Continuous enzymatic synthesis of lactulose in packed-bed reactor with immobilized Aspergillus oryzae β-galactosidase

Accepted Manuscript Continuous enzymatic synthesis of lactulose in packed-bed reactor with immobilized Aspergillus oryzae β-galactosidase Cecilia Guerrero, Felipe Valdivia, Claudia Ubilla, Nicolás Ramírez, Matías Gómez, Carla Aburto, Carlos Vera, Andrés Illanes PII: DOI: Reference:

S0960-8524(18)31662-6 https://doi.org/10.1016/j.biortech.2018.12.018 BITE 20775

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

25 October 2018 3 December 2018 6 December 2018

Please cite this article as: Guerrero, C., Valdivia, F., Ubilla, C., Ramírez, N., Gómez, M., Aburto, C., Vera, C., Illanes, A., Continuous enzymatic synthesis of lactulose in packed-bed reactor with immobilized Aspergillus oryzae β-galactosidase, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.12.018

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Continuous enzymatic synthesis of lactulose in packed-bed reactor with

2

immobilized Aspergillus oryzae β-galactosidase.

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Cecilia Guerrero1*, Felipe Valdivia1, Claudia Ubilla1, Nicolás Ramírez1, Matías Gómez1, Carla

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Aburto1, Carlos Vera2, Andrés Illanes1.

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1. School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso (PUCV), Valparaíso, Chile. 2. Department of Biology, Faculty of Chemistry and Biology, Universidad de Santiago de Chile (USACH), Santiago, Chile. *: corresponding author. Tel. 56 32- 2272035; E-mail address: [email protected]

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Abstract

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Lactulose synthesis from fructose and lactose in continuous packed-bed reactor operation

12

with glyoxyl-agarose immobilized Aspergillus oryzae β-galactosidase is reported for the first

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time. Alternative strategies to conventional batch synthesis have been scarcely explored for

14

lactulose synthesis. The effect of flow rate, substrates ratio and biocatalyst-inert packing

15

material mass ratio (MB/MIM) were studied on reactor performance. Increase in any of these

16

variables produced an increase in lactulose yield (YLu) being higher than obtained in batch

17

synthesis at comparable conditions. Maximum YLu of 0.6 g·g

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4.5, 50 % w/w total sugars, 15 mL·min-1, fructose/lactose molar ratio of 12 and MB/MIM of

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1/8 g·g-1; at such conditions yield of transgalactosylated oligosaccharides (YTOS) was 0.16

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g·g-1, selectivity (lactulose/TOS molar ratio) was 5.4 and lactose conversion (XLactose) was

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28 %. Reactor operation with recycle had no significant effect on yield, producing only some

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decrease in productivity.

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Keywords: β-galactosidase, lactulose, continuous operation, packed-bed reactor, glyoxyl agarose.

-1

was obtained at 50 °C, pH

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1. Introduction

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Lactulose (4-O-β-D-galactopyranosyl-D-fructose) is a synthetic non-digestible disaccharide

27

with interesting bioactive properties (Panesar & Kumari, 2011; Nooshkam et al., 2018) that

28

has received much attention in recent years due to its therapeutic and nutrition properties

29

(Wang et al., 2013; Wu et al., 2017; Nooshkam et al., 2018). Lactulose is produced

1

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industrially by alkaline isomerization of lactose (Zokaee et al., 2002; Aider & de Halleux,

31

2007). However, chemical synthesis has several drawbacks: a high amount of inorganic

32

catalyst is required, yields are moderate (0.2 to 0.8 g lactulose/g lactose), significant lactulose

33

degradation and unwanted byproduct formation occur due to the harsh reaction conditions

34

making product purification complex and costly (Hicks & Parrish, 2002; Aider & de Halleux,

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2007; Lima de Albuquerque et al., 2018). Therefore, developing an enzymatic process for

36

lactulose production aims to the purpose of increasing yield, reducing product degradation

37

and contamination with hard-to-remove byproducts, in compliance with the guidelines of

38

sustainable chemistry (Schumann, 2002; Panesar & Kumari, 2011; Guerrero et al., 2015a).

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So, production of lactulose by enzyme biocatalysis is a promising alternative for outpacing

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the inherent constraints of chemical synthesis (Schuster-Wolff-Bühring et al., 2010; Panesar

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& Kumari, 2011; Guerrero et al., 2011). Biocatalysis offers also the advantage that high

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purity lactose is not required as in the case of the less selective chemical synthesis (Guerrero

43

et al., 2017a). The most commonly used biocatalytic route for lactulose synthesis is the

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transgalactosylation of lactose with fructose catalyzed by β-galactosidase; however, yields

45

are quite lower than in chemical synthesis (Panesar & Kumari, 2011; Guerrero et al., 2017a).

46

Several strategies have been proposed for increasing lactulose yield, including the screening

47

for β-galactosidases from different sources (Lee et al., 2004; Guerrero et al., 2015a; Cardoso

48

et al., 2017), the use of different immobilized enzymes (Albayrak & Yang, 2002a; Bernal et

49

al., 2013; Urrutia et al., 2013; Guerrero et al., 2018; Rehbein et al., 2019) and the use of

50

different reaction modes of operation (Foda & Lopez-Leiva, 2000; Chockchaisawasdee et

51

al., 2004; Mayer et al., 2010; Guerrero et al., 2015b; Sitanggang et al., 2015; Guerrero et al.,

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2017b; Rehbein et al., 2019).

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The use of continuously operated reactors with immobilized β- galactosidases has been

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applied to the hydrolysis of lactose rather than to the synthesis of transgalactosylated

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oligosaccharides (Rodriguez-Colinas et al., 2016).

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Due to the kinetics of lactulose synthesis, continuous packed-bed reactors (CPBR) and batch

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reactors are more adequate than continuous stirred tank reactors (CSTR) for the synthesis of

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transgalactosylated oligosaccharides (TOS), namely galacto-oligosaccharides (GOS) and

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fructosyl-galacto-oligosaccharides (fGOS), since the high conversion required implies a low

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substrate concentration at reactor outlet. This means that in CSTR the enzyme will act at such

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low substrate concentrations disfavoring transgalactosylation (Foda & Lopez-Leiva, 2000;

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Albayrak & Yang, 2002a; Chockchaisawasdee et al., 2004; Vera et al., 2013). The synthesis

65

of transgalactosylated oligosaccharides in continuous mode of operation is quite useful since

66

higher efficiency can be obtained than in batch mode operation (Eskandarloo &

67

Abbaspourrad, 2018), which agrees with what was reported by Chockchaisawasdee et al.,

68

(2004) showing that, under the same operational conditions, a higher productivity of GOS

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synthesis was obtained with Kluyveromyces lactis β- galactosidases in continuous than in

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batch operation, while no significant difference was observed in product distribution.

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Use of CPBR is a rather unexplored field for lactulose synthesis, even though this type of

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reactor is usually employed for large-scale enzymatic processes because of its high

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efficiency, easy operation and favorable kinetic pattern. Efficiency of CPBR relates mainly

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to its high catalyst mass to reactor volume ratio (Nakkharat & Haltrich, 2007), easy retention

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of catalyst within the reactor and reduced shear stress, being therefore the most used reactor

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configuration (Hama et al., 2011; Illanes et al., 1992).

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The purpose of this work is the evaluation of lactulose synthesis from fructose and lactose in

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CPBR operation with glyoxyl-agarose immobilized Aspergillus oryzae β- galactosidase.

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Reactor performance is evaluated in terms of lactulose yield, productivity, selectivity and

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lactose conversion, considering flow rate, feed substrates ratio and immobilized enzyme-inert

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packing material mass ratio as variables, selecting the conditions at which lactulose

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concentration obtained is the highest. The effect of reactor geometry is assessed by working

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with reactors of different diameters and bed heights. Effect of recirculation rate is also

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evaluated in terms of lactulose yield and selectivity.

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2. Materials and Methods

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2.1. Materials

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Lactulose was supplied by Discovery Fine Chemicals (Wimborne, UK). Lactose

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monohydrate, fructose, glucose, galactose, o-nitrophenol (o-NP), o-nitrophenyl-β-D-

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galactopyranoside (o-NPG) and GOS standards were supplied by Sigma (St Louis, MO,

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USA). Agarose Bead Standard (6 % cross-linked with epichlorohydrin) and packed-bed

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reactor were purchased from Agarose Bead Technologies (Madrid, Spain). All other reagents

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were of the highest purity attainable and provided by Sigma or Merck (Darmstadt, Germany).

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The enzyme used was Enzeco™ Fungal Lactase Concentrate, a commercial preparation of

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Aspergillus oryzae β-galactosidase kindly donated by Enzyme Development Corporation,

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New York, USA.

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2.2. HPLC analysis of the reaction products

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Substrates and products of the reaction of lactulose synthesis were analyzed in a Jasco RI

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2031 HPLC equipment, provided with refractive index detector, isocratic pump (Jasco

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PU2080) and autosampler (Jasco AS 2055). BP-100 Ca++ columns (300 mm x 7.8 mm) for

105

carbohydrate analysis (Benson Polymeric, Reno, USA) were used. Samples were eluted with

106

milli-Q water at a flow rate of 0.5 mLmin-1. Column and detector temperatures were 80 and

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40 °C respectively. ChromPass software was used for integrating the chromatograms.

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Composition of samples was determined assuming that the area of each peak is proportional

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to the weight percentage of the respective sugar (Boon et al., 1999). Standards of galactose,

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fructose, lactulose, 4β-galactobiose and 3α-4β-3α galactotetraose were used to determine

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their retention times checking that measurements were in the linear range.

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2.3. Immobilization of Aspergillus oryzae β-galactosidase

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Immobilization of A. oryzae β-galactosidase in monofunctional glyoxyl-agarose supports

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was done following the procedure described by Guisán (1988) and Guerrero et al. (2017a).

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In order to determine the maximum hydrolytic potential of the biocatalyst, one international

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unit of hydrolytic activity (IUH) was defined as the amount of β-galactosidase that hydrolyzes

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1 μmol of o-NPG per minute at 45 mM o-NPG, 40 ºC and pH of 4.5 (Vera et al., 2011). The

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glyoxyl-agarose derivatives had a specific activity of 32,000 IUHg-1 at the above assay

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conditions.

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2.4. Continuous synthesis of lactulose in packed-bed reactor.

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Figure 1 shows a schematic representation of the experimental system used for the synthesis

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of lactulose in CPBR. The reactor had an effective volume (VE) of 45 mL, temperature was

124

kept at 50°C and pH at 4.5. A chromatographic furnace was used for temperature control

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(Figure 1) at 50 ºC in all experiments. To avoid catalyst bed compaction during operation,

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the reactor was packed with a mixture of immobilized enzymes and 0.75 mm glass beads as

127

inert material, in different proportions

128 129

Lactulose synthesis was performed in 45 mL packed-bed reactor at 50 °C, pH 4.5, 50% (w/w)

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total initial sugars concentration and different flow rates (1, 3, 5, 7, 9, 12 and 15 mL·min-1),

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fructose/lactose molar ratios (2, 4, 6, 8, 12, 16 and 20) and biocatalyst/inert packing material

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mass ratios (1/2, 1/5, 1/8, 1/11, 1/14 and 1/17). Reactor operations at different recirculation

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rates (0.25, 0.67, 1.5 and mLrecirculation·mLfeed -1) were also tested. Recirculation rate was

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defined as the ratio between recirculation flow and influent flow. Sugar substrates were

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dissolved in 100 mM citrate-phosphate buffer pH 4.5 previously heated at 95 °C and then

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cooled down to the reaction temperature. Different fructose/lactose molar ratios (F/L) were

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fed to the reactor using a Masterflex L/S 7525 (USA) pump and Masterflex 96400-14 silicone

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tubing cured in peroxide. 0.5 mL samples were taken every hour. Product distribution was

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determined by analyzing the amounts of lactulose, disaccharides, trisaccharides and

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tetrasaccharides produced. The assays were carried out in duplicate, with standard deviations

141

always below 5%. Quantification of carbohydrates was carried out as described in section

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2.2.

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Figure 1

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The reactions of synthesis were evaluated in terms of the following parameters:

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-Lactulose yield (YLu), which represents the average of the mass fraction of lactose entering

146

the reactor, which is converted into lactulose during the reactor operation: t

YLu =

∫0 F ∙ Cout Lu ∙ dt t

(Eq. 1)

out

∫0 F ∙ (Cin Lactose - CLactose ) ∙ dt

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-TOS yield (YTOS), which represents the average of the mass fraction of lactose that enters

148

the reactor, which is converted into TOS during the reactor operation: 5

t

YTOS =

∫0 F ∙ Cout TOS ∙ dt

(Eq. 2)

out

t

∫0 F ∙ (Cin Lactose - CLactose ) ∙ dt

149 150

- Selectivity (SLu/TOS), which represents the average of the ratio between the moles of

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lactulose and moles of TOS produced during the reactor operation: t

SLu/TOS =

∫0 F ∙ Nout Lu ∙ dt t

∫0 F ∙ Nout TOS ∙ dt

(Eq.3)

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- Specific productivity of lactulose (πLu), which represents the amount of lactulose produced

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(MLu) per unit mass of biocatalyst (MB) and unit reaction time (t) at the maximum lactulose

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concentration attained: t

πLu =

∫0 Cout Lu ∙ dt

(Eq.4)

t

MB ∙ ∫0 dt

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- Conversion of lactose (XLac), which represents the average of the mass fraction of lactose

156

that enters the reactor, which is reacted during reactor operation out

t

XLac =

in ∫0 F ∙ (CLactose - CLactose ) ∙ dt t ∫0 F∙Cin Lactose

∙dt

157

Where:

158

C

159

respectively (g·L-1).

160

C

ou t Lu

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C

ou t TO S

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F is the feed flow rate (mL·min-1).

163

NLouu tis the moles of lactulose at the reactor outlet (M).

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ou t NTos is the moles of TOS at reactor outlet (M).

in L actose and

C

ou t L actose are

(Eq.5)

the lactose concentrations at the inlet and outlet of the reactor

is the lactulose concentration at the reactor outlet (g·L-1). is the TOS concentration at the reactor outlet (g·L-1).

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MB is the total mass of biocatalyst in the reactor (g).

166

t is the reactor operation time (min).

167 168

3. Results and Discussion

169

3.1. Effect of the feed flow rate in the synthesis of lactulose in packed- bed reactor.

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Table 1 shows the effect of feed flow rate on YLu, πLu, SLu/TOS and lactose conversion in the

171

synthesis of lactulose in CPBR. The feed flow rates used corresponded to hydraulic residence

172

times (VE/F) between 3 and 45 mins.

173

YLu and YTOS increased with the increase in feed flow rate (Table 1). At 12 mL·min-1, YLu

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and YTOS obtained were 0.65 and 0.19 respectively, corresponding to a total

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transgalactosylation yield of 0.94, which means that 94 % of the reacted lactose was

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converted into transgalactosylated products and that synthesis prevailed over hydrolysis at

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such operation conditions. YLu and YTOS obtained are higher than the corresponding values

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reported for the synthesis of lactulose with glyoxyl-agarose immobilized A. oryzae β-

179

galactosidase in batch under similar reaction conditions, where YLu and YTOS reported were

180

0.35 and 0.1 respectively, with total transgalactosylation yield of 0.45 (Guerrero et al. 2017a).

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This means that 55 % of the lactose was hydrolyzed and that in batch mode of operation

182

hydrolysis strongly competes with transgalactosylation. However, in CPBR at 1 mL·min-1,

183

YLu and YTOS were 0.34 and 0.04 respectively, which are similar than obtained in batch;

184

therefore, at lower flow rates hydrolysis is favored over transgalactosylation very much as it

185

occurs in batch. In agreement with these results, Song et al., (2013) reported that CPBR

186

allowed obtaining higher YLu than in batch reactor, when the former is operated at high flow

187

rates.

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Table 1

189

As seen in Table 1, πLu increased with feed flow rate, which is consistent with the results

190

reported for the synthesis of GOS with glutaraldehyde-activated chitosan immobilized K.

191

lactis β- galactosidase in CPBR (Klein et al., 2013). Table 1 also shows that SLu/TOS and

192

XLactose decreased with feed flow rate, which agrees with the results reported by Mayer et al.

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193

(2010) for the synthesis of lactulose in CPBR with Pyrococcus furiosus β-galactosidase

194

immobilized onto an anion-exchange resin and onto Eupergit C, and also to those reported

195

by Kang (2013) for the hydrolysis of lactose with K. lactis immobilized in Duolite A568

196

where XLactose was significantly reduced by increasing the feed flow rate, which is due to the

197

reducing residence times, so that a substantial fraction of lactose remained unreacted.

198

As seen in Table 1, SLu/TOS in CPBR was 14 at 1 mL·min-1, which is higher than the value of

199

11 obtained in batch under similar conditions. But SLu/TOS decreased with feed flow rate being

200

only 5.5 at 12 mL·min-1 (Guerrero et al., 2017a) which reflects that at high feed flow rate a

201

higher fraction of the lactose fed remains unreacted due to the low residence time (45 mins

202

at 1 mL·min-1 and 3.8 mins at 12 mL·min-1. XLactose in batch (66 %) was significantly higher

203

than in CPBR at 12 mL·min-1 (26%); lactulose yield in CPBR, despite the low XLactose, is

204

higher than in batch which allows obtaining a higher lactulose concentration. At the low flow

205

rate of 1 mL·min-1, where XLactose obtained is high, lactulose yield is low and a lower lactulose

206

concentration is obtained than in batch.

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3.2. Effect of the fructose/lactose molar ratio (F/L) on the synthesis of lactulose in packed-

209

bed reactor.

210

Figure 2 shows the effect of F/L on the synthesis of lactulose in CPBR at different feed flow

211

rates (1, 3 and 5 mL·min-1).

212

Figure 2

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As seen, at higher F/L higher YLu, SLu/TOS and πLu were obtained at all the feed flow rates

214

evaluated, while YTOS decreased. The effect of F/L is similar than reported for batch synthesis

215

where this was the variable most influencing such parameters, being the one allowing to

216

control product distribution (Guerrero et al., 2011; Guerrero et al., 2017a). So, synthesis of

217

lactulose is favored over TOS at high F/L both for batch and CSTR operation, as shown in

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Table 2. When operating CPBR at a feed flow rate of 1 mL·min-1, YLu was lower than in

219

batch, but XLactose was similar at all substrates ratios evaluated. When increasing the feed

220

flow rate to 5 mL·min-1 YLu sharply increased being higher than in batch; however XLactose

8

221

decreased considerably due to the lower residence time. Similar results were reported by

222

Mayer et al. (2010) for the synthesis of lactulose in CPBR with Pyrococcus furiosus β-

223

galactosidase immobilized onto an anion-exchange resin and onto Eupergit C, and also to

224

those reported by Kang (2013) for the hydrolysis of lactose with K. lactis immobilized in

225

Duolite A568. In such cases, an increase in feed lactose concentration produced a decrease

226

in conversion at a given feed flow rate.

227

Table 2

228

As shown in Figures 2a and 2b, the higher the value of F/L the milder the effect of feed flow

229

rate on YLu and YTOS, being only severe in the F/L range from 2 to 6 at all feed flow rates

230

evaluated. XLactose (Figure 2e) was not affected significantly by variations in F/L at flow rates

231

under 3 mL·min-1, but at 5 mL·min-1 XLactose increased with the increase in F/L. A similar

232

pattern was observed in the batch synthesis of lactulose with soluble and glyoxyl-agarose

233

immobilized β-galactosidase (Guerrero et al., 2011; Guerrero et al., 2017a). Also, no

234

significant effect of F/L on XLactose was observed in the hydrolysis of lactose in CPBR with

235

immobilized K. marxianus cells (Panesar et al., 2010).

236

The biocatalyst remained fully active during the whole operation at the different substrates

237

ratios tested, which precluded an estimate of operation time for catalyst replacement.

238

However, Guerrero et al. (2017a) reported for the synthesis of lactulose in repeated batch

239

operation with the same biocatalyst here used that making an estimate of catalyst replacement

240

at one half-life, a total of 507 batches could be performed with a total operation time of 2823

241

h when operating the reactor at a F/L of 4, and 100 batches could be performed with a total

242

operation time of 940 h when operating the reactor at a F/L of 12. Similar total operating

243

times are to be expected in CPBR operation, allowing to produce a high mass of lactulose

244

per unit mass of biocatalyst so that a specific productivity much higher than in batch is

245

achievable with the consequent reduction in operating cost.

246

3.3. Effect of the biocatalyst/inert packing material mass ratio (MB/MIM) on the synthesis of

247

lactulose in packed- bed reactor.

248

Catalyst bed compaction during operation is one of the critical problems in CPBR, so in order

249

to reduce this effect the biocatalyst particles were mixed with particles of inert non-

9

250

compressible material (glass beads) in different proportions assessing the effect of MB/MIM

251

on reactor performance. Table 3 shows the effect of such ratio on YLu, YTOS, SLu/TOS, πLu and

252

XLactose.

253

Table 3

254

As seen in Table 3, the lower the MB/MIM the higher the values of YLu, YTOS and πLu obtained

255

in the synthesis of lactulose in CPBR. These results can be explained by the following

256

considerations: firstly, lower MB/MIM implies lower biocatalyst mass inside the reactor, since

257

the reactor volume was kept constant; secondly, the yield of transgalactosylation reactions

258

decreased at higher XLactose, because the transgalactosylation activity of β-galactosidases

259

declines at lower lactose concentrations and the presence of glucose and galactose also

260

reduces this activity (Albayrak & Yang, 2002b; Vera et al., 2002). Thus, at lower MB/MIM

261

lower XLactose conversions are reached in the reactor because there is less biocatalyst, then

262

higher YLu, YTOS and πLu are obtained because the transgalactosylation reactions prevail over

263

hydrolysis at lower XLactose- In agreement with these results, Neri et al. (2009) reported that

264

the reaction yield strongly decreased with the increase in XLactose in the case of GOS synthesis

265

with A. oryzae β-galactosidase.

266 267

3.4. Synthesis of lactulose in packed- bed reactor with recirculation.

268

The effect of recirculation on CPBR performance was evaluated in the synthesis of lactulose

269

at recirculation rates of 0.25, 0.67, 1.5 and 4 mLrecirculation·mLfeed -1 (Table 4).

270

Table 4

271

As shown in Table 4, YLu, YTOS and πLu decreased, while XLactose increased with the increase

272

in recirculation rate. This effect can be explained by considering that an increase in

273

recirculation rate will promote mixing, moving the flow pattern inside the reactor away from

274

ideal plug-flow regime (Fogler, 2017), which is the most favorable flow pattern for

275

transgalactosylation reactions according to its reaction kinetics (Splechtna et al., 2007)

276

Besides, increase in the recirculation rate implies higher concentrations of galactose and

277

glucose, which are inhibitors of the transgalactosylation activity of β-galactosidase inside the

10

278

reactor, with the consequent decrease in YLu, YTOS and πLu (Albayrak & Yang, 2002b; Vera

279

et al., 2011). It is worth noting that SLu/TOS increased at higher recirculation rates, which

280

reflects that the adverse effect on yields is stronger for the synthesis of TOS (GOS and fGOS)

281

than for lactulose. Recirculation rate is then a variable that these results suggest to be worthy

282

of optimization since it allows increasing SLu/TOS and XLactose at the expense of YLu and πLu.

283 284

4. Conclusions

285

Lactulose synthesis in CPBR is an interesting alternative to conventional batch synthesis

286

since it allows increasing lactulose and TOS yields. The effect of feed flow rate,

287

fructose/lactose molar ratio and biocatalyst to inert packing material mass ratio was evaluated

288

on reactor performance. Increase in any of these variables produced an increase in lactulose

289

and TOS yields, being them higher than reported for lactulose batch synthesis with the same

290

biocatalyst and at similar reaction conditions. The effect of reactor recirculation was also

291

assessed, but despite the increase in selectivity, no further increase in lactulose yield was

292

obtained and productivity decreased.

293 294

Acknowledgements

295

Work financed by Chilean Fondecyt Grant 1160216. We acknowledge generous donation of

296

β-galactosidase from Enzyme Development Corporation.

297 298

References

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413 414 415 416 417 418 419 420 421 422 423

Table 1: Effect of feed flow rate on lactulose and TOS yields (YLu and YTOS), lactulose productivity (πLu), selectivity (SLu/TOS) and lactose conversion (XLactose) in the synthesis of lactulose with glyoxyl-agarose immobilized A. oryzae β-galactosidase in CPBR. Operating conditions: 50 °C, pH 4.5, 50 % w/w total carbohydrates, feed fructose/lactose molar ratio of 12 and biocatalyst/inert packing material mass ratio of 1/8.

Feed Flow Rate (mL·min-1)

Y Lu

Y TOS

SLu/TOS

π Lu (g·min-1·g-1)

X Lactose

1 3 5 7 9 12 15

0.34 0.42 0.45 0.51 0.52 0.56 0.60

0.04 0.07 0.10 0.13 0.14 0.15 0.16

13.97 9.18 6.99 5.92 5.55 5.45 5.38

0.59 1.21 2.32 2.81 3.28 4.94 5.76

0.76 0.55 0.44 0.34 0.31 0.30 0.28

424 425 426 427 428 429 430 431 432

16

433 434 435 436 437 438 439 440 441 442 443 444 445 446

Table 2: Lactulose and transgalactosylated oligosaccharide yields (YLu and YTOS), selectivity (SLu/TOS) and lactose conversion (XLactose) in the synthesis of lactulose wit glyoxyl-agarose immobilized A. oryzae βgalactosidase in continuous packed-bed reactor (CPBR) and in batch reactor operation at 50 °C, pH 4.5, 50 % w/w total carbohydrates concentration. Reactions in CPBR were conducted at different fructose/lactose molar rations and feed flow rates at a biocatalyst/inert packing material mass ratio of 1/8.

Fructose/Lactose Molar Ratio

Type of Reactor Batch Reactor*

4

1 Packed-Bed Reactor Batch Reactor*

8

Feed Flow Rate (mL·min-1) -

Packed-Bed Reactor

5 1 5

Batch Reactor* 12

Packed-Bed Reactor

1 5

Batch Reactor* 16

Packed-Bed Reactor

1 5

Batch Reactor* 20

Packed-Bed Reactor

1 5

447

Y Lu

Y TOS

SLu/TOS

X Lactose

0.29

0.15

3

0.69

0.15

0.05

4.1

0.82

0.32

0.27

1.7

0.34

0.35

0.09

6

0.69

0.33

0.04

6.7

0.71

0.42

0.16

3.9

0.40

0.36

0.07

7.9

0.72

0.33

0.04

14

0.76

0.45

0.10

7

0.44

0.37

0.051

10.7

0.76

0.34

0.02

21.5

0.80

0.43

0.07

9.8

0.50

0.37

0.04

13.5

0.80

0.33

0.02

26.1

0.78

0.04

13.6

0.56

0.41 *Data reprocessed from Guerrero et al., 2017a.

448 449 450 451

17

452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468

Table 3: Effect of biocatalyst/inert packing material mass ratio (MB/MIM) on lactulose and TOS yields (YLu and YTOS), lactulose productivity (πLu), selectivity (SLu/TOS) and lactose conversion (XLactose) in the synthesis of lactulose with glyoxyl-agarose immobilized A. oryzae β-galactosidase in CPBR. Operating conditions: 50 °C, pH 4.5, 50 % w/w total carbohydrates, feed flow rate of 5 mL·min -1, fructose/lactose molar ratio of 12 and 45 mL of reactor.

Biocatalyst/inert packing material mass ratio (MB/MIM)

Y Lu

Y TOS

SLu/TOS

π Lu (g·min-1·g-1)

X Lactose

1/2 1/5 1/8 1/11 1/14 1/17

0.34 0.44 0.45 0.55 0.59 0.63

0.03 0.08 0.10 0.13 0.13 0.14

14.9 8.00 6.99 6.95 6.92 6.54

0.73 1.72 2.32 3.51 3.90 4.67

0.78 0.54 0.44 0.40 0.35 0.30

469 470 471 472 473 474 475 476 477

18

478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495

Table 4: Effect of recirculation rate on lactulose and TOS yields (YLu and YTOS), lactulose productivity (πLu), selectivity (SLu/TOS) and lactose conversion (XLactose) in the synthesis of lactulose with glyoxyl-agarose immobilized A. oryzae β-galactosidase in CPBR. Operating conditions: 50 °C, pH 4.5, 50 % w/w total carbohydrates, feed flow rate of 5 mL·min -1, fructose/lactose molar ratio of 12, and biocatalyst/inert packing material mass ratio (MB/MIM) of 1/8 g·g -1.

Recirculation rate

Y Lu

Y TOS

SLu/TOS

π Lu (g·min-1·g-1)

X Lactose

0 0.25 0.67 1.5 4

0.45 0.45 0.44 0.44 0.40

0.10 0.09 0.09 0.08 0.06

6.99 7.05 7.16 8.20 9,83

2.32 1.87 1.30 0.96 0.51

0.44 0.47 0.45 0.51 0.58

496 497 498 499 500 501 502 503

19

504 505 506 507

508 509 510 511 512 513 514 515 516 517 518 519

Figure 1: Experimental set-up for packed-bed reactor operation in the synthesis of lactulose with glyoxylagarose immobilized Aspergillus oryzae β-galactosidase. 1: Heating immersion circulator, 2: Magnetic stirring plate, 3: Substrates (fructose/lactose) mixture reservoir with 50 % w/w total sugars, 4: Feeding pump, 5: Product collecting reservoir, 6: Packed-bed reactor, 7: Chromatographic oven for temperature control. 8: Recirculation pump.

6

520 521 522 523 524

20

525 526 527 0.5

0.5

a)

0.4

b)

0.4

Y Lu

Y TOS

0.3

0.3

0.2

0.2

0.1

0.1

0.0

0.0 2

4

6

8

12

16

20

2

4

8

12

16

20

Fructose/Lactose Molar Ratio

Fructose/Lactose Molar Ratio 30

4

d)

c) π Lu (g·min-1·g-1)

25 20

SLu/TOS

6

15 10

3

2

1

5 0

0 2

4

6

8

12

16

20

2

4

Fructose/Lactose Molar Ratio 1.0

6

8

12

16

20

Fructose/Lactose Molar Ratio

e)

X Lactose

0.8 0.6 0.4

0.2 0.0 2

4

6

8

12

16

20

Fructose/Lactose Molar Ratio

528 529 530 531 532

Figure 2: Effect of feed fructose/lactose molar ratio on lactulose and TOS yields (YLu and YTOS), lactulose productivity (πLu), selectivity (SLu/TOS) and lactose conversion (XLactose) in the synthesis of lactulose with glyoxyl-agarose immobilized A. oryzae β-galactosidase in CPBR. Operating conditions: 50 °C, pH 4.5, 50 % w/w total carbohydrates, biocatalyst/inert packing material mass ratio of 1/8, and feed flowrates of 1 (), 3 () and 5 mL·min-1 ().

533 534

21

535 536 537 538 539

- A. oryzae β-gal immobilized on glyoxyl agarose was tested in CPBR for the first time

540 541

- Highest YLu obtained was 0.6 g·g -1 for continuous operation in a packed bed reactor

542 543

- Flow, substrate molar ratio and enzyme/inert support affected CPBR performance

544 545

- Lactulose synthesis in CPBR is a sound alternative to conventional batch synthesis

546 547 548

22

549 550

23