Food Chemistry 128 (2011) 773777

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Food Chemistry
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Short communication

Galactosyl oligosaccharide purication 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, India The Department of Chemical and Biomolecular Engineering, The University of Melbourne, Victoria 3010, Australia c The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria 3010, Australia d Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016, India e Innovative Food and Plants Division, South Australian Research and Development Institute, Regency International Centre, Days Road, Regency Park, SA 5010, Australia
b

a r t i c l e

i n f o

a b s t r a c t
Galactosyl oligosaccharides (GOS) are prebiotics commonly manufactured by b-galactosidase conversion of lactose, producing a mixture containing GOS, lactose, glucose and galactose. Enrichment of GOS in this mixture adds value to the product. This study aimed to determine whether the addition of ethanol to aqueous saccharide solutions could be used to selectively precipitate and enrich GOS from a reaction mixture. High concentrations of ethanol (>70% v/v) were required to induce precipitation. The total saccharide concentration was a signicant variable, with higher GOS enrichment occurring at lower total saccharide concentrations. Varying the temperature between 10 and 40 C had less impact than had changes in the concentration of saccharide or ethanol. GOS was enriched 2.3 (0.1) fold in the precipitate formed in a solution of 90% (v/v) ethanol with 28 g/L of total saccharide at 40 C. Performing two such precipitations sequentially reduced the monosaccharides from 48% (w/w) of the total saccharides to 4% (w/w). GOS precipitation has potential for industrial application as it is simple in operation and offers levels of purication similar to those by other techniques. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 9 August 2010 Received in revised form 10 February 2011 Accepted 16 March 2011 Available online 22 March 2011 Keywords: Galactosyl oligosaccharide GOS Prebiotic Saccharide precipitation

1. Introduction Galactosyl oligosaccharides (GOS) are an example of a prebiotic food ingredient that allows specic changes both in the composition and/or activity in the gastrointestinal microora that confers benets upon host well being and health (Macfarlane, Steed, & Macfarlane, 2008; Roberfroid, 2007). The global retail market for prebiotic foods is large and growing in size, with recent estimates of an annual 167,000 ton and 390 million Euro market (Siro, Kpolna, Kpolna, & Lugasi, 2008). GOS are produced from lactose by the enzyme b-galactosidase (Gosling, Stevens, Barber, Kentish, & Gras, 2010). This complex reaction system produces a mixture of GOS, along with the monosaccharides glucose and galactose, which are not considered prebiotic (Roberfroid, 2007). The product mixture typically also contains unreacted lactose. Isolating GOS would add value by increasing the prebiotic efcacy per unit mass while reducing caloric value and cariogenicity. Such purication would also change the functional properties of the product mixture by lowering hygroscopicity and

Corresponding author at: The Department of Chemical and Biomolecular Engineering, The University of Melbourne, Victoria 3010, Australia. Fax: +61 3 8344 4153. E-mail address: sgras@unimelb.edu.au (S.L. Gras).
0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.03.076

sweetness as well as increasing viscosity (Crittenden & Playne, 2002). Many GOS purication strategies have been reported. Large scale continuous ion-exclusion chromatography has long been applied to processing of sucrose (Bubnik et al., 2004) and purifying of lactose (Harju & Heikkila, 1990) and a semi-continuous ion-exclusion chromatography process has been patented for GOS purication (Sinclair, De Slegte, & Klarenbeek, 2008). Supercritical uid extraction (SFE) has achieved 75% (w/w) GOS purity with 94% (w/w) recovery (Montas, Olano, Reglero, Ibez, & Fornari, 2009). Nanoltration (NF) recovered 98% of the GOS from a commercial GOS syrup (Vivinal GOS) but also retained 18% of monosaccharide (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 present in saccharide mixtures, effectively enriching lactose and oligosaccharides. Exploiting differences in solubility to purify components of mixtures is a common approach, with the generic advantage over the above techniques of simplicity and cost effectiveness during scale up. This approach has been extensively studied for purifying lactose (Bourne, Hegglin, & Prenosil, 1983; Gnzle, Haase, & Jelen, 2008). GOS purication through the exploitation of differential solubility has not to our knowledge been reported.

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This study aimed to develop a simple, scalable method, to separate GOS from mono- and di-saccharides, based on precipitation. An ethanolwater system was selected as it allows the preparation of food-grade products. The enrichment and recovery of GOS, at varied saccharide concentrations and temperatures, was assessed at different ethanol concentrations. 2. Materials and methods 2.1. Enzymes and chemicals The b-galactosidase enzyme preparation, Biolacta FN5, was kindly supplied by Vitachem, Sydney, Australia. Lactose monohydrate, D-glucose (Chem Supply, Gillman, Australia), D-galactose (SigmaAldrich, Sydney, Australia) and ethanol (Merck, Kilsyth, Australia) were of analytical grade. Rafnose pentahydrate (SigmaAldrich, Sydney, Australia) was P99%. Milli Q water (resistivity < 18.2 Ohm) was used. 2.2. Preparation of the saccharide feedstock To produce a stable feedstock for solubility experiments, that was representative of GOS reaction products, Biolacta FN5 (1 g) was added to a 2 L solution of 10 mM sodium acetate buffer, at pH 6.6, containing 100 g/L of lactose monohydrate. The reaction was allowed to proceed for 17.5 h at 40.0 0.1 C. To stop the reaction, the enzyme was separated from the reaction mixture, using ultraltration in a cross-ow membrane module (Osmonics Sepa CF II Cell), equipped with a 15 kDa membrane (HFK-131 from Koch Membrane Systems, Australia). Ultraltration was performed until the retentate was concentrated by a volume factor (VCF) of 4 and the permeate containing the sugars further processed. An enzyme activity assay (Fujimoto, Miyasato, Ito, Sasaki, & Ajisaka, 1998) showed that all b-galactosidase had been removed from the permeate. A rotary evaporator was used to concentrate the permeate before freeze-drying (Dynavac FD5 operating below 10 Torr at 22 C). The reduced water content of the resultant saccharide syrup allowed experiments at high saccharide and ethanol concentrations. 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 diluted by the addition of 100 mL of water to 300 g of syrup, producing a feedstock, found by high performance liquid chromatography (HPLC), to contain 62 1% w/w of saccharides (composition shown in Table 1). Aqueous saccharide solutions, at three concentrations (280, 600 and 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 nal concentrations of 28, 60 and 81 g/L by adding 4.5 mL of ethanol and water. For example, when 90% (v/v) ethanol was required, 4.5 mL of neat ethTable 1 Composition of saccharide feedstock. Saccharide GOS Lactose Glucose Galactose Percentage of total saccharidea 16 37 30 18 (1.7) (2.1) (1.0) (0.8)

anol was added to the 0.5 mL saccharide solution aliquot. These solutions were incubated for 16 h at 40, 25 or 10 C, with orbital shaking at 60 rpm in sealed tubes to minimise evaporation. After incubation, samples were withdrawn and centrifuged at 13,000g for 3 min to separate any precipitated solids. The supernatant was diluted twofold with Milli Q water before analysis by HPLC. For sequential precipitations, solid material, precipitated in 90% (v/v) ethanol at 28 g/L of total saccharide and 40 C, was redissolved to 28 g/L of total saccharide with water before performing the precipitation again. 2.4. HPLC analyses HPLC analysis of carbohydrates was performed as previously described (Gosling et al., 2009). Saccharides were separated using a Shimadzu Prominence HPLC with a 300 7.8 mm Rezex RCMMonosaccharide Ca2+ column (Phenomenex). The Milli Q water mobile phase ow rate was 0.5 mL/min and saccharides were detected with a RID-10A refractive index detector. The column and detector cell were maintained at 80 and 40 C, respectively. Saccharides were quantied using external standards of analytical grade galactose, glucose and lactose. GOS was quantied using rafnose as a standard. 2.5. Calculations The mass and composition of the precipitate generated by each treatment were estimated by performing a mass balance (Eq. (1)). In order to calculate this mass balance, all test solutions were incubated in parallel to a reference solution containing 50% (v/v) ethanol. A preliminary study found that 50% (v/v) ethanol was insufcient to cause precipitation at any combination of temperature and saccharide concentration examined. The mass of each saccharide found in the solution phase of test solutions (Masstreated) was subtracted from the mass of saccharide found in the reference solution (Massreference) to give the mass of that individual saccharide in the precipitate (Massppt)

Massreference Masstreated Massppt

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

Percentage Recovery of GOS 100

GOS Massppt Initial GOS Mass

To calculate the fold enrichment (Eq. (3)), the mass fraction of GOS present in the saccharide feedstock (0.16 0.017 g of GOS per gram of total saccharide) (Table 1) was used in the denominator

Fold Enrichment of GOS GOS Massppt =Total Saccharide Massppt Initial GOS Mass=Initial Total Saccharide Mass GOS Massppt Lac Massppt 3

GOS : Lac 100

a Mean of six samples taken from the feedstock with standard deviations in parentheses.

Experimental error was assessed by performing triplicate precipitations at each of the saccharide concentrations (28, 60 and 81 g/L) and 40 C. The standard deviation of the mean concentration of GOS, lactose, glucose and galactose was less than 1.2 g/L for 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

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for the same saccharide concentration at the two other temperatures tested (10 and 25 C). Statistical signicance was determined by the Student t-test. 3. Results and discussion 3.1. General The aim of this study was to determine whether ethanol could be used to selectively precipitate and enrich GOS present in a reaction mixture of saccharides typical of those produced by b-galactosidase. The effects of varying ethanol concentration, saccharide concentration and temperature on precipitation were examined. 3.2. Effect of ethanol concentration on saccharide solubility Initial experiments showed that high concentrations of ethanol were required to cause saccharide precipitation. No solid material was formed after incubation of saccharides in 70% (v/v) ethanol, for any of the saccharide concentrations and temperatures tested. Precipitation was visually evident at 85% (v/v) ethanol for all temperatures tested (10, 25 and 40 C) at the two higher saccharide 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 all nine combinations of temperature and saccharide concentration tested. Recoveries of GOS were higher with 90% (v/v) ethanol than with 85% (v/v), so this ethanol concentration formed a focus for subsequent studies. The decrease in saccharide solubility with increased concentration of ethanol was consistent with the reported reduced solubility of glucose (Alves, Almeida e Silva, & Giulietti, 2007) and lactose (Machado, Coutinho, & Macedo, 2000) in ethanol/water mixtures with increasing ethanol concentration. The effect of ethanol concentrations higher than 90% (v/v) could not be tested as these would require greater concentration of the saccharide feedstock, which was not practically possible. 3.3. Effect of saccharide concentration on GOS purication by precipitation The GOS enrichment (Eq. (3)), achieved by precipitation in 90% (v/v) ethanol, increased signicantly (p < 0.05) with decreasing saccharide concentration at all temperatures tested (Fig. 1). At 40 C, for example, GOS was enriched in the precipitate by factors of 1.2 (0.00), 1.4 (0.01) and 2.3 (0.10) at total saccharide concentrations of 81, 60 and 28 g/L, respectively. Similarly, the GOS:Lac ratio (Eq. (4)) also increased signicantly (p < 0.05) with decreasing saccharide concentration for all temperatures tested (Fig. 2). Conversely, GOS recovery increased signicantly (p < 0.05) with increasing saccharide concentration (Fig. 3). These observations are consistent with previous reports that an increase in the total saccharide concentration reduces the solubility of individual saccharides within a mixture (Nickerson & Moore, 1972). For example, the solubility of lactose is reduced by the presence of other sugars, such as sucrose (Hartel & Shastry, 1991) or mixtures of glucose and galactose (Bourne et al., 1983). The total saccharide concentration therefore determines the proportion of an individual saccharide in the solution and solid phases, with a higher proportion of saccharides present in the precipitate rather than in solution when the total saccharide concentration is high and a higher proportion of saccharides present in solution when the total saccharide concentration is low. The partitioning of saccharides between the solution and solid phases differed between saccharides when the total saccharide concentration was low,
Fold Enrichment of GOS

20

40

60

80

100

Saccharide concentration (g/L)

Fig. 1. The effect of saccharide concentration on the enrichment of GOS in the precipitate 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 the percentage of the value estimated as experimental error from triplicate experiments performed for each saccharide concentration at 40 C (see Section 2.5).

100

80

GOS to lactose ratio

60

40

20

0 0 20 40 60 80 100

Saccharide concentration (g/L)

Fig. 2. The effect of saccharide concentration on the GOS to lactose ratio in the precipitate 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 the percentage of the value estimated as experimental error from triplicate experiments performed for each saccharide concentration at 40 C (see Section 2.5).

affording some selectivity in precipitation, with a higher proportion of the total mass of lactose, glucose and galactose in solution compared to GOS. This resulted in a higher GOS enrichment but a lower percentage recovery of GOS, as there was more GOS in solution and less GOS in the solid precipitate under these conditions. 3.4. Effect of temperature on GOS purication by precipitation The variation in temperature, between 10 and 40 C, had less inuence on the purication of GOS by precipitation than had variation in the saccharide concentration. Increases in temperature gave slight increases in the GOS:Lac ratio at 28 g/L of total saccharide (Fig. 4), with a signicant difference between 10 and 40 C. There was no signicance in the differ-

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that the ethanol and total saccharide concentrations used were more important in affecting solubility behaviour between 10 and 40 C than was the temperature.

80

Percentage recovery of GOS

3.5. GOS purication

60

40

20

0 0 20 40 60 80 100

Saccharide concentration (g/L)

Fig. 3. The effect of saccharide concentration on the percentage recovery of GOS in precipitate 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 the percentage of the value estimated as experimental error from triplicate experiments performed for each saccharide concentration at 40 C (see Section 2.5).

100

80

60

40

20

0 10 15 20 25
o

30

35

40

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

The purication efcacy of ethanol precipitation can be increased by performing repeated precipitations. Two sequential precipitations increased the percentage of GOS from 15% (w/w) in the feedstock to 75% (w/w) in the product while reducing the monosaccharide concentration from 48% to 4% (w/w) (Fig. 5). However, only 6% (w/w) of the mass of GOS initially present was recovered after these two precipitation steps. Purication of GOS by a single step ethanol precipitation, using the optimal conditions tested here (28 g/L saccharide concentration and 40 C) compared favourably with other techniques. Data from a published study examining common laboratory scale GOS purication techniques (Hernndez, Ruiz-Matute, Olano, Moreno, & Sanz, 2009) were used for the calculation of GOS enrichment given by Eq. (3) (Section 2.5). The GOS enrichments of charcoal adsorption, selective monosaccharide fermentation by S. cerevisiae and size-exclusion chromatography were 2.4-, 1.3- and 1.0-fold, respectively. A single step ethanol precipitation achieved 2.3-fold GOS enrichment, which compares very favourably with charcoal adsorption. However, GOS recovery with charcoal adsorption was 69%, 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, ethanol precipitation offers advantages over other techniques used to purify food grade GOS. For example, supercritical uid extraction (SFE) gives excellent separation (Montas et al., 2009) but can be very expensive. Production scale chromatography can also be costly. Selective fermentation of monosaccharides introduces fermentation end-products, such as ethanol or lactic acid, which alters product composition, nutrition and taste. Selective fermentation also generally fails to remove lactose, which may reduce applications for GOS ingredients. A disadvantage of nanoltration is that it requires high pressure to achieve a moderate ux on the permeate side, making the process energy intensive. Ethanol precipitation can be used as an alternative to the competing technologies above or in combination with these methods. For instance, it could be used as a step to enrich and concentrate GOS prior to chromatography. Conversely, nanoltration could be used to concentrate the sugar solution prior to a nal precipitation step.

ences of the GOS:Lac ratio with varied temperature, at either 60 or 81 g/L of total saccharide. The temperature had no signicant effect on either enrichment (Eq. (2)) or percentage recovery (Eq. (1)) of GOS achieved by precipitation with 90% (v/v) ethanol at a given total saccharide concentration. Temperature is known to affect the solubility of saccharides in both aqueous and ethanol water solutions. Lactose, glucose and galactose mixtures in water show decreased solubility with decreasing temperature (Bourne et al., 1983). A decrease in temperature is also known to decrease the concentration at which glucose becomes saturated in ethanol water mixtures with ethanol concentrations up to 80% (v/v) (Alves et al., 2007). However, the solubility of lactose did not signicantly change in 90% (v/v) ethanol between 60 and 25 C (Machado et al., 2000). It seems likely

GOS to lactose ratio

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

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The complete data set, containing the mass of saccharides measured in the solution and solid phases in this study, is available in the Supplementary material to aid further work and the application 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. GOS enrichment was highest under conditions expected to give the highest saccharide solubility. The combination of the lowest saccharide concentration tested (28 g/L) and highest temperature tested (40 C) gave 2.3 0.1-fold GOS enrichment and a GOS:Lac ratio 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 Government Department of Biotechnology under the Indo-Australian Biotechnology Fund (vide sanction letter no. BT/PR9547/ICD/16/754/ 2006 of DBT/Indo-Aus/01/35/06 dated July 02, 2007) and the Australian Government Department of Innovation, Industry, Science and Research Australia India Strategic Research Fund BF010024. We also acknowledge the Particulate Fluids Processing Centre, 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, in the online version, at doi:10.1016/j.foodchem.2011.03.076. References
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