Production of a lactose-free galacto-oligosaccharide mixture by usingselective enzymatic oxidation of lactose into lactobionic acid

Barbara Splechtna, Inge Petzelbauer, Ursula Baminger, Dietmar Haltrich,Klaus D. Kulbe, Bernd Nidetzky*

Division of Biochemical Engineering, Institute of Food Technology, Universitat fur Bodenkultur Wien (BOKU), Muthgasse 18, A-1190 Wien,Vienna, Austria

Received 24 January 2001; revised 2 July 2001; accepted 10 July 2001

Abstract

We report a novel and efficient way of producing lactose-derived galacto-oligosaccharides (GOS) that do not contain remaining lactoseand monosaccharides. The initial sugar mixture was obtained by enzymatic transformation at 70°C of a lactose solution of 270 g/liter usingrecombinant �-glycosidase from the Archaeon Sulfolobus solfataricus. At the optimum reaction time for kinetically controlled transgalac-tosylation, it contained 46% monosaccharides, 13% lactose and 41% GOS. Lactose was selectively oxidised into lactobionic acid by usingfungal cellobiose dehydrogenase which displays a � 100-fold preference for reaction with lactose compared to reaction with GOS.Oxidation of lactose was coupled to reduction of 2,6-dichloro-indophenol which was added in catalytic concentrations. The oxidised redoxmediator was regenerated continuously by fungal laccase-catalysed reduction of molecular oxygen into water. Ion exchange chromatog-raphies were employed to remove lactobionic acid, other ions and monosaccharides. The final product contained 97% GOS, 1.2% lactoseand 2.1% monosaccharides. The yield accounted for 25% of original lactose. An enzymatic assay for lactose has been developed. It is robustand allows sensitive quantification of the analyte in complex sugar mixtures containing large excesses of monosaccharides and GOS.© 2001 Elsevier Science Inc. All rights reserved.

1. Introduction

The transfer of galactose to acceptors other than water iscommonly observed during a �-glycosidase-catalysed trans-formation of lactose. It leads to the formation of new �-D-galactosides as kinetic reaction intermediates. When theenzymatic process is carried out at a high sugar concentra-tion, transgalactosylation to sugar hydroxyls often prepon-derates over complete hydrolysis of lactose. Galacto-oligo-saccharides (GOS) are thus formed in a kineticallycontrolled reaction. GOS produced from lactose throughenzymatic transgalactosylation are known as a divergentmixture of saccharides which differ in monosaccharidecomposition, degree of polymerisation and glycosidic link-age (see Scheme 1). �-Glycosidases from different sourceshave been extensively characterised pertaining to theirtransgalactosylation properties, and some are currently usedin Japan and Europe for industrial GOS production. GOS

yield depends on the enzyme and the conditions used in theprocess, and values ranging from 25% to 50% of total sugarhave been reported [1]. Composition of GOS is chieflydetermined by the specificity of the �-glycosidase employedin the reaction. GOS produced by transgalactosylation al-ways contain considerable amounts of non-reacted lactoseand monosaccharides [1–4].

In recent years, GOS have been discussed much pertain-ing to their use as non-digestible, carbohydrate-based foodingredients in human nutrition, which may have positive,health-related physiological activities. The ability of GOSto promote the proliferation of intestinal bifidobacteria andlactobacilli has been recognised. The predominance of bi-fidobacteria in the colon has been suggested to cause ben-eficial effects for maintaining human health, providing pro-tection from infection and facilitating the normal functionsof the gut. Apart from their proposed effects on health, GOShave certain other useful properties. Their stability underacidic conditions during food processing make them poten-tially applicable as ingredients for a wide variety of foodproducts. Their excellent taste quality and relatively lowsweetness make GOS interesting functional sweeteners.

* Corresponding author. Tel.: �43-1-36006-6274; f ax: �43-1-36006-6251.

E-mail address: nide@edv2.boku.ac.at (B. Nidetzky).

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0141-0229/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.PII: S0141-0229(01)00412-4

They pass the small intestine without being digested andare, therefore, of low caloric value. In addition, GOS cannotbe metabolised by microorganisms of the oral cavity and arethus not implicated in the formation of dental caries [5–8].

The efficient removal of sugar components other thanGOS, especially lactose is an important factor of the com-mercialisation of GOS-based products. Lactose-free GOSare of interest considering that 70% of the world populationlack �-galactosidase in the small intestine and are thereforesensitive to lactose. GOS depleted of monosaccharides areexpected to display a more selective functional effect thanGOS containing large amounts of D-glucose. Unfortunately,the separation of lactose from the disaccharide fraction ofGOS has proven to be difficult and leads to large losses ofproduct. In this study, a new approach was taken to produceGOS which do not contain appreciable amounts of lactoseand D-glucose. It is based on the selective enzymatic oxi-dation of lactose into lactobionic acid using a fungal cello-biose dehydrogenase. Lactobionic acid is easily separatedfrom non-ionic sugars by using anion exchange chromatog-raphy. Monosaccharides are subsequently removed by asingle chromatographic step, yielding purified GOS withonly minor losses of the main components of the targetproduct. An enzymatic lactose assay was developed, and weshow its utility for measuring precisely the lactose concen-tration in the presence of large excesses of GOS andmonosaccharides.

2. Materials and methods

2.1. Enzymes

The gene encoding �-glycosidase from Sulfolobus solfa-taricus was overexpressed in Escherichia coli BL21 (DE3)by using the plasmid vector pT7BM1 [9]. The enzyme(Ss�Gly) was purified by precipitating mesophilic bacterialprotein at 80°C [10]. It was kindly provided by Dr. M.Moracci (CNR, Naples, Italy).

Cellobiose dehydrogenase (CDH) from Sclerotium rolf-

sii CBS 191.62 was prepared as previously reported [11].The enzyme had a specific activity of 26 U/mg and wasdevoid of �-glucosidase and �-galactosidase activity.

Laccase from Trametes pubescens MB 89 was preparedaccording to Baminger et al. [11].

2.2. Assays

�-Glycosidase activity was measured at 80°C using lac-tose as the substrate [12].

The assay for CDH activity was essentially that described inBaminger et al. [13]. It used 0.3 mM 2,6-dichloro-indophenol(DCIP), 30 mM lactose and 1% ethanol in a 100 mM sodium-acetate buffer, pH 4. The reduction of DCIP was monitoredcontinuously by absorbance at 520 nm. The molar extinc-tion coefficient of DCIP under these conditions was deter-mined experimentally to be 6.8 mM�1. cm�1. One unit ofCDH activity is the amount of enzyme that reduces 1 �molof DCIP per minute under these conditions

The reaction mixture for assaying laccase activity con-tained 5 mM 2,2�-azinobis(3-ethylbenzthiazoline-sulfonicacid) (ABTS; �436 � 29,300 M�1cm�1) in 20 mM acetatebuffer, pH 3.5 [14]. One unit of laccase activity was defined asthe amount of enzyme oxidising 1 �mol of ABTS per minuteat 25°C.

Protein was determined with the Bio Rad (Hercules, CA,USA) Coomassie Blue reagent using BSA as the standard.

2.3. Enzymatic procedure for quantification of lactose

The method is based on the enzymatic oxidation oflactose. DCIP was dissolved in ethanol (10% of the finalvolume) and subsequently diluted with water to a finalconcentration of 3 mM. The sample was diluted appropri-ately to a lactose concentration in the range 0.3–3.0 mM.One hundred microliter of diluted sample and 100 �l of a 3mM DCIP solution were added to 780 �l of 100 mMsodium acetate buffer, pH 4, and mixed well. The absor-bance at 520 nm was recorded (A1). The reaction wasstarted by adding 20 �l (0.6 U) of CDH. After 20 min at30°C, the absorbance was measured again (A2) and thedecrease in absorbance (A1–A2) was determined. A reactionlacking lactose served as the control, and correction forblank readings was made as required. Each mole of reducedDCIP corresponds to one mole of oxidised lactose [11].Calibration was carried out using 10 lactose solutions ofknown concentration (0.3–3.0 mM).

2.4. Further analyses

D-Glucose and D-galactose were quantified by enzymaticmethods [12]. Lactobionic acid was analysed by HPLCusing an Ostion LGKS 0800 Ca2� column (250 � 8 mm;Watrex, Prague, Czech Republic) at 80°C with 10 mMCa(NO3)2 as eluent (0.7 ml/min) and refractive index de-tection.

Scheme 1. Structures of lactose (I), �-D-Galp-(136)-D-Glc (allolactose,II), �-D-Galp-(136)-D-Gal (III), and �-D-Galp-(136)-D-Gal-(134)-D-Glc (IV).

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2.5. Thin layer chromatography (TLC)

TLC was carried out using high-performance TLC silicaplates (Kieselgel 60 F245, Merck, Darmstadt, Germany).An appropriately diluted sample containing �20 g/liter ofsugar was applied to the plate (1.2 �l) and eluted twice inascending mode with isobutanol/pyridine/water (6/4/3).Thymol reagent was used for detection.

2.6. Capillary electrophoresis (CE)

This was executed on a HP-3D-CE unit (Hewlett Pack-ard, Naldbronn, Germany) with a built-in diode array de-tector, using the procedure of derivatisation and separationin a phosphate buffer system described by Petzelbauer et al.[10]. Quantification was done via internal lactose standards.

2.7. Oxidation of lactose to lactobionic acid usingcellobiose dehydrogenase

In a batchwise reaction CDH was used to oxidise lactoseto lactobionic acid while at the same time, the redox medi-ator DCIP was reduced. Laccase was used to re-oxidiseDCIP in the presence of molecular oxygen. Therefore, onlycatalytic concentrations of DCIP were required. The follow-ing reactant concentrations were used: 182 mM sodiumacetate buffer, pH 5.0, 5.45 mM DCIP, 1 U/ml CDH, and2.5 U/ml laccase. NaOH was added during the reaction asrequired to maintain a constant pH value of 5. This and theaddition of reactants caused a maximum 1.3-fold dilution ofthe original sugar mixture. The reaction was performed at30°C using 100-ml beakers with a working volume of 50ml. The reaction system was stirred and oxygenated bysparging the solution with oxygen (3.5 vol. per vol. solutionper min). The dissolved oxygen concentration was mea-sured by using a microoxygen electrode (Microelectrodes,Londonderry, NH, USA). pH control was achieved auto-matically (Dulcometer from ProMinent, Heidelberg, Ger-many). At the times stated, 0.5 ml samples were taken,boiled and centrifuged. The supernatant was used to deter-mine the course of reaction by HPLC. The batch was rununtil no more oxygen was consumed and the pH was con-stant. This indicated that the reaction was complete which inthis case took approximately 140 min. The produced solu-tion was heat-treated (100°C, 1 min), and used for down-stream processing.

2.8. Removal of lactobionic acid and salts

This was done by applying the centrifuged and filteredproduct solution to serial ion exchange chromatography.Two XK 16/20 columns (Amersham/Pharmacia/Biotech,Uppsala, Sweden) were used. The first column contained7 g of anion exchange material Dowex 1 � 8 (Fluka, Buchs,Switzerland) and was connected to a second column, con-taining 2 g of cation exchange material Amberlite CG-120-

II1 � 8 (Fluka, Switzerland). It was shown experimentallythat the anion and cation exchange resins have bindingcapacities of at least 1.9 mmol anions/g and 3.12 mmolcations/g, respectively. Approximately 5 g sugar (in 29 ml)were applied to the first column. Elution was done withwater at a flow rate of 1 ml/min. Fractions of 5 ml werecollected and analysed by TLC. Fractions containing car-bohydrates were analysed further by using HPLC, underconditions described above. D-glucose, D-galactose, and lac-tobionic acid could be quantified, lactose co-eluted withGOS. Pooled fractions were lyophilised.

2.9. Removal of monosaccharides

Separation of GOS from D-glucose and D-galactose wasdone by using the strongly acidic cation exchange resinUnibead UBK-530 (Mitsubishi Chemical Corporation, To-kyo, Japan) as described by Matsumoto et al. [15]. Thefreeze dried sample was dissolved in water to contain 50%(w/v). One ml of the solution was applied to a column witheffective dimensions of 9 � 580 mm. It was equipped witha heat jacket for thermostatisation from an external water-bath. The operating temperature was 70°C and elution wascarried out with water at a flow rate of 0.3 ml/min. Fractionsof 0.5 ml were collected and analysed by TLC. Fractionsdevoid of monosaccharides were pooled and gave the endproduct. D-glucose, D-galactose and lactose were quantified.

3. Results and discussion

3.1. Enzymatic production of GOS

Enzymatic conversion of lactose (273 g/liter) was carriedout at 70°C in 20 mM sodium citrate buffer, pH 5.5, usingSs�Gly (20 U/ml). After a reaction time of four hours, thefollowing sugar mixture was obtained: 83 g/liter (�33%,per weight) D-glucose, 33 g/liter (�13%, per weight) D-galactose, 102 g/liter (�41%, per weight) oligosaccharidesand 33 g/liter (�13%, per weight) remaining lactose. It hasbeen shown recently that GOS produced by Ss�Gly consistof di-, tri- and tetrasaccharides, mainly containing �(133)and �(136) glycosidic linkages [16]. Note that for conve-nience we use the term “oligosaccharides” for all trans-galactosylation products including disaccharides.

3.2. Enzymatic lactose assay using cellobiosedehydrogenase

It is difficult to determine the concentration of lactosethat remains in sugar mixtures obtained through �-glycosi-dase-catalysed transformation of lactose. The commondrawback of HPLC or even CE is that other disaccharidesoften co-elute with lactose and so quantification is seriouslyflawed. Commercial test kits may also give incorrect fig-ures: applying an enzymatic kit based on �-galactosidase

436 B. Splechtna et al. / Enzyme and Microbial Technology 29 (2001) 434–440

and galactose dehydrogenase (Boehringer Mannheim,Mannheim, Germany) to a sample containing 33 g/literlactose and 102 g/liter GOS overestimated the true value forlactose by more than 200%. The �-galactosidase is notspecific enough and hydrolyses both lactose and GOS.

A lactose assay based on using CDH from Sporotrichumthermophile was described by Canevascini et al. [17]. Theseauthors have found that a large molar excess of monosac-charides interferes with the determination of lactose. Wehave re-assessed the utility of CDH-catalysed oxidation oflactose as means of measuring colorimetrically the concen-tration thereof in the presence of GOS and monosacchar-ides. When the observed decrease in absorbance at 520 nmdue to enzymatic reduction of DCIP is plotted against thelactose concentration in the sample, Fig. 1 is obtained. Thelinear relationship vindicates the method for analysing purelactose solutions. In an effort to validate the lactose assayfor complex mixtures of sugars, we studied systematicallythe effect on �A520nm when a solution of constant lactoseconcentration was charged with increasing concentrationsof D-glucose or GOS. Results are shown in Fig. 2. Theyindicate that the true value for the lactose concentration of0.7 g/liter is overestimated by less than 10% if a 15-foldexcess of GOS is present. D-glucose hardly interferes withthe determination unless its concentration exceeds that oflactose by a factor of 50. Therefore, we conclude that thecolorimetric lactose assay based on oxidation of the analyteby S. rolfsii CDH is applicable to sugar mixtures commonlyobtained through transformation of lactose by a �-glycosi-dase.

3.3. Oxidation of lactose using cellobiose dehydrogenase

A reaction system composed of CDH, laccase and theredox mediator DCIP was used. Fig. 3 illustrates how thelaccase-catalysed reduction of molecular oxygen is em-ployed to achieve a continuous regeneration of oxidisedDCIP which is consumed stoichiometrically in the enzy-matic oxidation of lactose. The equilibrium of the overallreaction is completely on the side of the products. Fig. 4shows the course of reaction which was monitored by re-cording the decrease in pH, and measuring with HPLC theconcentrations of lactose and lactobionic acid. The endpoint of the reaction was also indicated by the absence offurther oxygen consumption. Using the enzymatic lactoseassay, the lactose content of the initial GOS mixture wasshown to be reduced by about 96%. In Fig. 5, which com-pares the integrated CE data before and after the enzymaticoxidation, it can be seen that the lactose peak was onlyreduced by about 80%. This result indicates that there isanother disaccharide co-eluting with lactose in CE, whichwas not identified.

Fig. 1. Calibration line for the enzymatic lactose assay. Reactions werecarried out at pH 4 and 30°C using the conditions specified under Materialsand methods. The absorbance at 520 nm was monitored and the end pointafter � 20 min recorded.

Fig. 2. Validation of the lactose assay for use with common sugar solutionsobtained through enzymatic transformation of lactose.

Fig. 3. Reaction scheme for the oxidation of lactose into lactobionic acidusing continuous regeneration of oxidised DCIP by the laccase-catalysedreduction of dioxygen.

437B. Splechtna et al. / Enzyme and Microbial Technology 29 (2001) 434–440

The similarity of the electropherograms in Fig. 5 dem-onstrates that overall, the CDH-catalysed oxidation of lac-tose has little effect on GOS yield and composition. Adetailed comparison at the level of individual saccharidecomponents suggests losses of � 25% for the trisaccharide�-D-Gal-(136)-�-D-Galp-(134)-D-Glc and each of thethree tetrasaccharides. In accordance to Baminger et al. [18]CDH proved to be specific for di- and oligosaccharideswhich possess a �-(134)-linked sugar residue at the reduc-ing end. The material balance of the enzymatic oxidation oflactose is shown in Table 1.

3.4. Removal of lactobionic acid and salts

Serial ion exchange chromatographies were used to re-move all ionic components, including lactobionic acid, andDCIP. HPLC analysis showed that lactobionic acid andGOS had been separated completely. Under the conditionsemployed, monosaccharides eluted at a rate slightly slowerthan that of GOS elution. Therefore, 10–15% of monosac-charides were found in the last few, almost GOS-free frac-tions and could be separated from the mix. Unfortunately,about 20% of total sugar (referring to the initial amount oflactose) were lost during these processing steps, approximatelyhalf of that being valuable GOS. That means a loss of about25% of total GOS obtained after the � -glycosidase treatmentof the lactose solution. Further investigation will be necessaryto find and eliminate the reason for this problem.

3.5. Removal of monosaccharides

D-Glucose and D-galactose were separated from the GOSto an extent of � 95% by using a single step of cationexchange chromatography with Unibead UBK-530. TLC ofcarbohydrate-containing fractions was used to monitor the

Fig. 4. Batchwise enzymatic oxidation of lactose into lactobionic acidusing a substrate solution containing lactose, monosaccharides and GOS.The reaction mixture consisted of CDH (1 U/ml), laccase (2.5 U/ml), DCIP(5.45 mM), and sodium acetate buffer (183 mM, pH 5). It was flushed withpure oxygen to supply the substrate of laccase. See the Materials andmethods section for further details of the experimental procedure. Symbols:-● - lactobionic acid [mM], -f- lactose [mM], -Œ-pH.

Fig. 5. Capillary electropherograms of GOS (A) before oxidation with CDH and (B) after oxidation with CDH. Numbering refers to 1 � lactose, 2 � �-D-Galp-(133)-D-Glc, 3 � �-D-Galp-(136)-D-Glc (allolactose), 4 � �-D-Galp-(136)-D-Gal, 5 � unknown trisaccharide, 6 � �-D-Galp-(136)-�-D-Galp-(134)-D-Glc, 7 � �-DGalp-(133)-�-D-Galp-(134)-D-Glc, 8 � trisaccharide, 9–11 � tetrasaccharides.

Table 1Composition of GOS before and after oxidation with CDH

Composition of GOS [%]* Total loss[%]*

Beforeoxidation

Afteroxidation

Disaccharides 37 40 6Trisaccharides 47 47 14Tetrasaccharides 16 13 25

* Percentage values were calculated by weight.

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separation. Pooling those fractions that were rich in GOSbut low in D-glucose gave an end product of high purity(97%). It contained 0.9% D-glucose, 1.2% D-galactose and1.2% lactose. Table 2 shows the material balance for theproduction and downstream processing of GOS. The overallyield was 25% of original lactose or 65% of GOS in the�-glycosidase-treated mixture.

When comparison is made pertaining to the content oflactose and monosaccharides, the product obtained by theprocedure reported here is of considerably higher puritythan GOS mixtures available on the market (Table 3). Es-pecially the low lactose content is remarkable. Matsumotoet al. [15] describe a procedure of producing GOS with areduced lactose content. As was done here, Unibead UBK-530 was used for the purification of GOS. However, thelarge content of lactose in the GOS mixture made it neces-sary to go through several steps of re-chromatography em-ploying a cyclic operating method. Nevertheless, the result-ing GOS product still contained 14% lactose and 1%monosaccharides. Using the here described method of oxi-dising lactose into lactobionic acid and removal thereof byanion exchange chromatography a product containing only1.2% lactose is obtained and a near-complete removal ofmonosaccharides is achieved in a single step of cationexchange chromatography.

4. Conclusions

(1). In this study we have developed a new method ofproducing lactose-free GOS in high purity (97%). There-fore, the outlined procedure may be specially suited forthe production of physiologically active GOS designedfor nutrition of lactose-intolerant consumers.(2). Lactobionic acid, a by-product of the described pro-cess, is of value for medical purposes. As an example, itis used in the “Wisconsin transplantation solution” be-cause of its excellent metal-chelating properties that re-duce oxidative damage to tissue during storage of organscaused by certain metal ions [19]. In food technologylactobionic acid may find applications due to its ability toform mineral salt complexes [20] and its presumed pre-biotic effect [21]. Therefore, the technical and economicfeasibility of the recovery of lactobionic acid from theanion exchange resin constitutes an interesting field forfurther investigation.(3). The here developed lactose assay using CDH is asimple and robust means of determining lactose in thepresence of GOS.

Acknowledgments

Financial support from the European Commission isgratefully acknowledged (grant EC FAIR CT 96-1048). Dr.Marco Moracci (CNR, Naples, Italy) is thanked for provid-ing Ss�Gly; Christiane Galhaup and Roland Ludwig(BOKU, Vienna) for supplying the laccase and CDH, re-spectively. The measurements by CE were kindly supportedby Dr. Sabine Baumgartner (IFA Tulln, BOKU, Vienna).

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Table 2Material balance of GOS production from lactose

Processing steps Compositionof product [%]*

Purificationfold

Yield[%]*

Monosacch. Lactose GOS

Initial solution 0 100 0After treatment with

Ss�Gly46 13 41 92

After oxidation withCDH

56 1 43 1.1 75

Final product 2 1 97 2.4 25

* Percentage values were calculated by weight.

Table 3Comparison of compositions (by weight) of GOS mixtures available onthe market and described in the literature

Company/Reference Product GOS[%]

Lactose[%]

Monosaccharides[%]

Borculo WheyProducts1

Elix’or® 60 20 20

Yakult Honsha2 Oligomate 50® 50–52 10–13 36–39Europ. Patent

0272095385 14 1

Product describedhere

97 1 2

1 Data from product information brochure (1996), Borculo Whey Prod-ucts.

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