Food Chemistry 128 (2011) 773–777

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Food Chemistry

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Short communication

Galactosyl oligosaccharide purification by ethanol precipitation

Dwaipayan Sen a, Aaron Gosling b,c, Geoff W. Stevens b, Prashant K. Bhattacharya d, Andrew R. Barber e,Sandra E. Kentish b, Chiranjib Bhattacharjee a, Sally L. Gras b,c,⇑a The Department of Chemical Engineering, Jadavpur University, Kolkata 700 032, Indiab The Department of Chemical and Biomolecular Engineering, The University of Melbourne, Victoria 3010, Australiac The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria 3010, Australiad Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016, Indiae Innovative Food and Plants Division, South Australian Research and Development Institute, Regency International Centre, Days Road, Regency Park, SA 5010, Australia

a r t i c l e i n f o

Article history:Received 9 August 2010Received in revised form 10 February 2011Accepted 16 March 2011Available online 22 March 2011

Keywords:Galactosyl oligosaccharideGOSPrebioticSaccharide precipitation

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.03.076

⇑ Corresponding author at: The Department ofEngineering, The University of Melbourne, Victoria8344 4153.

E-mail address: sgras@unimelb.edu.au (S.L. Gras).

a b s t r a c t

Galactosyl oligosaccharides (GOS) are prebiotics commonly manufactured by b-galactosidase conversionof lactose, producing a mixture containing GOS, lactose, glucose and galactose. Enrichment of GOS in thismixture adds value to the product. This study aimed to determine whether the addition of ethanol toaqueous saccharide solutions could be used to selectively precipitate and enrich GOS from a reaction mix-ture. High concentrations of ethanol (>70% v/v) were required to induce precipitation. The total saccha-ride concentration was a significant variable, with higher GOS enrichment occurring at lower totalsaccharide concentrations. Varying the temperature between 10 and 40 �C had less impact than hadchanges in the concentration of saccharide or ethanol. GOS was enriched 2.3 (±0.1) fold in the precipitateformed in a solution of 90% (v/v) ethanol with 28 g/L of total saccharide at 40 �C. Performing two suchprecipitations sequentially reduced the monosaccharides from 48% (w/w) of the total saccharides to4% (w/w). GOS precipitation has potential for industrial application as it is simple in operation and offerslevels of purification similar to those by other techniques.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Galactosyl oligosaccharides (GOS) are an example of a prebioticfood ingredient that allows specific changes ‘‘both in the composi-tion and/or activity in the gastrointestinal microflora that confersbenefits upon host well being and health’’ (Macfarlane, Steed, &Macfarlane, 2008; Roberfroid, 2007). The global retail market forprebiotic foods is large and growing in size, with recent estimatesof an annual 167,000 ton and 390 million Euro market (Siro, Kápol-na, Kápolna, & Lugasi, 2008).

GOS are produced from lactose by the enzyme b-galactosidase(Gosling, Stevens, Barber, Kentish, & Gras, 2010). This complexreaction system produces a mixture of GOS, along with the mono-saccharides glucose and galactose, which are not considered prebi-otic (Roberfroid, 2007). The product mixture typically also containsunreacted lactose. Isolating GOS would add value by increasing theprebiotic efficacy per unit mass while reducing calorific value andcariogenicity. Such purification would also change the functionalproperties of the product mixture by lowering hygroscopicity and

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Chemical and Biomolecular3010, Australia. Fax: +61 3

sweetness as well as increasing viscosity (Crittenden & Playne,2002).

Many GOS purification strategies have been reported. Largescale continuous ion-exclusion chromatography has long been ap-plied to processing of sucrose (Bubnik et al., 2004) and purifying oflactose (Harju & Heikkila, 1990) and a semi-continuous ion-exclu-sion chromatography process has been patented for GOS purifica-tion (Sinclair, De Slegte, & Klarenbeek, 2008). Supercritical fluidextraction (SFE) has achieved 75% (w/w) GOS purity with 94%(w/w) recovery (Montañés, Olano, Reglero, Ibáñez, & Fornari,2009). Nanofiltration (NF) recovered 98% of the GOS from a com-mercial GOS syrup (Vivinal� GOS) but also retained 18% of mono-saccharide (Goulas, Kapasakalidis, Sinclair, Rastall, & Grandison,2002). Microbes such as Saccharomyces cerevisiae (Goulas, Tzortzis,& Gibson, 2007) and Zymomonas mobilis (Crittenden & Playne,2002) selectively consume up to 90% of monosaccharides presentin saccharide mixtures, effectively enriching lactose andoligosaccharides.

Exploiting differences in solubility to purify components of mix-tures is a common approach, with the generic advantage over theabove techniques of simplicity and cost effectiveness during scaleup. This approach has been extensively studied for purifying lac-tose (Bourne, Hegglin, & Prenosil, 1983; Gänzle, Haase, & Jelen,2008). GOS purification through the exploitation of differential sol-ubility has not to our knowledge been reported.

774 D. Sen et al. / Food Chemistry 128 (2011) 773–777

This study aimed to develop a simple, scalable method, to sep-arate GOS from mono- and di-saccharides, based on precipitation.An ethanol–water system was selected as it allows the preparationof food-grade products. The enrichment and recovery of GOS, atvaried saccharide concentrations and temperatures, was assessedat different ethanol concentrations.

2. Materials and methods

2.1. Enzymes and chemicals

The b-galactosidase enzyme preparation, Biolacta FN5, waskindly supplied by Vitachem, Sydney, Australia. Lactose monohy-drate, D-glucose (Chem Supply, Gillman, Australia), D-galactose(Sigma–Aldrich, Sydney, Australia) and ethanol (Merck, Kilsyth,Australia) were of analytical grade. Raffinose pentahydrate (Sig-ma–Aldrich, Sydney, Australia) was P99%. Milli Q water (resistiv-ity < 18.2 Ohm) was used.

2.2. Preparation of the saccharide feedstock

To produce a stable feedstock for solubility experiments, thatwas representative of GOS reaction products, Biolacta FN5 (1 g)was added to a 2 L solution of 10 mM sodium acetate buffer, atpH 6.6, containing 100 g/L of lactose monohydrate. The reactionwas allowed to proceed for 17.5 h at 40.0 ± 0.1 �C. To stop the reac-tion, the enzyme was separated from the reaction mixture, usingultrafiltration in a cross-flow membrane module (Osmonics SepaCF II Cell), equipped with a 15 kDa membrane (HFK-131 from KochMembrane Systems, Australia). Ultrafiltration was performed untilthe retentate was concentrated by a volume factor (VCF) of 4 andthe permeate containing the sugars further processed. An enzymeactivity assay (Fujimoto, Miyasato, Ito, Sasaki, & Ajisaka, 1998)showed that all b-galactosidase had been removed from the per-meate. A rotary evaporator was used to concentrate the permeatebefore freeze-drying (Dynavac FD5 operating below 10 Torr at22 �C). The reduced water content of the resultant saccharide syrupallowed experiments at high saccharide and ethanol concentra-tions. It also provided a mimic for commercial GOS products (e.g.Vivinal� GOS), which are supplied as concentrated syrups.

2.3. Saccharide precipitation

The saccharide syrup, obtained after freeze-drying, was dilutedby the addition of 100 mL of water to 300 g of syrup, producing afeedstock, found by high performance liquid chromatography(HPLC), to contain 62 ± 1% w/w of saccharides (composition shownin Table 1).

Aqueous saccharide solutions, at three concentrations (280, 600and 810 g/L) were prepared, with densities of 1.08 ± 0.02,1.21 ± 0.05 and 1.30 ± 0.07 g/mL, respectively. Aliquots (0.5 mL)of each saccharide solution were diluted to the final concentrationsof 28, 60 and 81 g/L by adding 4.5 mL of ethanol and water. Forexample, when 90% (v/v) ethanol was required, 4.5 mL of neat eth-

Table 1Composition of saccharide feedstock.

Saccharide Percentage oftotal saccharidea

GOS 16 (1.7)Lactose 37 (2.1)Glucose 30 (1.0)Galactose 18 (0.8)

a Mean of six samples taken fromthe feedstock with standard devia-tions in parentheses.

anol was added to the 0.5 mL saccharide solution aliquot. Thesesolutions were incubated for 16 h at 40, 25 or 10 �C, with orbitalshaking at 60 rpm in sealed tubes to minimise evaporation.

After incubation, samples were withdrawn and centrifuged at13,000g for 3 min to separate any precipitated solids. The superna-tant was diluted twofold with Milli Q water before analysis byHPLC.

For sequential precipitations, solid material, precipitated in 90%(v/v) ethanol at 28 g/L of total saccharide and 40 �C, was redis-solved to 28 g/L of total saccharide with water before performingthe precipitation again.

2.4. HPLC analyses

HPLC analysis of carbohydrates was performed as previouslydescribed (Gosling et al., 2009). Saccharides were separated usinga Shimadzu Prominence HPLC with a 300 � 7.8 mm Rezex RCM-Monosaccharide Ca2+ column (Phenomenex). The Milli Q watermobile phase flow rate was 0.5 mL/min and saccharides were de-tected with a RID-10A refractive index detector. The column anddetector cell were maintained at 80 and 40 �C, respectively.

Saccharides were quantified using external standards of analyt-ical grade galactose, glucose and lactose. GOS was quantified usingraffinose as a standard.

2.5. Calculations

The mass and composition of the precipitate generated by eachtreatment were estimated by performing a mass balance (Eq. (1)).In order to calculate this mass balance, all test solutions were incu-bated in parallel to a reference solution containing 50% (v/v) etha-nol. A preliminary study found that 50% (v/v) ethanol wasinsufficient to cause precipitation at any combination of tempera-ture and saccharide concentration examined. The mass of each sac-charide found in the solution phase of test solutions (Masstreated)was subtracted from the mass of saccharide found in the referencesolution (Massreference) to give the mass of that individual saccha-ride in the precipitate (Massppt)

Massreference �Masstreated ¼Massppt ð1Þ

The mass of saccharide present in the precipitate was then usedto calculate metrics for assessing each treatment (Eqs. (2)–(4))

Percentage Recovery of GOS ¼ 100� GOS Massppt

Initial GOS Massð2Þ

To calculate the fold enrichment (Eq. (3)), the mass fraction ofGOS present in the saccharide feedstock (0.16 ± 0.017 g of GOSper gram of total saccharide) (Table 1) was used in thedenominator

Fold Enrichment of GOS

¼ GOS Massppt=Total Saccharide Massppt

Initial GOS Mass=Initial Total Saccharide Massð3Þ

GOS : Lac ¼ 100� GOS Massppt

Lac Masspptð4Þ

Experimental error was assessed by performing triplicate pre-cipitations at each of the saccharide concentrations (28, 60 and81 g/L) and 40 �C. The standard deviation of the mean concentra-tion of GOS, lactose, glucose and galactose was less than 1.2 g/Lfor each saccharide. The percentage of the mean GOS recovery,enrichment and GOS:Lac, represented by the standard deviation,was used as an estimate of the experimental error of those metrics

3

D. Sen et al. / Food Chemistry 128 (2011) 773–777 775

for the same saccharide concentration at the two other tempera-tures tested (10 and 25 �C). Statistical significance was determinedby the Student t-test.

0 20 40 60 80 100

1

2

Fol

d E

nric

hmen

t of G

OS

Saccharide concentration (g/L)

Fig. 1. The effect of saccharide concentration on the enrichment of GOS in theprecipitate formed at different temperatures in the presence of 90% (v/v) ethanol.Temperatures were 40 �C (}), 25 �C (�) and 10 �C (M). The error bars show thepercentage of the value estimated as experimental error from triplicate experi-ments performed for each saccharide concentration at 40 �C (see Section 2.5).

0 20 40 60 80 1000

20

40

60

80

100

GO

S to

lact

ose

ratio

Saccharide concentration (g/L)

Fig. 2. The effect of saccharide concentration on the GOS to lactose ratio in theprecipitate formed at different temperatures in the presence of 90% (v/v) ethanol.Temperatures were 40 �C (}), 25 �C (�) and 10 �C (M). The error bars show thepercentage of the value estimated as experimental error from triplicate experi-ments performed for each saccharide concentration at 40 �C (see Section 2.5).

3. Results and discussion

3.1. General

The aim of this study was to determine whether ethanol couldbe used to selectively precipitate and enrich GOS present in a reac-tion mixture of saccharides typical of those produced by b-galacto-sidase. The effects of varying ethanol concentration, saccharideconcentration and temperature on precipitation were examined.

3.2. Effect of ethanol concentration on saccharide solubility

Initial experiments showed that high concentrations of ethanolwere required to cause saccharide precipitation. No solid materialwas formed after incubation of saccharides in 70% (v/v) ethanol, forany of the saccharide concentrations and temperatures tested.

Precipitation was visually evident at 85% (v/v) ethanol for alltemperatures tested (10, 25 and 40 �C) at the two higher saccha-ride concentrations examined (60 and 81 g/L of total saccharide).GOS was also enriched in the precipitates of these samples.

Precipitation occurred in the 90% (v/v) ethanol solutions at allnine combinations of temperature and saccharide concentrationtested. Recoveries of GOS were higher with 90% (v/v) ethanol thanwith 85% (v/v), so this ethanol concentration formed a focus forsubsequent studies.

The decrease in saccharide solubility with increased concentra-tion of ethanol was consistent with the reported reduced solubilityof glucose (Alves, Almeida e Silva, & Giulietti, 2007) and lactose(Machado, Coutinho, & Macedo, 2000) in ethanol/water mixtureswith increasing ethanol concentration.

The effect of ethanol concentrations higher than 90% (v/v) couldnot be tested as these would require greater concentration of thesaccharide feedstock, which was not practically possible.

3.3. Effect of saccharide concentration on GOS purification byprecipitation

The GOS enrichment (Eq. (3)), achieved by precipitation in 90%(v/v) ethanol, increased significantly (p < 0.05) with decreasingsaccharide concentration at all temperatures tested (Fig. 1). At40 �C, for example, GOS was enriched in the precipitate by factorsof 1.2 (±0.00), 1.4 (±0.01) and 2.3 (±0.10) at total saccharide con-centrations of 81, 60 and 28 g/L, respectively. Similarly, the GO-S:Lac ratio (Eq. (4)) also increased significantly (p < 0.05) withdecreasing saccharide concentration for all temperatures tested(Fig. 2). Conversely, GOS recovery increased significantly(p < 0.05) with increasing saccharide concentration (Fig. 3).

These observations are consistent with previous reports that anincrease in the total saccharide concentration reduces the solubil-ity of individual saccharides within a mixture (Nickerson & Moore,1972). For example, the solubility of lactose is reduced by the pres-ence of other sugars, such as sucrose (Hartel & Shastry, 1991) ormixtures of glucose and galactose (Bourne et al., 1983). The totalsaccharide concentration therefore determines the proportion ofan individual saccharide in the solution and solid phases, with ahigher proportion of saccharides present in the precipitate ratherthan in solution when the total saccharide concentration is highand a higher proportion of saccharides present in solution whenthe total saccharide concentration is low. The partitioning of sac-charides between the solution and solid phases differed betweensaccharides when the total saccharide concentration was low,

affording some selectivity in precipitation, with a higher propor-tion of the total mass of lactose, glucose and galactose in solutioncompared to GOS. This resulted in a higher GOS enrichment but alower percentage recovery of GOS, as there was more GOS in solu-tion and less GOS in the solid precipitate under these conditions.

3.4. Effect of temperature on GOS purification by precipitation

The variation in temperature, between 10 and 40 �C, had lessinfluence on the purification of GOS by precipitation than had var-iation in the saccharide concentration.

Increases in temperature gave slight increases in the GOS:Lacratio at 28 g/L of total saccharide (Fig. 4), with a significant differ-ence between 10 and 40 �C. There was no significance in the differ-

0 20 40 60 80 1000

20

40

60

80

100

Saccharide concentration (g/L)

Per

cent

age

reco

very

of G

OS

Fig. 3. The effect of saccharide concentration on the percentage recovery of GOS inprecipitate formed at different temperatures in the presence of 90% (v/v) ethanol.Temperatures were 40 �C (}), 25 �C (�) and 10 �C (M). The error bars show thepercentage of the value estimated as experimental error from triplicate experi-ments performed for each saccharide concentration at 40 �C (see Section 2.5).

10 15 20 25 30 35 400

20

40

60

80

100

GO

S to

lact

ose

ratio

Temperature ( oC)

Fig. 4. Effects of temperature on GOS to lactose ratio at different saccharideconcentrations in the precipitate formed in the presence of 90% (v/v) ethanol. Totalsaccharide concentrations were 81 (}), 60 (�) and 28 g/L (M). The error bars showthe percentage of the value estimated as experimental error from triplicateexperiments performed for each saccharide concentration at 40 �C (see Section 2.5).

Fig. 5. Composition of saccharides (% w/w) in solid fraction from two sequentialprecipitations.

776 D. Sen et al. / Food Chemistry 128 (2011) 773–777

ences of the GOS:Lac ratio with varied temperature, at either 60 or81 g/L of total saccharide. The temperature had no significant effecton either enrichment (Eq. (2)) or percentage recovery (Eq. (1)) ofGOS achieved by precipitation with 90% (v/v) ethanol at a given to-tal saccharide concentration.

Temperature is known to affect the solubility of saccharides inboth aqueous and ethanol – water solutions. Lactose, glucose andgalactose mixtures in water show decreased solubility withdecreasing temperature (Bourne et al., 1983). A decrease in tem-perature is also known to decrease the concentration at which glu-cose becomes saturated in ethanol – water mixtures with ethanolconcentrations up to 80% (v/v) (Alves et al., 2007). However, thesolubility of lactose did not significantly change in 90% (v/v) etha-nol between 60 and 25 �C (Machado et al., 2000). It seems likely

that the ethanol and total saccharide concentrations used weremore important in affecting solubility behaviour between 10 and40 �C than was the temperature.

3.5. GOS purification

The purification efficacy of ethanol precipitation can be in-creased by performing repeated precipitations. Two sequentialprecipitations increased the percentage of GOS from 15% (w/w)in the feedstock to 75% (w/w) in the product while reducing themonosaccharide concentration from 48% to 4% (w/w) (Fig. 5). How-ever, only 6% (w/w) of the mass of GOS initially present was recov-ered after these two precipitation steps.

Purification of GOS by a single step ethanol precipitation, usingthe optimal conditions tested here (28 g/L saccharide concentra-tion and 40 �C) compared favourably with other techniques. Datafrom a published study examining common laboratory scale GOSpurification techniques (Hernández, Ruiz-Matute, Olano, Moreno,& Sanz, 2009) were used for the calculation of GOS enrichment gi-ven by Eq. (3) (Section 2.5). The GOS enrichments of charcoaladsorption, selective monosaccharide fermentation by S. cerevisiaeand size-exclusion chromatography were 2.4-, 1.3- and 1.0-fold,respectively. A single step ethanol precipitation achieved 2.3-foldGOS enrichment, which compares very favourably with charcoaladsorption. However, GOS recovery with charcoal adsorption was69%, higher than GOS recovery by ethanol precipitation at 47%(Fig. 3, 28 g/L saccharide concentration and 40 �C).

In addition to comparable enrichment performance, ethanolprecipitation offers advantages over other techniques used to pur-ify food grade GOS. For example, supercritical fluid extraction (SFE)gives excellent separation (Montañés et al., 2009) but can be veryexpensive. Production scale chromatography can also be costly.Selective fermentation of monosaccharides introduces fermenta-tion end-products, such as ethanol or lactic acid, which alters prod-uct composition, nutrition and taste. Selective fermentation alsogenerally fails to remove lactose, which may reduce applicationsfor GOS ingredients. A disadvantage of nanofiltration is that it re-quires high pressure to achieve a moderate flux on the permeateside, making the process energy intensive.

Ethanol precipitation can be used as an alternative to the com-peting technologies above or in combination with these methods.For instance, it could be used as a step to enrich and concentrateGOS prior to chromatography. Conversely, nanofiltration could beused to concentrate the sugar solution prior to a final precipitationstep.

D. Sen et al. / Food Chemistry 128 (2011) 773–777 777

The complete data set, containing the mass of saccharides mea-sured in the solution and solid phases in this study, is available inthe Supplementary material to aid further work and the applica-tion of this technique.

4. Conclusion

GOS can be enriched from a reaction mixture of GOS, lactose,glucose and galactose by precipitation in 90% (v/v) ethanol. GOSenrichment was highest under conditions expected to give thehighest saccharide solubility. The combination of the lowest sac-charide concentration tested (28 g/L) and highest temperaturetested (40 �C) gave 2.3 ± 0.1-fold GOS enrichment and a GOS:Lacratio of 99.8 ± 5.5%. By contrast, GOS recovery was highest(97.5 ± 0.3%) at the highest saccharide concentration (81 g/L)studied.

Acknowledgements

This study was jointly funded by the Indian GovernmentDepartment of Biotechnology under the Indo-Australian Biotech-nology Fund (vide sanction letter no. BT/PR9547/ICD/16/754/2006 of DBT/Indo-Aus/01/35/06 dated July 02, 2007) and the Aus-tralian Government Department of Innovation, Industry, Scienceand Research Australia – India Strategic Research Fund BF01-0024. We also acknowledge the Particulate Fluids Processing Cen-tre, a Special Research Centre of the Australian Research Council(ARC), for their support.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.foodchem.2011.03.076.

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