Optimisation of synthesis of oligosaccharides derived from lactulose (fructosyl-galacto-oligosaccharides) with β-galactosidases of different origin

Food Chemistry 138 (2013) 2225–2232

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Optimisation of synthesis of oligosaccharides derived from lactulose (fructosyl-galacto-oligosaccharides) with b-galactosidases of different origin Cecilia Guerrero, Carlos Vera, Andrés Illanes ⇑ School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Avenida Brasil 2147, Valparaíso, Chile

a r t i c l e

i n f o

Article history: Received 24 August 2012 Received in revised form 20 October 2012 Accepted 24 October 2012 Available online 10 November 2012 Keywords: Lactulose Prebiotic b-Galactosidase Fructosyl-galacto-oligosaccharides Transgalactosylation Galacto-oligosaccharides

a b s t r a c t Batch synthesis of fructosyl-galacto-oligosaccharides from lactulose was performed with commercial b-galactosidase preparations from Aspergillus oryzae, Kluyveromyces lactis and Bacillus circulans. The enzyme from A. oryzae produced the highest yield and specific productivity of synthesis, being selected for further studies. Optimization of fructosyl-galacto-oligosaccharides synthesis was carried out using response surface methodology, considering temperature and initial sugar concentration as variables and yield and specific productivity as response parameters. Maximum yield of 0.41 g g1 fructosylgalacto-oligosaccharides was obtained at 70 °C and 60% w/w lactulose concentration, while maximum specific productivity of 1.2 g h1 mg1 was obtained at 70 °C and 40% w/w lactulose concentration. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Prebiotics are increasingly being considered as health-promoting food components (Wang, 2009). Most prebiotics are non-digestible oligosaccharides (NDO) and, among them, galacto-oligosaccharides (GOS), fructo-oligosaccharides (FOS), inulin and lactulose have been conclusively proven to exert prebiotic effects (Rycroft, Jones, Gibson, & Rastall, 2001), while other NDO are considered as health-promoting agents complying with some, but not all, of the requirements to be considered as prebiotics (Gänzle, Haase, & Jelen, 2008; Klewicki, 2007; Li et al., 2009). Prebiotic effects on the colonic microbiota depend on the chemical structure of the oligosaccharides (number and type of monomers; type, position and conformation of the glycosidic linkages) (Hernández-Hernández, Montañes, Clemente, Moreno, & Sanz, 2011; Martínez-Villaluenga et al., 2008; Olano & Corzo, 2009). Therefore, evaluating new NDO structures is an open field of research as some of them may lead to better prebiotics (Cardelle-Cobas et al., 2011). b-Galactosidases are important biocatalysts for industry, traditionally used for their hydrolytic activity to reduce the lactose content in foods and process wastewaters (Illanes, 2011; Tuure & Korpela, 2004), and, more recently, as catalysts for transgalactosylation reactions leading to the synthesis of GOS, lactulose and lactosucrose (Albayrak & Yang, 2002; Guerrero, Vera, Plou, &

⇑ Corresponding author. Tel.: +56 32 2273642; fax: +56 32 2273803. E-mail address: [email protected] (A. Illanes). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.10.128

Illanes, 2011; Kim, Park, & Oh, 2006; Lee, Kim, & Oh, 2004; Li et al., 2009). The mechanism of the reaction catalyzed by b-galactosidase was described more than 50 years ago as a transglycosylation reaction in which the enzyme catalyzes the transfer of a galactose moiety in a non reducing b-galactoside (donor) to an acceptor containing a hydroxyl group (Prenosil, Stuker, & Bourne, 1987). However, the potential technological value of such a reaction began to be explored more than three decades later for the synthesis of transgalactosylated oligosaccharides, once these compounds acquired interest as potential prebiotics. Among them, GOS and lactulose stand out for their scientifically proven prebiotic condition. GOS are composed of a variable number of galactose units (usually from two to ten) and a terminal glucose unit, mostly b-(1 ? 4)and b-(1 ? 6)- linked; lactulose is a disaccharide (4-O-b-D-galactopyranosyl-D-fructose). In the synthesis of GOS, lactose plays both the role of donor and acceptor of the galactosyl residue, so forming trisaccharides which in turn can act as acceptors forming tetrasaccharides and so on (Albayrak & Yang, 2002; Vera, Guerrero, & Illanes, 2011). In the enzymatic synthesis of lactulose, lactose is the galactose donor, fructose acting as acceptor, but since lactose can also act as acceptor, a mixture of lactulose and GOS will be produced (Guerrero et al., 2011; Kim et al., 2006; Lee et al., 2004). The synthesis of GOS and lactulose is strongly determined by the origin of the b-galactosidase (Guerrero et al., 2011; Kim et al., 2006; Lee et al., 2004; Sanz-Valero, 2009), so that product composition, yield and specific productivity will vary accordingly. Most studies have been focused in increasing yield that, as said above, is strongly dependent on the enzyme source, since it results from

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the balance between transgalactosylation and hydrolytic activities (Bucke, 1996; Kasche, 1986). Another key variable is the initial lactose concentration; high concentrations favoring synthesis over hydrolysis so increasing yield (Albayrak & Yang, 2002; Guerrero et al., 2011; Sanz-Valero, 2009). Maximum allowable concentration of lactose is determined by its solubility, which is strongly dependent on temperature and so this also becomes a key variable (Vera, Guerrero, Conejeros, & Illanes, 2012). Temperature also influences the ratio of transglycosylation to hydrolysis rates (Gosling, Stevens, Barber, Kentish, & Gras, 2010). Other operational variables, such as pH and enzyme concentration, have been shown to have little influence on GOS and lactulose yields, though they affect volumetric productivity (Guerrero et al., 2011; Vera et al., 2011a). Specific productivity of GOS and lactulose synthesis, though being pH and temperature dependent (these variables affect transgalactosylation and hydrolysis rates differently), is not affected by the enzyme to substrate ratio (Bucke, 1996; Guerrero et al., 2011; Kasche, 1986; Vera, Guerrero, Illanes, & Conejeros, 2011). It has been recently demonstrated that b-galactosidase can accept lactulose as donor and acceptor of transgalactosylated galactose, leading to the synthesis of galacto-oligosaccharides derived from lactulose (Martínez-Villaluenga et al., 2008). These oligosaccharides represent a new type of compounds that are likely to have improved prebiotic activity compared to GOS and lactulose (Cardelle-Cobas et al., 2011; Martínez-Villaluenga et al., 2008; Olano & Corzo, 2009). Some authors (Cardelle-Cobas, MartinezVillaluenga, Villamiel, Olano, & Corzo, 2008), have suggested that the definition of GOS should be extended to include this new type of NDO that differs from them only in the terminal sugar moiety (fructose instead of glucose or galactose), and have described them as fructosyl-galactooligosaccharides (fGOS). The synthesis of fGOS with b-galactosidases is a little-explored field, in which the effect of key variables on yield and specific productivity of synthesis has not been clearly established and whose prebiotic effect is still to be scientifically confirmed (Cardelle-Cobas et al., 2011). The mechanism proposed for the synthesis of fGOS holds that one molecule of lactulose binds to the enzyme while one molecule of fructose is released, the galactosyl-enzyme complex so formed reacts with another molecule of lactulose leading to the formation of the trisaccharide (fGOS-3), which in turn can also act as acceptor of the galactosyl-enzyme complex leading to fGOS-4 and so on, extending the length of the oligosaccharide chain. Released galactose, as a consequence of lactulose hydrolysis, usually acts as a competitive inhibitor of b-galactosidases (Jurado, Camacho, Luzón, & Vicaria, 2002), which stems from the fact that the galactose moiety is the one recognized by the enzyme. Since the origin of the enzyme is one of the most important variables in the synthesis of transgalactosylated oligosaccharides, three different sources of b-galactosidases, namely Aspergillus oryzae, Kluyveromyces lactis and Bacillus circulans were evaluated as catalysts for the synthesis of fGOS. All of them are readily available commercial preparations previously used as catalysts for the synthesis of GOS. In this way, product distribution, substrate into product yield (YfGOS) and specific productivity (pfGOS) of fGOS synthesis can be compared and, based on the results obtained, the best enzyme preparation may be selected for the optimization of fGOS synthesis. The selected enzyme may be characterized in terms of the effect of lactulose and fructose on its hydrolytic activity, determining the Michaelis constant for lactulose and possible inhibition constant by fructose; additionally, the effect of galactose and fructose on the reaction of transgalactosylation can be determined so as to assess their impact on the synthesis of fGOS. The synthesis of fGOS should be able to be optimized using response surface methodology, considering temperature and initial concentration of lactulose as variables and YfGOS and pfGOS as evaluation parameters.

2. Materials and methods 2.1. Materials Lactulose (4-O-b-D-galactoypyranosyl-D-fructose) was provided by Discovery Fine Chemicals (Wimborne, UK); o-nitrophenol (o-NP), o-nitrophenyl-b-D-galactopyranoside (o-NPG) and GOS standards (4b-galactobiose and 3a-4b-3a galactotetraose) were supplied by Sigma (St. Louis, MO, USA. D-(+)-galactose, D-fructose and all other reagents were analytical grade and provided either by Sigma or Merck (Darmstadt, Germany). Three commercial b-galactosidases from different origin were tested. A commercial b-galactosidase preparation of A. oryzae (AOG), marketed under the trade name EnzecoÒ Fungal Lactase Concentrate, was kindly donated by Enzyme Development Corporation (New York, USA). The enzyme preparation had a specific activity of 196,000 IU g1, where one international unit of activity (IUH) is equivalent to the amount of enzyme hydrolyzing 1 lmole of o-NPG per minute at pH 4.5, 40 °C and 30 mmol L1 o-NPG. b-Galactosidase from K. lactis (KLG) traded as Lactozym Pure 6500 L was kindly supplied by Novozymes Latin America S.A. (Araucária, Brazil); the enzyme had a specific activity of 4836.3 IU g1, where one international unit of activity (IUH) is equivalent to the amount of enzyme hydrolyzing 1 lmole of o-NPG per minute at pH 6.5, 40 °C, 1.6 mmol L1 MgCl2 and 30 mmol L1 o-NPG. b-Galactosidase from B. circulans (BCG) traded under the name Biolactasa-NTL was a product from Biocon (Barcelona, Spain); the enzyme had a specific activity of 3182.7 IU g1, where one international unit of activity (IUH) is equivalent to the amount of enzyme hydrolyzing 1 lmole of o-NPG per minute at pH 6, 40 °C and 30 mmol L1 o-NPG. The enzymes were stored at 4 °C and remained fully active throughout the work. 2.2. HPLC analysis of the reaction products Substrates and products of synthesis were analyzed in a Jasco RI 2031 HPLC machine, provided with a refractive index detector, an isocratic pump (Jasco PU2080) and autosampler (Jasco AS 2055), using BP-100 Ca++ columns (300  7.8 mm) for carbohydrate analysis (Benson Polymerics, Reno, USA). Samples were eluted with mili-Q water at a flow-rate of 0.5 mL min1. Column and detector temperatures were 80 and 40 °C respectively. Chromatograms were integrated using the software ChromPass. Composition of samples was determined by assuming that the area of each peak is proportional to the weight percentage of the respective sugar. The accuracy of this assumption was checked by a material balance according to Boon, Janssen, and van der Padt (1999). Standards of galactose, fructose, lactulose, 4b-galactobiose and 3a-4b-3a galactotetraose were used to determine their retention times and check the linear range of the measurements. 2.3. Selection of the enzyme Syntheses of fGOS were conducted at 40% w/w lactulose, 40 °C, enzyme to substrate ratio (E/S) of 200 IUH g1 and at the optimum pH for each enzyme (4.5 for AOG, 6 for BCG and 6.5 for KLG). In the case of KLG, MgCl2 was added as cofactor at 1.6 mmol L1 concentration. Reactions were carried out in 100 mL Erlenmeyer flasks by dissolving 40 g of lactulose into 50 g of 100 mmol L1 McIlvaine citrate–phosphate buffer at the corresponding pH. Substrate was dissolved by heating the solution at a temperature of 95 °C (no degradation of sugars was detected by HPLC analysis) and then, after cooling to the reaction temperature, 10 g of a properly diluted enzyme solution was added to start the reaction. During synthesis, 0.5 mL samples were taken at regular intervals and the reaction

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was arrested by adding 0.5 mL of 200 mmol L1 NaOH in the case of AOG, and by heating at 90 °C for 30 s in the case of BCG and KLG. Then, samples were filtered through 0.22 lm Durapore membranes (Filterpore, Chile) prior to assay. Time course of the reaction was followed during 10 h. Product distribution was then determined by analyzing the amounts of lactulose and fGOS-2, fGOS-3, fGOS-4 and fGOS-5 synthesized, numbers meaning the monosaccharide units of each fGOS. The assays were carried out in duplicate, with standard deviations never exceeding 5%. The quantification of sugars was carried out as described in Section 2.2. 2.4. Synthesis of galacto-oligosaccharides (GOS) Synthesis of GOS was performed with the three b-galactosidases as background information for the synthesis of fGOS. Syntheses were carried out as described in Section 2.3 but using lactose instead of lactulose as substrate. 2.5. Effect of lactulose and fructose in the hydrolysis activity of b-galactosidase Having selected the enzyme, the effect of substrate (lactulose) and their products of hydrolysis (galactose and fructose) on its activity on the artificial substrate o-NPG was assessed. The effect of galactose has been previously determined and reported (Vera et al., 2011a). The hydrolysis reaction of o-NPG was carried out at 40 °C, pH 4.5 in 100 mmol L1 citrate–phosphate buffer and 30 mmol L1 o-NPG, at 0, 250, 500, 1000 y 1500 mmol L1 fructose concentrations. Initial rates of hydrolysis were determined by measuring the amount of o-NP released in the first 5 min of the reaction. o-NP was determined by measuring the absorbance at 420 nm in a Jenway 6715 spectrophotometer, using an extinction coefficient of 253.5 mol L1 cm1 under the assay conditions. The assays were carried out in triplicate with less than 2% difference among samples.

(0, 100, 200, 400, 600, 800, 1200 and 1600 mmol kg1). The experiments were carried out at pH 4.5 and 40 °C as described in Section 2.3. All the experiments were carried out in duplicate, with differences never exceeding 5%. As previously reported (Vera et al., 2011a), the effect of galactose on transgalactosylation activity can be determined from Eq. (1) that considers galactose as a total competitive inhibitor of the enzyme:



1

v transgal

1þ



Transgalactosylation activity of AOG in the synthesis of fGOS was determined as previously reported for the synthesis of GOS (Vera et al., 2011a). One international unit of b-galactosidase transgalactosylation activity (IUT) was defined as the amount of enzyme that catalyzes the transglycosylation of 1 lmol of galactose per min at 40% w/w initial lactulose concentration at pH 4.5 and 40 °C. The transgalactosylated galactose was determined considering the following material balance: 1 mol of galactose is transgalactosylated during the formation of 1 mol of trisaccharides, 2 mol of galactose are transgalactosylated during the formation of 1 mol of tetrasaccharides and 3 mol of galactose are transgalactosylated during the formation of 1 mol of pentasaccharides. The enzyme to substrate ratio in the reaction vessel was 20 IUH g1 in order to provide a linear range of transgalactosylation up to 15 min of reaction. The assay was carried out at 40% w/w of lactulose, pH 4.5 and 40 °C, as described in Section 2.3. Nine samples of 0.5 mL were taken at regular intervals and the reaction was stopped by adding 0.5 mL of 200 mmol L1 NaOH. The assays of transgalactosylation activity were carried out in triplicate, with differences never exceeding 5%.

   KM 1 KM   Gal þ VM Lo Lo  V M  K Gal

ð1Þ

where vtransgal is the specific transgalactosylation rate (lmol L1 min1 g1), KM the Michaelis constant (mmol L1), Lo the initial lactulose concentration (mmol L1), VM the maximum reaction rate (lmol L1 min1 g1), KGal the inhibition constant by galactose (mmol L1) and Gal the galactose concentration (mmol L1). Results corresponding to Section 2.5 showed that fructose is a total competitive inhibitor of b-galactosidases; therefore, its effect on transgalactosylation activity was determined, as in the case of galactose, by using Eq. (1). 2.8. Effect of operational variables in the synthesis of fructosyl-galactooligosaccharides Syntheses were conducted as described in Section 2.3, at varying temperatures (30, 40, 50, 60 and 70 °C) and initial lactulose concentrations (30, 40, 50, 60 and 70 % w/w). Assays were carried out in duplicate with less than 5% difference among samples. The following parameters were defined to evaluate the synthesis of fGOS and GOS: – Substrate conversion (XL) is defined by Eq. (2), which represents the fraction of the initial mass of substrate that is transformed during the reaction.

XL ¼ 2.6. Determination of transgalactosylation activity for the synthesis of fGOS

2227

ðLo  LÞ Lo

ð2Þ

where Lo and L represent the mass of substrate at the beginning and at different times of the reaction, respectively, Lo and L being either lactulose in the synthesis of fGOS or lactose in the synthesis of GOS. – Yield of transgalactosylated oligosaccharides (Y) is given by Eq. (3), which represents the fraction of the initial mass of substrate that is converted into transgalactosylated oligosaccharides (OT), be it fGOS or GOS (with polymerization degrees between 2 and 5).

Y¼

OT L0

ð3Þ

– Specific productivity (p) is given by Eq. (4), which represents the amount of OT produced per unit mass of enzyme preparation (ME) and unit time (t), evaluated at the maximum yield.

p¼

OT ME  t

ð4Þ

2.9. Optimization of synthesis of fructosyl-galacto-oligosaccharides 2.7. Effect of galactose and fructose on the transgalactosylation activity for the synthesis of fGOS Activity of transgalactosylation for the synthesis of fGOS was determined in the presence of different initial concentrations of galactose (0, 50, 100, 200, 400, and 600 mmol kg1) and fructose

Optimization of fGOS synthesis with AOG was conducted using an experimental design based on response surface methodology for two variables at three levels. Variables were the reaction temperature (T) and initial concentration of lactulose (Lo), being YfGOS and pfGOS the objective functions. Temperature range was from

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respectively; the level of lactulose hydrolysis obtained with KLG was the highest, but YfGOS was lower than previously reported by Hernández-Hernández et al. (2011). Cardelle-Cobas (2009) reported a YfGOS of 0.28 g g1 for the synthesis of fGOS with K. lactis b-galactosidase. Of the b-galactosidases tested, the highest values of YfGOS and pfGOS were obtained with AOG (0.39 g g1 and 0.57 g h1 mg1 respectively). The value reported by Cardelle-Cobas (2009) for the synthesis of fGOS with A. oryzae b-galactosidase at pH 4.5, 50 °C and 450 g L1 of lactulose is slightly higher (YfGOS 0.43 g g1), which may be simply an artifact arising from the different methodology employed for the determination of sugars. Yield obtained in this work with AOG (0.39 g g1) is significantly higher than the value reported for the synthesis of fGOS with Aspergillus aculeatus b-galactosidase at pH 4.5 (0.27 g g1) (Cardelle-Cobas, 2009; Cardelle-Cobas et al., 2008). Fig. 2a and b present a comparison of the values of yield and specific productivity obtained in the synthesis of fGOS and GOS with the three enzymes. Highest YGOS was obtained with BCG, reaching a value of de 0.41 g g1, while pGOS was 0.033 g h1 mg1. The corresponding values for AOG and KLG were 0.28 g g1 and 0.188 g h1 mg1, and 0.31 g g1 and 0.146 g h1 mg1, respectively. Values of YGOS obtained are within the ranges usually reported (Boon, Janssen, & van´t Riet, 2000; Chockchaisawasdee, Athanasopoulos, Niranjan, & Rastall, 2005; Gosling et al., 2010; Martínez-Villaluenga et al., 2008; Sanz-Valero, 2009). However, yield and specific productivity obtained with the different b-galactosidases under study differ markedly from the corresponding values obtained in the synthesis of GOS, which highlights the strong dependence of these parameters from the nature of the donor and the acceptor of the transgalactosylated galactose. It is worthwhile noting that when using lactulose as substrate for fGOS synthesis with BCG, both YfGOS and pfGOS were reduced by 66% with respect to the corresponding values obtained when using lactose as substrate for GOS synthesis. In contrast, with AOG, YfGOS and pfGOS increased by 39% and 217%, respectively.

Table 1 Central composite design for the optimization of fGOS synthesis with A. oryzae bgalactosidase, considering two variables and three levels. Lactulose concentration (% w/w)

Temperature (°C)

YfGOS (g fGOS/g lactulose)

pfGOS

60 50 40 50 35.86 64.14 60 50 50 50 50 40 50

50 45.86 50 60 60 60 70 60 74.14 60 60 70 60

0.404 0.409 0.390 0.398 0.380 0.397 0.405 0.396 0.398 0.399 0.396 0.378 0.397

0.372 0.518 0.958 0.734 0.934 0.326 0.830 0.731 0.816 0.736 0.731 1.394 0.733

(g fGOS mg1 h1)

40 to 60 °C and the initial concentration range of lactulose was between 40 and 60 % w/w (see Table 1). Analysis of the synthesis of fGOS by response surface experimental design and the ANOVA test of experimental results was done using Software Design Expert Statistical, Trial version 8.0, Stat-Ease Inc. 3. Results and discussion 3.1. Selection of the enzyme Fig. 1 shows the sugar profiles during fGOS synthesis for the three enzymes evaluated. Lowest YfGOS and pfGOS were obtained with BCG (0.14 g g1 and 0.011 g h1 mg1, respectively). These results cannot be properly analyzed in comparative terms since no previous reports on the synthesis of lactulose derived oligosaccharides have been reported previously with BCG. Values of YfGOS and pfGOS obtained with KLG were 0.2 g g1 and 0.055 g h1 mg1

Sugar Concentration (% w/w)

Sugar Concetration (% w/w)

100

(a)

80 60 40 20

100

(b) 75

50

25

0

0 0

200

400

600

0

200

Time (min)

400

600

Time (min)

Sugar Concentration (% w/w)

125

(c)

100 75 50 25 0 0

200

400

600

Time (min) Fig. 1. Enzymatic synthesis of transgalctosylated oligosaccharides form lactulose at 40% w/w and 40 °C. (a) A. oryzae b-galactosidase at pH 4.5; (b) B. circulans b-galactosidase at pH 6.0; (c) K. lactis b-galactosidase at pH 6.5.  Lactulose, e total fGOS, 4 galactosa, Nfructosa.

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0.6

(a) π (g ·h-1·mg -1 )

Y (g·g -1)

(b)

0.5

0.4

0.3

0.2

0.4 0.3 0.2

0.1

0.1 0

0 B. circulans

K. lactis

B. circulans

A. oryzae

K. lactis

A. oryzae

Fig. 2. Comparison of yield (a) and specific productivity (b) of the reaction of synthesis of galacto-oligosaccharides from lactose (h) and fructosyl-galacto-oligosaccharides from lactulose ( ) at 40 °C, pH 4.5, 40% w/w substrate initial concentration and E/S of 200 IUH g1 substrate.

Table 2 Kinetic parameters of the reactions of hydrolysis of o-NPG and transgalactosylation of lactose and lactulose. Parameter

Value

Confidence interval (95%)

Units

Experiment

VMH KGal VMH KLactulose VMH KFru VMT KGal VMT KGal VMT KGal

194.7 23.54 212.9 90 213.0 2560 59716 3.86 122000 3.68 122000 64.5

±7.68 ±2.56 ±9.38 ±4.3 ±9.15 ±23.3 ±2860 ±0.56 ±2209 ±0.28 ±3098 ±7.01

lM min 1 g1

Hydrolysis of o-NPG in the presence of galactose (a)

mM

lM min1 g1 mM lM min1 g1 mM lM min1 g1 mmol kg of water1 lM min1 g1 mmol kg of water1 lM min1 g1 mmol kg of water1

Hydrolysis of o-NPG in the presence of lactulose (b) Hydrolysis of o-NPG in the presence of fructose (b) Transgalactosylation of lactose in the presence of galactose in the synthesis of GOS (a) Transgalactosylation of lactulose in the presence of galactose in the synthesis of fGOS (b) Transgalactosylation of lactulose in the presence of fructose in the synthesis of fGOS (b)

(a) Data from Vera et al., 2004. (b) Data obtained in this work.

These results highlight the strong effect of the type of substrate on the reactions of transgalactosylation and hydrolysis, since the parameters of synthesis depend on the ratio of these rates (Bucke, 1996; Kasche, 1986; Mladenoska, Winkelhausen, & Kuzmanova, 2008). Product distribution (with respect to polymerization degree) of the oligosaccharides formed from lactulose and lactose at the point of maximum yield also varied significantly depending on the enzyme source. In the synthesis of fGOS with BCG, the main products of synthesis were disaccharides, while in the synthesis of GOS the main products were tri and tetrasaccharides, which is in agreement with previously reported results for the latter (Sanz-Valero, 2009; Vera et al., 2011b). In the case of KLG, products of synthesis were mostly di and trisaccharides, both in the synthesis of fGOS and GOS, which for the latter case agrees with previously reported results (Cardelle-Cobas, 2009; Chockchaisawasdee et al., 2005; Sanz-Valero, 2009). In the case of AOG, tri- and tetrasaccharides were the main products of synthesis in both reactions which in the case of GOS agrees with previously reported results (Cardelle-Cobas, 2009; Sanz-Valero, 2009; Vera et al., 2011b). Based on the above results, AOG is the enzyme best suited for fGOS synthesis and was therefore selected for further studies. 3.2. Effect of lactulose and fructose on the hydrolytic activity of b-galactosidase With the purpose of explaining the differences observed in the synthesis of fGOS and GOS with AOG in terms of enzyme kinetics, the Michaelis constant for lactulose was determined. Since lactulose is a substrate for b-galactosidase, when the hydrolysis of o-NPG is conducted in the presence of lactulose, its effect on the

hydrolysis of o-NPG corresponds to a competitive inhibitor (Table 2). A value of 90 mmol L1 was obtained, which is similar to the value of 94 mmol L1 reported for the Michaelis constant of AOG for its natural substrate (Jurado et al., 2002; Vera et al., 2011a). In addition, the effect of fructose on the hydrolysis of o-NPG was assessed and its effect on the Michaelis constant for o-NPG was quite mild, and it had no effect on the maximum reaction rate. Therefore, as it occurs with galactose, the effect of fructose was modeled as a total competitive inhibition, with an inhibition constant of 2560 mmol L1 (see Table 2).

3.3. Determination of the effect of galactose and fructose on the activity of transgalactosylation for the synthesis of fGOS Transgalactosylation activity (TA) of AOG using lactulose as substrate was 109,000 UIT g1, which is higher than the value of 55,900 UIT g1 reported when using lactose as substrate (Vera et al., 2011a). The effect of galactose and fructose on TA was evaluated, both sugars being inhibitors of AOG, as seen in Fig. 3. Results were fitted to Eq. (1) and the values obtained for the inhibition constants were 3.68 and 64.5 mmol kg1 of water, respectively. Maximum specific TA was 1.22 105 lmol L1 min1 g1. Value of the inhibition constant by galactose on TA in the synthesis of fGOS is similar to the one obtained in the synthesis of GOS, while maximum specific rate of transgalactosylation was 95% of the value reported for the synthesis of GOS (Vera et al., 2011a). Inhibitor concentrations producing a 50% reduction on TA (IC50) in the synthesis of fGOS were 56 and 1350 mmol kg1 for galactose and fructose, respectively, meaning that galactose is a much stronger inhibitor than is fructose.

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Transgalactosylation activity (µmol·L-1·min-1·g -1)

1.2E+05 1.0E+05 8.0E+04 6.0E+04 4.0E+04 2.0E+04 0.0E+00 0

200

400

600

800

1000

1200

1400

1600

1800

Galactose and Fructose (mmol/kg of water) Fig. 3. Effect of galactose () and fructose (e) on transgalactosylation activity with A. oryzae b-galactosidase at 40% w/w lactulose, 40 °C and pH 4.5.

From the data in Table 2, the catalytic efficiencies (VMax/KM) of AOG in the transgalactosylation reactions were 1.36 106 min1 g1 for the synthesis of fGOS and 0.64 106 min1 g1 in the synthesis of GOS, i.e., 113% higher for fGOS than for GOS. 3.4. Effect of operation variables on the synthesis of fructosyl-galactooligosaccharides with A. oryzae b-galactosidase Fig. 4 shows the effect of temperature and initial concentration of lactulose on YfGOS and pfGOS. At all temperatures tested, profiles of total fGOS synthesized were similar, maximum YfGOS being reached at XL close to 70%. However, as seen in Fig. 4a, YfGOS moderately decreased with temperature from 0.40 g g1 at 30 °C, to 0,377 g g1 at 70 °C. This behavior has been reported by CardelleCobas (2009) for the synthesis of fGOS with AOG, who observed a 16% decrease in YfGOS with an increase in temperature from 50 to 60 °C. This effect has been also reported for the synthesis of GOS with AOG (Albayrak & Yang, 2002; Sanz-Valero, 2009; Vera et al., 2012). This behavior is contrary to that reported for A. aculeatus and K. lactis b-galactosidases, where YfGOS increased by 33% and 26% with an increase in temperature from 40 to 60 °C and

(b)

0.4

Y fGOS (g·g-1)

Y fGOS (g· g-1)

(a)

40–50 °C, respectively (Cardelle-Cobas et al., 2008). Since the synthesis of fGOS is a kinetically controlled reaction, these results suggest that increasing the temperature selectively favors hydrolysis over synthesis, so explaining the decrease in YfGOS with temperature. A similar result has been reported for the synthesis of GOS with AOG, where it has been observed that the activation energy for hydrolysis reactions is 6% higher than for transgalactosylation reactions (Vera et al., 2011a). As seen in Fig. 4b, YfGOS is affected by the initial concentration of lactulose; a slight increase in YfGOS with lactulose concentration is observed in the range from 30% to 50% w/w, while a steeper decrease is observed at higher concentrations (greater than 50% w/w). Likewise, Cardelle-Cobas et al. (2008) reported a slight increase in YfGOS with lactulose concentration in the range 250–650 gL1 with A. aculeatus b-galactosidase and a sharp decrease at 850 g L1. However, the YfGOS is unaffected by an increase in initial lactulose concentration from 450 to 650 g L1 when K. lactis b-galactosidase is used as catalyst (Cardelle-Cobas, 2009). A slight increase in YGOS with lactose concentration has also been previously reported in the synthesis of GOS in the 30–50% w/w range (Albayrak & Yang, 2002; Sanz-Valero, 2009); however, data at higher concentrations are not feasible to obtain because of spontaneous precipitation of lactose over 50% w/w (Vera et al., 2012). Fig. 4c and d show the effects of temperature and initial concentration of lactulose on pfGOS. As seen in Fig. 4c, pfGOS increased fourfold when the temperature was increased from 30 °C (0.329 g h1 mg1) to 70 °C (1.4 g h1 mg1). These results are in agreement with previously reported data on the synthesis of fGOS and synthesis of GOS with AOG, where the increase in reaction rate was clearly reflected in the increase in pGOS (Albayrak & Yang, 2002; Cardelle-Cobas, 2009; Sanz-Valero, 2009). On the other hand, as seen in Fig. 4d, the increase in lactulose concentration from 30% to 70% w/w produced a 93% decrease in pfGOS, from 0.68 to 0.048 g h1 mg1, which agrees with results reported for the synthesis of GOS with AOG (Vera et al., 2012). Similar effects of temperature and lactulose concentration in pfGOS was reported by Cardelle-Cobas (2009) in the synthesis of fGOS with A. aculeatus and K. lactis b-galactosidases.

0.32 0.24 0.16

0.32 0.24 0.16 0.08

0.08 0

0.4

30

40

50

60

0

70

0.8 0.4 0

30

40

50

60

70

Temperature ( C)

·mg-1·h-1)

1.2

(d)

0.8

π fGOS (g

·h-1)

1.6

π fGOS (g·mg-1

(c)

30

40

50

60

70

Total sugar concentration (% w/w)

Temperature ( C)

0.4

0.6

0.2

0 30

40

50

60

70

Total sugar concentration (% w/w)

Fig. 4. Effect of temperature and initial concentration of lactulose on yield and specific productivity of fructosyl-galacto-oligosaccharides synthesis at pH 4.5 and E/S of 200 IUHg1. (a) Yield at 40% w/w initial lactulose concentration at different temperatures. (b) Yield at 40 °C at different initial lactulose concentration. (c) Specific productivity at 40% w/w initial lactulose concentration at different temperatures. (d) Specific productivity at 40 °C at different initial lactulose concentration.

C. Guerrero et al. / Food Chemistry 138 (2013) 2225–2232

3.5. Optimization of synthesis of fructosyl-galacto-oligosaccharides In order to assess the potential of this process at productive level, synthesis of fGOS was optimized with the most suitable enzyme. According to the results previously obtained, the parameters of synthesis of fGOS (YfGOS and pfGOS) varied with temperature and initial concentration of lactulose; therefore, the synthesis of fGOS with AOG was optimized in terms of those variables using response surface methodology. This methodology leads to the determination of the interaction and values of these variables that maximize the parameters of synthesis of fGOS. As a result of the experimental design, the following third-order polynomial equations were obtained for YfGOS and pfGOS:

Y fGOS ¼ 0:45 þ 0:025  ðLo Þ þ 0:022  ðTÞ  5:7  104  ðLo  TÞ  1:1  104  ðL2o Þ  1:8  104  ðT 2 Þ þ 1  106  ðL2o  TÞ þ 4:2  106  ðLo  T 2 Þ

compromise to be reached between both parameters. Therefore, optimal reaction conditions should be determined from an economic analysis to evaluate the influence of both at industrial scale.

Acknowledgements Work financed by Fondecyt Grant 1100050 from Conicyt, Chile and Project DII 037.112/2008 from the Pontificia Universidad Católica de Valparaíso, Chile. Doctoral Fellowships Conicyt 21080173 and Mecesup2 UCV0608 are acknowledged. We acknowledge the generous donations of A. oryzae b-galactosidase by Enzyme Development Corporation (New York, USA).

References

ð5Þ

pfGOS ¼ 2:1 þ 0:41  ðLo Þ  0:16  ðTÞ  3:1  103  ðLo  TÞ  7  103  ðL2o Þ þ 3:9  103  ðT 2 Þ þ 1:2  104  ðL2o  TÞ  7:3  105  ðLo  T 2 Þ

2231

ð6Þ

Correlation coefficients for Eqs. (5) and (6) were 99.5 and 97.9, respectively. The polynomial equations show the interaction of both variables and their effect on YfGOS and pfGOS. Temperature had a mild effect on YfGOS, while YfGOS slightly increased with initial lactulose concentration. On the other hand, both variables had a strong effect on pfGOS. From such equations, optimal operation conditions maximizing YfGOS and pfGOS were obtained and validated experimentally. Maximum YfGOS was 0.41 g g1, obtained at 70 °C and 60% w/w initial lactulose concentration. Maximum pfGOS was 1.2 g h1 mg1, obtained at 70 °C and 40% w/w initial lactulose concentration.

4. Conclusions The main purpose of this work was to analyze in comparative terms the effect of the galactose donor and acceptor molecules in the synthesis of transgalactosylated oligosaccharides with three commercial b-galactosidase preparations of different origin. In addition, the effect of enzyme source, temperature and initial sugars concentrations was evaluated in the synthesis of fGOS. This is the first report in which these variables have been studied simultaneously. Yield and specific productivity of the enzymatic synthesis of fGOS were strongly dependent on the source of the enzyme. AOG was the source producing the highest yield and specific productivity, and was therefore selected for further studies. The yield of synthesis of fGOS with AOG was strongly dependent on the initial concentration of lactulose, but was only slightly affected by temperature within the ranges studied. Meanwhile, specific productivity was strongly dependent on both variables. Compared to the synthesis of GOS from lactose, yield was 46% higher in the synthesis of fGOS from lactulose, which is explained considering that the catalytic efficiency of transgalactosylation in the latter reaction was higher. Synthesis of fGOS has been optimized with the best suited of the enzyme preparations studied as a step forward for the assessment of the technological potential of the enzyme production of fGOS. Therefore, optimum reaction conditions for the synthesis of fGOS were determined by response surface methodology and validated experimentally. For both parameters (yield and specific productivity) the optimal temperature was 70 °C, while the optimal initial lactulose concentration was 40 and 60 % w/w for specific productivity and yield, respectively. This shows that there is a

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