclusions and future prospects radically new approaches to lactose utilization are needed to
achieve a ‘‘quantum leap’’ in converting this unique carbohydrate
Lactose is presently a most underutilized dairy component. The to new industrially attractive products. Some of the lactose
continued struggle to find new uses for the cheese whey is tied to derivatives discussed above are just a few examples of the new
finding new uses for lactose, the main whey component. It trends in surplus lactose management. Additional major oppor-
becomes increasingly difficult to dispose of whey or any other tunities may arise in using lactose (with or without prior
process streams containing high amounts of lactose from dairy hydrolysis) for various fermentation applications. Lactose used
and cheese operations as: (i) environmental discharge standards to be one of the cheapest fermentation carbohydrate on the
are getting higher and more difficult to comply with, (ii) market for some time. It is not inconceivable that lactose, in its
authorities are becoming more vigilant in all regions, (iii) crudest form as whey or UF permeate, could become an
ARTICLE IN PRESS
economical source of biogas or bioethanol. Unfortunately, the Fox, P. F., & McSweeney, P. L. H. (1998). Dairy chemistry and biochemistry. London,
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Garman, J., Coolbear, J., & Smart, J. (1996). The effect of cations on the hydrolysis of
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‘‘final solution’’ of the whey (and thus lactose) problem. strains of lactic acid bacteria. Applied Microbiology and Biotechnology, 46,
Finally, addressing the widespread problem of lactose intoler- 22–27.
Geciova, J., Giesova, M., & Jelen, P. (2002). Disruption of Streptococcus thermophilus
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lactose. Bulletin 289, pp. 33–37. Brussels, Belgium: International Dairy
benefit the industrial dairy processors in opening up new
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