Synthesis of β-Galactooligosaccharides
`from Lactose Using Microbial β-Galactosidases
`
`Daniel Obed Otieno
`
`Abstract: Galactooligosaccharides (GOSs) are nondigestible oligosaccharides and are comprised of 2 to 20 molecules of
`galactose and 1 molecule of glucose. They are recognized as important prebiotics for their stimulation of the proliferation
`of intestinal lactic acid bacteria and bifidobacteria. Therefore, they beneficially affect the host by selectively stimulating
`the growth and/or activity of a limited number of gastrointestinal microbes (probiotics) that confer health benefits.
`Prebiotics and probiotics have only recently been recognized as contributors to human health. A GOS can be produced
`by a series of enzymatic reactions catalyzed by β-galactosidase, where the glycosyl group of one or more D-galactosyl
`units is transferred onto the D-galactose moiety of lactose, in a process known as transgalactosylation. Microbes can be
`used as a source for the β-galactosidase enzyme or as agents to produce GOS molecules. Commercial β-galactosidase
`enzymes also do have a great potential for their use in GOS synthesis. These transgalactosyl reactions, which could find
`useful application in the dairy as well as the larger food industry, have not been fully exploited. A better understanding
`of the enzyme reaction as well as improved analytical techniques for GOS measurements are important in achieving this
`worthwhile objective.
`
`Introduction
`Galactooligosaccharides (GOSs) are regarded as an emerging
`special class of prebiotics that is primarily synthesized from lac-
`tose. It is the main milk sugar, and is associated with dairy prod-
`ucts but could also be made into pure solutions. It is the main
`substrate for GOS synthesis, through a reaction process referred
`to as transgalactosylation. Transgalactosylation is the process by
`which the enzyme β-galactosidase hydrolyzes lactose and, instead
`of transferring the galactose unit to the hydroxyl group of water,
`the enzyme transfers galactose to another carbohydrate, in this
`case lactose, to result in oligosaccharides with a higher degree
`of polymerization (DP) (Kim and others 1997). These molecules
`formed by galactose transfer contain one or more galactosides that
`are named (GOSs) (Petzelbauer and others 2000a). Since trans-
`galactosylation products are substrates of β-glycosidase-catalyzed
`hydrolysis, the composition of the product mixture changes quite
`significantly with progressing reaction time, hence the significance
`of an optimized reaction process to monitor GOS yield with reac-
`tion time (Boon and others 1999). The other reaction that lactose
`easily undergoes is hydrolysis in which the molecule is split into
`the monomeric forms of glucose and galactose. Lactose hydroly-
`sis and transgalactosylation are concomitant reactions catalyzed by
`β-glycosidase, resulting in monomeric products, as well as many
`
`MS 20100193 Submitted 2/22/2010, Accepted 4/10/2010. Author is with Bioen-
`ergy and Bioproducts Engineering Laboratories-BSEL, Washington State Univ., TriC-
`ities Campus, 2710 Univ. Drive, Richland, WA 99354, U.S.A. Direct inquiries to
`author Otieno (E-mail: dotieno@tricity.wsu.edu).
`
`newly formed β-glycosides, mainly di-, tri-, and tetrasaccharides
`(Prenosil and others 1987).
`Prebiotics are nondigestible food ingredients that reach the
`colon thus stimulating the growth or activity of bacteria in the
`digestive system that are beneficial to the health of the body
`(Gibson and Roberfroid 1995). Extensive studies have revealed
`that oligosaccharides, which may reach the lower digestive tract
`without being absorbed, can be utilized by bifidobacteria as an en-
`ergy source and promote the proliferation of intestinal bifidobac-
`teria (Onishi and Tanaka 1995). Equally, GOS can also serve as
`an important growth factor in the proliferation of other probiotic
`intestinal bacteria such as Lactobacillus acidophilus and L. casei. These
`oligosaccharides are also collectively referred to as bifidus factors
`or prebiotics. They have been known to promote and sustain the
`growth of beneficial bacteria, especially bifidobacteria within the
`colon (Tomomatsu 1994). The observation that GOS can stimulate
`the growth of bifidobacteria and other health-promoting bacteria
`(Rabiu and others 2001) has generated interest in the transferase re-
`action of β-galactosidases (Cho and others 2003). Also, it has been
`reported that GOS in a commercial milk powder can support the
`in vitro growth of 2 strains of probiotic bacteria, Bifidobacterium lactis
`DR10 and L. rhamnosus DR20. These studies therefore confirm
`the positive effects of GOS on digestive health upon consumption.
`Among the breast-fed infants, where the intestinal microflora is
`not yet well developed, their improved colonic health has been
`attributed to GOS in their mothers’ milk (Boehm and others
`2004). Among older consumers, other health benefits linked to
`GOS consumption include reduction in colon cancer risk and
`enhanced immunity (Crittenden and Playne 1996). Short-chain
`fatty acids (SCFAs) are key products of GOS fermentation in the
`
`c(cid:2) 2010 Institute of Food Technologists®
`doi: 10.1111/j.1541-4337.2010.00121.x
`
`Vol.9,2010 r Comprehensive Reviews in Food Science and Food Safety 471
`
`

`

`Synthesis of β-GOS from lactose . . .
`
`colon and the profiles of propionate and butyrate concentrations
`vary from one oligosaccharide to another. Propionate and butyrate
`have been confirmed as having positive implications in the preven-
`tion of colon cancer (Cummings 1981). Currently, there is much
`interest in the concept of active management of colonic microflora
`with the aim of improving human health. This has been attempted
`by the consumption of live microbial food components or sup-
`plements, known as probiotics. An alternative approach, however,
`is the consumption of prebiotics that provide nourishment to in-
`testinal bacteria thus promoting their proliferation.
`β-Galactosidase, also known as lactase, is a hydrolase that attacks
`the O-glucosyl group of lactose. The enzyme is derived from var-
`ious microorganisms, including fungi and bacteria, and has been
`found to perform different degrees of transgalactosyl bioconver-
`sions leading to variations in the level and composition of GOSs
`synthesized. Probiotic microorganisms might therefore be used to
`produce GOS structures that could have special prebiotic effects,
`specifically targeting colonic probiotic strains (Rastall and Maitin
`2002) and consequently improve the host’s immunity. Ideally, the
`use of probiotic microorganisms with high β-galactosidase activ-
`ity could be very important in the development of functional
`compounds such as GOS. According to the current definition
`adopted by the Food and Agriculture Organization (FAO) and
`World Health Organization (WHO), probiotics are: “Live mi-
`croorganisms which when administered in adequate amounts con-
`fer a health benefit on the host” (Joint FAO/WHO Report 2009).
`Lactic acid bacteria (LAB) and bifidobacteria are the most com-
`mon types of microbes used as probiotics; but certain yeasts and
`bacilli may also be important. Probiotics are therefore commonly
`consumed as part of fermented foods with specially added active
`live cultures, such as in yogurt, soy yogurt, or as dietary supple-
`ments. In the case of use as enzyme sources for GOS synthesis,
`they could provide the double advantage as probiotics as well as in
`prebiotic synthesis.
`Prenosil and others (1987) showed that of all β-galactosidases
`from fungal sources, that from Aspergillus oryzae produced the
`highest concentration of GOS compared with that from A. niger,
`Kluyveromyces lactis, and K. fragilis. The yeast Sterigmatomyces elviae
`CBS8119 has been found to be the highest producer of GOS
`among yeasts (Onishi and Tanaka 1995). Earlier, Nakanishi and
`others (1983) had shown that β-galactosidase from Bacillus circulans
`was the best for GOS production among bacterial sources when
`compared with that from Escherichia coli and yeasts.
`Chemical synthesis of GOS is also possible but it requires many
`reaction steps due to the necessary selective protection of the
`hydroxyl groups, which is not the case with enzymatic synthe-
`sis. Also, the environmental impact of toxic reagents would be
`far greater with chemical than with enzymatic synthesis (Hans-
`son and others 2001). Therefore, enzymatic processes are more
`feasible, more environmentally useful, and less costly than the
`chemical processes. Consequently, there remains an untapped po-
`tential as yet for GOS (bio)synthesis (Gekas and Lopez-Leiva 1985)
`from pure lactose solutions and low-value whey lactose from the
`dairy industry to create high-value functional food products. Apart
`from GOS, the other carbohydrates reported to have prebiotic
`properties are the isomaltooligosaccharides (IMO), fructooligosac-
`charides (FOS),
`lactulose (Gibson and others 1999), and xy-
`looligosaccharides (Suwa and others 1999).
`In this review, the biosynthesis of the prebiotic GOS using β-
`galactosidase of bacterial, fungal, and yeast origins is extensively
`examined and a concise summary is provided to stimulate further
`interest in research and industrial applications in this area.
`
`Progress in the Understanding of Transgalactosyl
`Reactions
`Lactose hydrolysis and transgalactosylation are complex pro-
`cesses involving a multitude of sequential reactions with saccha-
`rides as intermediate products as well as the formation of GOS
`apart from glucose and galactose as shown in Figure 1. As can be
`seen, the normal function of β-glycosidases is to hydrolyze sub-
`(cid:3)
`strates formed by a monosaccharide coupled by a β bond (β1
`4
`(cid:3)
`(cid:3)
`(cid:3)
`6 more common and β1
`2 and β1
`and β1
`3 rarely) to another
`polyol (Figure 1). However, under certain conditions, the same
`enzymes also catalyze the transgalactosylation reaction and syn-
`thesize GOS. The main drawback of oligosaccharide synthesis by
`these enzymes is that the reaction equilibrium is shifted to favor
`hydrolysis over synthesis in aqueous systems, which leads to a low
`yield in GOS production (Petzelbauer and others 2000b).
`Hydrolysis and possibly the transgalactosyl reaction of lactose
`by E. coli β-galactosidase was first postulated by Wallenfells and
`Malhotra (1960). They first suggested that glucose liberation was
`due to the hydrolysis of the D-pyranoside ring as opposed to the
`glycosidic bond. The untouched glycoside bond could, however,
`cause inhibition by blocking the active site of the enzyme. The au-
`thors were only able to analyze monomeric sugars; however, they
`eventually quantified sugar oligomers beginning with allolactose
`as a primary intermediate product. Allolactose, being similar to
`(cid:3)
`(cid:3)
`lactose, except having a β1
`6 instead of a β1
`4 glycoside bond,
`led to the conclusion that the enzyme had an extra role in bond
`modification. Further investigation of the transgalactosyl reaction
`led Jobe and Bourgeois (1972) to conclude that β-galactosidase
`(cid:3)
`(cid:3)
`4 and 1
`6 lactose iso-
`could have different binding sites for the 1
`mers. Other investigators, such as Nishizawa (1960) and Nisizawa
`and Hashimoto (1970), postulated that hydrolytic potential of lac-
`tose depended on the isomeric orientation of lactose and that it
`(cid:3)
`(cid:3)
`(cid:3)
`increased in the order of 1
`3, 1
`4, and 1
`6. Further investigation on
`the kinetics of the lactose reaction included studies on the specific
`rotation of galactose (Huber and others 1981). But it was Flaschel
`and others (1982) who analyzed the specific rotation of both α-
`and β-glucopyranose in lactose as it approached the equilibrium
`between its α- and β-anomeric forms, and they concluded that
`β-galactosidase from E. coli hydrolyzed α-lactose 2 times faster
`than the β form. Escherichia coli, whose plasmids can also be used
`to up-regulate the production of β-forms of GOS from lactose
`(Ji and others 2005) apparently possess higher α-lactase activity
`than β-lactase activity. Between lactose and galactose, the latter
`was found to be a stronger inhibitor than the former for the active
`sites of β-galactosidase by Flaschel and others (1982), who also
`found that α-galactose was a more significantly stronger inhibitor
`than the β-form.
`According to Prenosil and others (1987), β-galactosidase action
`on lactose and the products of its hydrolysis brought much insight
`into the reaction mechanism, but, on the other hand, made the
`classical kinetic modeling approach impractical due to the com-
`plexity of the equations and the number of the kinetic constants
`involved. Until the recent past, the reaction mechanism for GOS
`synthesis had not been fully understood (Vasella and others 2002).
`Reactions involving lactose and β-galactosidase are initiated by a
`covalent bonding of the galactosyl moiety to the active site of the
`enzyme, thus releasing the glucose moiety. Two types of reactions
`can possibly occur, namely, hydrolysis and transgalactosylation. In
`the case of GOS synthesis, transgalactosyl reaction is desired as
`opposed to hydrolysis. The determinant of either reaction is the
`galactosyl acceptor in the reaction system. When the acceptor is a
`sugar, then there is a galactosyl transfer favoring GOS formation.
`
`472 Comprehensive Reviews in Food Science and Food Safety r Vol. 9, 2010
`
`c(cid:2) 2010 Institute of Food Technologists®
`
`

`

`Synthesis of β-GOS from lactose . . .
`
`CH2OH
`
`OH
`
`O
`
`CH2OH
`
`O
`
`Figure 1–Transgalactosylation and hydrolytic
`processes involving lactose and
`β-galactosidase to produce forms of
`galactooligosaccharides.
`
`OH
`
`OH
`
`OH
`
`OH
`
`OH
`
`Hydrolysis
`
`OH
`Galactose
`
`OH
`
`OH
`Glucose
`
`CH2OH
`
`CH2OH
`
`O
`
`OH
`
`O
`
`OH
`
`OH
`
`OH
`Lactose
`
`O
`
`OH
`
`Transgalactosylation
`
`CH2OH
`
`OH
`
`O
`
`O
`
`CH2
`
`OH
`
`OH
`
`O
`
`CH2OH
`
`O
`
`OH
`
`O
`
`OH
`
`OH
`
`OH
`
`1'6 linkage
`n
`
`OH
`
`CH2OH
`
`OH
`
`O
`
`OH
`
`CH2OH
`
`CH2OH
`
`O
`
`O
`
`OH
`
`OH
`
`O
`
`CH2OH
`
`OH
`
`O
`
`OH
`
`O
`
`OH
`
`OH
`
`1'4 linkage
`n
`
`CH2OH
`
`OH
`
`CH2OH
`
`OH
`
`OH
`
`O
`
`O
`
`1'3 linkage
`n
`
`OH
`
`OH
`
`O
`
`OH
`
`O
`
`OH
`
`OH
`
`OH
`
`1'6
`1'4
`
`1'3
`1'2
`
`Common
`
`Rarely forms
`
`Galactooligosaccharides, galactose molecules in brackets, n = 1 to 4
`
`Conversely, when the acceptor is water, then hydrolysis occurs and
`lactose is broken down into its constituent monomeric glucose and
`galactose. The transgalactosyl reaction model can be applicable in
`the dairy industry where milk sugar can be used as suitable sub-
`strate for GOS synthesis, thus expanding the niche of valuable
`products obtained from milk. A recent study by Curda and others
`(2006) established that GOS can be obtained and purified from
`dried buttermilk using a purified β-galactosidase. Varying con-
`centrations of the enzyme (Maxilact LX 5000) ranging from 0.1%
`
`−1) can be used to obtain a maximum of 31.4% GOS
`
`to 2% (vv
`−1).
`(ww
`
`Substrate Sources for GOS Synthesis
`Lactose accumulates worldwide at an estimated 3.3 million met-
`ric tons annually, as a component of dairy byproducts, especially
`from cheese whey; and approximately half of that is used for hu-
`man consumption or animal feed, and the remainder is discarded,
`thus causing environmental problems (Pesta and others 2007).
`
`c(cid:2) 2010 Institute of Food Technologists®
`
`Vol. 9, 2010 r Comprehensive Reviews in Food Science and Food Safety 473
`
`

`

`Synthesis of β-GOS from lactose . . .
`
`Table 1–Characteristics of β-galactosidases from fungal, yeast, and bacterial sources.
`
`pHopt
`
`Topt
`
`Lactose
`Km (mM) M, kD
`
`Activator
`
`Inhibitor
`
`Reference
`
`58
`55
`
`37
`30
`
`60
`
`40
`50
`55
`55
`45
`50
`50
`70
`45
`60
`65
`55
`
`85
`50
`
`124
`90
`
`115
`35
`0.23-0.99 200
`(oNPG)
`–
`
`–
`
`2
`700
`2
`6
`13
`31
`580
`2.96
`800
`2.6
`41.7
`50
`
`540
`–
`220
`540
`35
`72
`53
`70
`362
`470
`67
`430
`
`–
`–
`
`–
`–
`
`Aehle (2004)
`Aehle (2004)
`
`K+, Mg2+
`–
`
`Ca2+, Na2+, Zn, Cu
`SDS
`
`Aehle (2004)
`Jurado and others (2004)
`
`Fe2+, Zn2+, Cu2+
`
`–
`
`Onishi and Tanaka (1998)
`
`Na+, K+
`
`Mg2+
`
`–
`
`–
`Na+, K+, and Mn2+
`Na+, K+, and Mn2+
`–
`–
`
`EDTA
`Na+, K+
`
`EDTA
`
`–
`
`–
`–
`
`Aehle (2004)
`Aehle (2004)
`Aehle (2004)
`Aehle (2004)
`–
`Fe2+, Ca2+, Cu2+, and Zn2+
`Nguyen and others (2006)
`Fe2+, Ca2+, Cu2+, and Zn2+
`Nguyen and others (2006)
`Ag3+, SDS
`Cho and others (2003)
`Fe2+, Zn2+, Cu2+, Pb2+, and Sn2+
`Chen and others (2008)
`Zn2+, Mn2+ Co2+, Ca2+, Sn2+
`Dumortier and others (1994)
`Cr3+,EDTAc,urea, galactose PCMB Hung and Lee (2002)
`–
`Nakanishi and others (1983)
`Fe3+, Cu2+, Zn2+,and galactose
`Batra and others (2005)
`
`Origin
`Fungal
`Aspergillus niger
`Aspergillus oryzae
`Yeast
`Kluyveromyces lactis
`Kluyveromyces fragilis
`
`3.5
`5.0
`
`6.5
`6.6
`
`6.0
`
`Sterigmatomyces elviae
`CBS8119
`Bacterial
`7.2
`Escherichia coli
`6.5
`Bacillus subtilis
`6.2
`Bacillus stearothermophilus
`6.2
`Lactobacillus thermophilus
`8.0
`Lactobacillus reuteri L103
`6.5
`Lactobacillus reuteri L461
`Bullera singularis KCTC 7534 5
`7.0
`Bacillus stearothermophilus
`4.8
`Bifidobacterium bifidum
`5.0
`Bifidobacterium infantis
`6.0
`Bacillus circulans
`6.5
`Bacillus coagulans
`
`kD = kiloDalton; oNPG = o-nitrophenyl β - D galactopyranoside; SDS = sodium dodecyl sulfate; EDTA = ethylene diamine tetraacetic acid; PCMB = p-chloromercuribenzoate.
`
`Table 2–GOS yield as influenced by purified β-galactosidases from various microorganisms and lactose concentrations.
`
`Microorganism
`Rhodotorula minuta
`Sterigmatomyces elviae
`Sterigmatomyces elviae CBS8119
`Penicillium sp. KFCC 10888
`Bacillus circulans
`Saccharopolyspora rectivirgular
`Bullera singularis
`Sulfolobus solfataricus
`Aspergillus oryzae
`Escherichia coli
`Aspergillus oryzae
`Bullera singularis ATCC 24193
`Lactobacillus reuteri
`Talaromyces thermophilus CBS 23658
`Thermus sp. Z-1
`
`Initial lactose
`concentration (g/L)
`200
`200
`360
`400
`47
`600
`180
`—
`—
`—
`—
`—
`—
`80
`158.4
`
`Galactooligosaccharide
`−1%)
`(conversion yield, ww
`38%
`39%
`63%
`40%
`41%
`44%
`50%
`48%
`36%
`32%
`22%
`54%
`36%
`40%
`40%
`
`Reference
`Onishi and Yokozeki (1996)
`Onishi and Tanaka (1995)
`Onishi and Tanaka (1998)
`In and Chae (1998)
`Mozaffar and others (1986)
`Nakao and others (1994)
`Cho and others (2003)
`Reuter and others (1999)
`Reuter and others (1999)
`Reuter and others (1999)
`Matella and others (2006)
`Shin and others (1998)
`Splechtna and others (2007)
`Nakkharat and Haltrich (2007)
`Akiyama and others (2001)
`
`A dash indicates that lactose concentration is not provided in the original referenced source.
`
`According to Miller (2003), lactose from whey has increased expo-
`nentially in recent years due to the high demand for production of
`cheese and whey protein concentrate. This has been compounded
`by low demand and as yet limited applications for lactose. As a
`result, GOS manufacture from whey lactose is considered a value-
`adding process and a commercially feasible process (Albayrak and
`Yang 2002).
`Dairy products containing lactose as well as pure lactose so-
`lutions are therefore the main substrates from which GOS can
`be synthesized using β-galactosidase. For example, yogurt can be
`used as a basic material for the production of GOS via the use of
`yogurt-based starter cultures during the incubation period. Yogurt
`containing B. infantis has been reported to have a higher con-
`centration of GOS than yogurt containing other bifidobacteria
`(Lamoureux and others 2002), thus implying a possible higher
`transgalactosyl reaction by β-galactosidase from this microorgan-
`ism.
`Galactosyl transferase synthesis using nucleotide phospho-sugars
`as substrates has also been used to produce GOS (Prenosil and
`others 1987). In addition, specific enzyme substrates such as o-
`nitrophenyl galactopyranoside (oNPG) could be used for GOS
`synthesis using β-D-galactosidase. According to Kim and others
`
`(1997), GOSs have been produced from specific substrates en-
`hanced by special ion activators. For example, the authors showed
`that β-galactosidase from K. lactis were able to produce GOS
`from oNPG with the addition of Mg2+ and Mn2+. They also re-
`ported that the Co2+, Zn2+, and Ni2+ were able to activate the
`oNPG-hydrolyzing activity of the enzyme while inhibiting the
`lactose-hydrolyzing ability. Table 1 shows a summary of fungal
`and bacterial β-galactosidases, their optimum transferase and hy-
`drolytic conditions on lactose, as well as activators and inhibitors.
`Other activators that are proven to enhance GOS synthesis from
`lactose include K+, Na+, Fe2+, and ethylene diamine tetraacetic
`acid (EDTA) also summarized in Table 1. Therefore, what acti-
`vates the hydrolyzing and transgalactosyl activity of 1 microorgan-
`ism could be inhibitory to another at different reaction conditions.
`Thus, the activation-inhibition effect during reactions is specific
`to a microorganism and also the reaction conditions.
`
`GOS Yields Using Different Enzyme Sources
`GOS yield is mainly dependent on reaction factors such as
`substrate concentration (lactose), reaction temperature, enzyme
`activity, source of enzyme, and the length of reaction. In general,
`the yield of GOS would be likely higher with an increase in
`
`474 Comprehensive Reviews in Food Science and Food Safety r Vol. 9, 2010
`
`c(cid:2) 2010 Institute of Food Technologists®
`
`

`

`Synthesis of β-GOS from lactose . . .
`
`Table 3–Galactooligosaccharide (GOS) yield from lactose solutions by batch and continuous processes.
`
`Reaction conditions
`Source
`◦
`38% lactose, 40
`C and pH 4.5
`Aspergillus oryzae
`30 ˚C and pH 4.6 Lactose conc. not given
`Aspergillus oryzae ATCC
`20423
`◦
`4.0% lactose, 40
`C and pH 4.5
`Aspergillus oryzae
`◦
`4.56% lactose, 40
`C and pH 6.0
`Bacillus circulans
`◦
`4.56% lactose, 40
`C and pH 6.0
`Bacillus circulans
`◦
`30% lactose, 45
`C and pH 3.7
`Bacillus singularis
`◦
`10% lactose, 45
`C and pH 4.8
`Bacillus singularis
`◦
`16% lactose, 70
`Thermus aquaticus YT-1
`C and pH 4.0
`Not given
`Lactobacillus reuteri
`Not given
`Lactobacillus reuteri
`−1) lactose, 95
`70% (wv
`Pyrococcus furiosus (F426Y)
`−1) lactose, 50
`60% (wv
`Penicillium simplicissimum
`−1) lactose, 40
`25% (wv
`Kluyveromyces lactis
`(Lactozym 3000 L HP G)
`◦
`−1) lactose, 50
`20% (wv
`Bullera singularis KCTC 7534
`C and pH 5.0
`◦
`−1) lactose, 70
`60% (wv
`C, and pH 6.0
`Saccharopolyspora
`rectivirgula
`
`−1) lactose, 55◦
`5 to 30% (ww
`C and pH 7.5
`Bifidobacterium bifidum
`BB-12
`−1) lactose, 40 – 45
`◦
`45-50% (wv
`Bifidobacterium bifidum
`NCIMB 41171
`◦
`−1) lactose, 55
`5 to 30% (ww
`C and pH 7.5
`Bifidobacterium angulatum
`−1) lactose, 55
`◦
`5 to 30% (ww
`C and pH 7.5
`Bifidobacterium infantis
`−1) lactose, 55
`◦
`5 to 30% (ww
`C and pH 7.5
`Bifidobacterium
`pseudolongum
`−1) lactose, 55
`5 to 30% (ww
`Batch (shaking)
`Bifidobacterium adolescentis
`aGOS yield is a weight percentage of oligosaccharides based on total saccharides in the reaction medium.
`SE = soluble enzyme; IE = immobilized enzyme; CSTR = continuous stirred tank reactor; PBR = packed bed reactor.
`
`Mode
`Batch (SE)
`Batch (IE)
`
`Continuous (IE, PBR)
`Batch (SE)
`Continuous (IE, CSTR)
`Batch (IE)
`Continuous (IE, PBR)
`Batch (IE)
`Batch
`Continuous (CSTR)
`Batch
`Batch
`Batch
`
`Batch
`Batch
`
`Batch (shaking)
`
`Continuous
`
`Batch (shaking)
`Batch (shaking)
`Batch (shaking)
`
`◦
`◦
`◦
`
`C and pH 5.0
`C and pH 6.5
`C and pH 6.5
`
`C and pH 6.8
`
`◦
`
`C and pH 7.5
`
`Productivity
`(GOS yield)a
`32%
`26%
`
`26.6%
`24%
`40%
`54%
`55%
`34.8%
`36%
`30%
`45%
`30.5%
`17.1%
`
`50%
`41%
`
`Reference
`Iwasaki and others (1995)
`Sheu and others (1998)
`
`Albayrak and Yang (2002)
`Mozaffar and others (1986)
`Mozaffar and others (1986)
`Shin and others (1998)
`Shin and others (1998)
`Berger and Venhaus (1992)
`Splechtna and others (2007)
`Splechtna and others (2007)
`Hansson and others (2001)
`Cruz and others (1999)
`Martinez-Villaluenga and
`others (2008)
`Cho and others (2003)
`Nakao and others (1994)
`
`37.6%
`
`Rabiu and others (2001)
`
`36 – 43% Goulas and others (2007)
`
`43.8%
`47.6%
`26.8%
`
`43.1%
`
`Rabiu and others (2001)
`Rabiu and others (2001)
`Rabiu and others (2001)
`
`Rabiu and others (2001)
`
`lactose concentration and vice versa. As shown in Table 2, the
`influence of β-galactosidases from yeasts and bacteria determines
`GOS yields. On the other hand, Table 3 highlights the effects of
`batch and continuous processes on GOS yields. In a continuous
`process, there is no limitation factor of accumulating end products
`during a reaction, which favors transgalactosylation and therefore
`leads to higher GOS yields. The element of shaking or mixing
`enhances enzyme performance, thus favoring higher GOS yields
`compared to a batch process in which there is no mixing.
`In a study by Dumortier and others (1994), 60% of the ini-
`tial lactose concentration and B. bifidum cells yielded 29% GOS.
`In previous studies involving wild-type β-glycosidases, transgalac-
`tosyl reactions from lactose resulted in maximal GOS yields of
`40% to 42% (Nakao and others 1994; Onishi and Tanaka 1995).
`These yields could represent the upper limit for what is possible
`to achieve by wild-type β-glycosidases.
`
`Glycosyl-Linkage Analysis of GOSs
`For a complete understanding of the sugar structure, the study
`should involve aspects such as sugar identification, stereochem-
`istry, types of linkages, ring structures, anomeric configurations,
`and the sugar sequences in oligosaccharides. Glycosyl linkage anal-
`ysis, also referred to as methylation analysis, is one of the impor-
`tant procedures in the determination of structural composition of
`polysaccharides (Rabiu and others 2001). The interpretation of
`glycosyl linkages is based on electron impact mass spectra of par-
`tially O-methylated, and partially O-acetylated alditol derivatives
`from gas chromatography-mass spectrometry (GC-MS). Briefly,
`the main steps involve derivatization of the sugars in the samples
`through partial methylation followed by partial acetylation. The
`sequential reaction of the sample material with methanolic acid,
`tert-butanol, and dissolution in methanol leads to the formation
`of a methyl glycoside. The methyl glycosides are then dissolved
`in methyl sulfoxide and potassium methylsulfinyl-methanide to
`deprotonate most of the hydroxyl groups in the sample before
`addition of iodomethane to obtain a partially methylated methyl
`
`glycoside. Acetylation with addition of acetic anhydride is a very
`slow reaction process, thus requiring the use of 1-methylimidazole
`as a catalyst. Extraction of the partially methylated, partially acety-
`lated methyl glycoside is achieved through extraction in water and
`dichloromethane and evaporated in a stream of filtered air to re-
`move excess dichloromethane. The partially methylated, partially
`acetylated methyl glycosides are then hydrolyzed using trifluo-
`roacetic acid and subsequently reduced by addition of sodium
`tetradeuteroborate (NaBD4) to form partially methylated alditol
`acetates (PMAAs). The acetylation of PMAAs is attained by disso-
`lution in glacial acetic acid, ethyl acetate, and acetic anhydride in
`a specific ratio. The acetylation reaction can be slow, therefore has
`to be catalyzed by addition of a strong acid such as 60% perchloric
`acid. After sequential extraction with dichloromethane, the ex-
`tracted layer is injected for analysis in GLC and GLC-MS. A mass
`spectrum (spectrum of masses of intact and fragmented original
`species) of PMAAs is used to identify glycosyl residue linkages. A
`beam of electrons breaks the PMAAs apart, generating fragment
`ions in the gas phase and the ions are projected into a mass analyzer,
`and the mass of the fragments are counted. Thus, it is possible to
`characterize the GOS synthesized based on galactosyl bond types
`during transgalactosylation.
`
`Determinants of GOS Composition, DP and Glycosyl
`Linkages during Synthesis
`From the early 1950s, different chain lengths of oligosaccharides
`were obtained by β-galactosidases from different microorganisms.
`For example, 4 different oligosaccharides were obtained using en-
`zyme from K. fragilis, while 3 oligosaccharides were obtained us-
`ing β-galactosidase from E. coli (Aronson 1952). Pazur (1954)
`repeated the same experiment using lactase from K. fragilis and
`found similar results, while others (Roberts and Pettinati 1957;
`van der Meulen and others 2004) found that by increasing the
`initial lactose concentration, a much greater variety (11 types) of
`oligosaccharides was be formed. During transgalactosyl reactions,
`there are species of oligosaccharides that tend to be dominant in
`
`c(cid:2) 2010 Institute of Food Technologists®
`
`Vol. 9, 2010 r Comprehensive Reviews in Food Science and Food Safety 475
`
`

`

`Synthesis of β-GOS from lactose . . .
`
`Table 4–Types and degree of polymerization of synthesized galactooligosaccharides produced by different microorganisms.
`
`Types of linkages
`
`4)-Glc
`4)-Glc
`(cid:3)
`4)-Glc
`6)-Gal(β1
`(cid:3)
`(cid:3)
`6)- Gal(β1
`6)-Gal(β1
`
`4)-Glc
`
`Reference
`Goulas and others (2007)
`Goulas and others (2007)
`
`Goulas and others (2007)
`Goulas and others (2007)
`Dumortier and others (1990)
`
`Microorganism
`Bifidobacterium bifidum
`NCIMB 41171
`
`Bifidobacterium bifidum
`DSM 20456
`
`Sulfolobus solfataricus (SsbGly)
`
`Pyrococcus furiosus (CelB)
`
`Polymerization
`and composition
`Disaccharide
`Trisaccharides
`
`Tetrasaccharide
`Pentasaccharide
`Disaccharides
`
`Trisaccharides
`
`Tetrasaccharides
`Pentasaccharide
`Hexasaccharide
`Heptasaccharide
`Disaccharide
`Trisaccharide
`Disaccharide
`Trisaccharide
`
`(cid:3)
`6)-Gal
`Gal(α1
`(cid:3)
`(cid:3)
`Gal(β1
`6)-Gal(β1
`(cid:3)
`(cid:3)
`Gal(β1
`3)-Gal(β1
`(cid:3)
`(cid:3)
`Gal(β1
`6)-Gal(β1
`(cid:3)
`(cid:3)
`Gal(β1
`6)-Gal(β1
`(cid:3)
`Gal(β1
`6)-Glc
`(cid:3)
`Gal(β1
`3)-Glc
`(cid:3)
`Gal(β1
`6)-Gal
`(cid:3)
`(cid:3)
`Gal(β1
`4)-Glc
`3)-Gal(β1
`(cid:3)
`(cid:3)
`Gal(β1
`6)-Gal(βI
`4)-Glc
`(cid:3)
`(cid:3)
`Gal(β1
`2)-Gal(βI
`6)-Glc
`(cid:3)
`(cid:3)
`(cid:3)
`Gal(β1
`3)-Gal(βl
`3)-Gal(βI
`(cid:3)
`(cid:3)
`(cid:3)
`Gal(βl
`3)-Gal(β1
`3)-Gal(βI
`(cid:3)
`(cid:3)
`(cid:3)
`Gal(βI
`3)-Gal(βl
`3)-Gal(β1
`(cid:3)
`(cid:3)
`(cid:3)
`Gal(βl
`3)-Gal(β1
`3)-Gal(βI
`(cid:3)
`Gal(β1
`6)-Glc
`(cid:3)
`(cid:3)
`Gal(β1
`6)-Gal (β1
`6)-Glc
`(cid:3)
`Gal(β1
`3)-Glc
`(cid:3)
`(cid:3)
`Gal(β1
`3)- Gal(β1
`
`4)-Glc
`(cid:3)
`4)-Glc
`3)-Gal(β1
`(cid:3)
`(cid:3)
`4)-Glc
`3)-Gal(βI
`3)-Gal(β I
`(cid:3)
`(cid:3)
`(cid:3)
`3)-Gal(β1
`3)-Gal(βI
`3)-Gal(β1
`
`4)-Glc
`
`Petzelbauer and others (2000a)
`
`Petzelbauer and others (2000a)
`
`3)-Glc
`
`the solution, for example, allolactose as reported in the study by
`Huber and others (1976). Using β-galactosidase from K. lads on a
`known lactose solution, Dickson and others (1979) also detected
`allolactose, galactobiose, and tri- and tetra-oligosaccharides. In the
`1980s, Toba and others (1981) also confirmed that β-galactosidase
`from different microorganisms yielded varying concentrations
`and compositions of oligosaccharides. In standardized large-scale
`productions, using the β-galactosidase derived from B. circulans,
`more than 55% of the lactose was reportedly converted to GOS
`(Mozaffar and others 1986). Although tri- to hexa-saccharides
`with 2 to 5 galactose units are the main products of this reaction,
`disaccharides consisting of galactose and glucose with different
`β-glycoside bonds from lactose were also produced (Sako and
`others 1999). In another study by Hsu and others (2007), the
`production of GOS using β-galactosidase from B. longum BCRC
`15708 resulted in 2 types of GOSs, tri and tetra-saccharides, from
`−1). In this study, trisaccha-
`a lactose concentration of 40% (wv
`rides were the major type of GOS formed. A maximum yield
`−1) GOS could be achieved from a 40% (wv
`−1)
`of 32.5% (ww
`◦
`C and pH 6.8. Table 4 shows a summary
`lactose solution at 45
`of types of galactosyl linkages formed during transgalactosylation
`by β-galactosidase from different microorganisms. As shown in
`Table 4, synthesis of GOS from lactose and whey permeate us-
`ing the whole cells of B. bifidum NCIMB 41171 gave rise to a
`variety of oligosaccharides with different degrees of polymeriza-
`tion (DP > 3) and transgalactosylated disaccharides (Goulas and
`others 2007). Therefore, whereas different microorganisms seem-
`ingly yield different compositions of oligosaccharides with varying
`degrees of polymerization, it appears that the primary determi-
`nants are the actual reaction conditions. In addition, for reasons
`that are still not very clear, certain microorganisms express en-
`zymes that only synthesize certain types of GOS. For example,
`it has been established that β-galactosidases derived from B. cir-
`culans (Mozaffar and others 1986) or Cryptococcus laurentii (Ozawa
`(cid:3)
`(cid:3)
`and others 1989) synthesize mainly β1
`4 bonds (4
`-GOS) during
`lactose hydrolysis. Conversely, when enzymes derived from A.
`oryzae or Streptococcus thermophilus (Matsumoto 1990) were used,
`(cid:3)
`(cid:3)
`β1
`6 bonds (6
`-GOS) were formed. It is possible that the predom-
`inant transgalactosyl enzymes in these microorganisms, owing to
`their specificity, simply respond to the stereochemical configura-
`tions of the glycosyl substrates in the reaction. Therefore, at this
`point in time, it appears that GOS composition, the galactosyl
`
`bond types, and the DP may be determined by a combination of
`factors including reaction temperature, the purity of the enzyme,
`pH, initial lactose concentration (water activity), and the source
`and form of the enzyme. Interestingly, Goulas and others (2007)
`reported that crude endogenous enzymes produced mixed com-
`positions of GOS, containing both α and β bonds. This scenario
`is unlikely when pure β-galactosidase or α-galactosidase is used as
`opposed to cru