chitosan support for β-galactosidase immobilization for application in dairy technology

Accepted Manuscript Highly stable novel silica/chitosan support for β-galactosidase immobilization for application in dairy technology Natália Carminatti Ricardi, Eliana Weber de Menezes, Edilson Valmir Benvenutti, Jéssie da Natividade Schöffer, Camila Regina Hackenhaar, Plinho Francisco Hertz, Tania Maria Haas Costa PII: DOI: Reference:

S0308-8146(17)31836-8 https://doi.org/10.1016/j.foodchem.2017.11.026 FOCH 22003

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

6 July 2017 6 November 2017 8 November 2017

Please cite this article as: Ricardi, N.C., de Menezes, E.W., Valmir Benvenutti, E., da Natividade Schöffer, J., Hackenhaar, C.R., Hertz, P.F., Costa, T.M.H., Highly stable novel silica/chitosan support for β-galactosidase immobilization for application in dairy technology, Food Chemistry (2017), doi: https://doi.org/10.1016/ j.foodchem.2017.11.026

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Highly stable novel silica/chitosan support for β-galactosidase immobilization for application in dairy technology

a

a

a

Natália Carminatti Ricardi , Eliana Weber de Menezes , Edilson Valmir Benvenutti , b

b

b

Jéssie da Natividade Schöffer , Camila Regina Hackenhaar , Plinho Francisco Hertz , a,

Tania Maria Haas Costa *

a

Instituto de Química (IQ), Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre,

RS, Brazil b

Instituto de Ciência e Tecnologia de Alimentos (ICTA), Universidade Federal do Rio Grande do

Sul (UFRGS), Porto Alegre, RS, Brazil

Abstract β-D-Galactosidase is an important enzyme in the dairy industry, and the enzyme from the yeast Kluyveromyces lactis is most widely used. Here, we report immobilization of the enzyme on a silica/chitosan composite support, devised to have 10% and 20% chitosan (SiQT10 and SiQT20, respectively). Morphological and textural characterizations showed that chitosan is dispersed in micrometric regions in silica. For comparison, a silica organofunctionalized with 3aminopropyltrimethoxysilane (SiO2aptms) was prepared. Performance of the biocatalysts was tested for lactose hydrolysis, and the enzyme immobilized in SiQT10 and SiQT20 composites showed higher efficiency (62% and 47%, respectively) compared with the enzyme in SiO2aptms. Operational stability in this system was evaluated for the first time. After 200 hours of continuous use in a fixed-bed reactor, SiQT10 remained with approximately 90% activity. Thus, in addition to demonstrating compatibility for food processing, these results align the enzyme stabilization properties of chitosan with the mechanical resistance of silica. Keywords: inorganic-organic composite materials, lactose hydrolysis reaction, heterogeneous biocatalyst, continuous operation system, biocatalysis, enzyme immobilization

* Corresponding author: Tania Maria Haas Costa E-mail address: [email protected] Phone: +55 (51) 3308 6279

1

1. Introduction Enzymes are widely used in various sectors of the food industry due to their applications in different purposes. An important application is in the dairy industry, where βgalactosidase (EC 3.2.1.23) catalyzes lactose hydrolysis reaction. Among undesirable characteristics, such as the tendency to cause crystallization of dairy products, lactose also causes health disorders in people who suffer from intolerance to this carbohydrate. Lactose hydrolysis can minimize these problems by preventing sandiness in milk derivatives and enabling the consumption of dairy products by lactose-intolerant people (Grosová, Rosenberg, & Rebroš, 2008; Mlichová & Rosenberg, 2006). Moreover, in the presence of concentrated lactose, this enzyme synthesizes galactooligosaccharides (GOS), an important prebiotic food ingredient that is naturally present in human milk, which has had increasing application in functional foods (Pereira-Rodríguez et al., 2012). Enzymes are typically used in their free form, preventing their recovery and reuse. In biocatalysis, there is increasing use of immobilized enzymes due to their advantages over free enzymes, such as ease of enzyme separation from the reaction solution and capacity for reuse, thereby simplifying the recovery and recycling of biocatalysts and reducing operational costs (Liu et al., 2012). The good performance of these systems is directly related to the type of support used in the immobilization process. In this sense, it is of fundamental importance to identify new supports that combine mechanical properties and biocompatibility and can also improve enzyme characteristics (Bolivar, Consolati, Mayr, & Nidetzky, 2013; Garcia-Galan, Berenguer-Murcia, Fernandez-Lafuente, & Rodrigues, 2011). Furthermore, the use of enzymes immobilized on solid supports enables the development of continuous operation systems, thereby expanding the potential application of enzyme technology in industrial processes (Li, Yang, Yang, Zhu, & Wang, 2012). Immobilization of β-galactosidase is currently being studied in different carriers to improve its stability in processes where it is applied (Grosová, Rosenberg, & Rebroš, 2008). Immobilization can involve organic and inorganic supports, and various methods, such as encapsulation, crosslinking, physical adsorption or covalent binding. Among these immobilization strategies, covalent attachment of enzymes on the functionalized surfaces of solid supports has been widely investigated, since the activity and stability of the immobilized

2

enzyme is directly related to the properties of the support, such as pore size, chemical stability and binding affinity to the enzyme. Therefore, the development of a suitable step using supports is an important part of the process (Mateo, Palomo, Fernandez-Lorente, Guisan, & FernandezLafuente, 2007; Zucca & Sanjust, 2014). Among various organic materials used as supports for enzyme immobilization, chitosan is interesting due to such properties as non-toxicity, biocompatibility and biodegradability, and it also presents reactive functional groups available for direct reactions with enzymes and functionalization (Krajewska, 2004). Due to these chitosan properties, a recent study featured βgalactosidase immobilized in chitosan-grafted hydrogels and tested in controlled release for low-dosage lactose foods in lactose-intolerant individuals (Facin, Moret, Baretta, Belfiore, & Paulino, 2015). Other studies addressed the use of hollow spheres of chitosan as supports for immobilizing enzymes, showing satisfactory results for use in biocatalysis, such as increasing catalyst thermal stability and the possibility of operation over wider pH ranges (Klein et al., 2012; Lorenzoni, Aydos, Klein, Rodrigues, & Hertz, 2014; Schöffer, Klein, Rodrigues, & Hertz, 2013). However, the morphological and mechanical properties of chitosan may be improved for application in reactors and continuous processes, where the compaction and fragmentation of the supports were deemed to be responsible for the performance of these materials, hindering application to the food industry. Silica-based supports may be used for enzyme immobilization due to their excellent biocompatibility, mechanical properties and non-toxicity. Due to these properties, inorganic materials are notably suitable for industrial purposes, since they are stable over a wide range of pressure, temperature and pH (Rimola, Costa, Sodupe, & Ugliengo, 2013; Zucca & Sanjust, 2014). An interesting characteristic of silica matrices is the presence of surface silanol groups, which allow modification of surface properties and functionalization with cross-linking agents while maintaining the mechanical strength of the matrix (Acres et al., 2012; Bernal, Sierra, & Mesa, 2014; Giacomini, Villarino, Franco-fraguas, & Batista-viera, 1998; Song, Lee, Kang, & Kim, 2010). Alternative materials suggested as supports for enzyme immobilization include organicinorganic composites or hybrid materials prepared by incorporation of organic moieties on silica matrices, allowing the formation of materials with better mechanical and chemical properties

3

and porosity control (Cong & Yu, 2009; Portier, Choy, & Subramanian, 2001; Zhu & Row, 2012). Such polymers as chitosan can be proposed to form composites with silica, showing a porous microstructure with higher mechanical resistance, and accompanied by NH2 groups present in chitosan molecules that provide a compatible environment with biomolecules and serve as an adequate support for enzyme immobilization, eliminating surface functionalization steps (Singh, Srivastava, Singh, Singh, & Malviya, 2016; Zhao, Cui, Shah, Xu, & Wang, 2016). In the literature, most work related to the use of such composite material for enzyme immobilization are referred to for the preparation of electrochemical biosensors (Ramos et al., 2015). To the best of our knowledge, only two works using silica/chitosan composites have been shown to have application in enzymatic catalysis. However, these composites were not applied to the immobilization of β-galactosidase. One composite involves the immobilization of diglycosidase, where 80% of reactivity was retained for up to one hour (Piñuel, Mazzaferro, & Breccia, 2011). The other work addresses the immobilization of lipase applied to biodiesel synthesis (Silva, Oliveira, Giordani, & Castro, 2011). Additionally, there was a lack of structural, morphological and textural studies to evaluate the synthesis methods and the location and distribution of chitosan in the silica moiety. Since the behavior of each enzyme is very specific, a thorough study for β-galactosidase immobilization in silica/chitosan composite is necessary. The preparation of silica/chitosan hybrids or composite materials is not a simple task because the dissolution of chitosan is only possible in acidic systems, and adjustment of this solution to the synthesis system, while maintaining solubility is very difficult to obtain. This fact hinders the control of morphological and textural properties, which depends on the homogeneity of the initial system. To the best of our knowledge, the materials obtained to date were neither well-characterized nor show an adequate level of dispersion of the chitosan on silica. Therefore, the morphological characterization of the silica/chitosan system requires further study. One method to obtain these silica/chitosan composites with a high grade of chitosan is the sol-gel method based on hydrolysis and condensation reactions of molecular precursors. This method allows the planned physicochemical characteristics of the obtained materials as a result of synthesis conditions (Benvenutti, Moro, & Gallas, 2009; Ciriminna et al., 2013).

4

From this perspective, this work describes the development of inorganic-organic composite materials based on silica/chitosan using the sol-gel method, as well as the morphological and textural characterization of the obtained materials. The materials will be applied as an innovative support for β-galactosidase immobilization with the purpose of improving immobilized enzyme activity by combining the mechanical properties of silica and the benefits of chitosan. This approach will allow the use of composite materials in continuous systems, which have not been explored to date. For comparison, silica material was organofunctionalized with APTMS.

2. Materials and Methods

2.1.

Materials K. lactis β-D-galactosidase (Maxilact LX-5000) was obtained from Globalfood (Brazil),

and chitosan (≥75% deacetylated), o-Nitrophenyl β-D-galactopyranoside (o-NPG), tetraethyl orthosilicate 98% (TEOS), 3-aminopropyltrimethoxysilane 97% (APTMS) and glutaraldehyde 25%, were obtained from Sigma-Aldrich (Brazil). A D-glucose determination kit was purchased from In Vitro Diagnóstica (Brazil) and D-(+)-lactose was obtained from Dinâmica. All solvents and other chemicals were of analytical grade.

2.2.

Preparation of silica/chitosan composite materials (SiQT) Two samples of composite material were synthesized by the sol-gel method using 10%

and 20% w/w of silica and chitosan. The material was prepared in two steps: (a) dissolution of chitosan and (b) TEOS hydrolysis. In step (a), 0.13 g of chitosan was dissolved in 1.3 mL of water, 0.8 mL of acetic acid and 5.0 mL of ethanol under constant stirring until homogenization. In step (b), the sample containing 10% of chitosan underwent hydrolysis of the inorganic precursor via the addition of 5.0 mL of TEOS in a beaker containing 5.0 mL of ethanol. Next, 0.3 mL of water and 0.05 mL of HCl (37%) were added. For the sample containing 20% of chitosan,

5

the inorganic precursor was hydrolyzed by adding 2.5 mL of TEOS in a beaker containing 2.5 mL of ethanol. Subsequently, 0.1 mL of water and 0.05 mL of HCl (37%) were added to this solution. The solutions (b) were kept under magnetic stirring for 1 hour and slowly added to the (a) solutions under strong agitation. To this mixture were added 10 drops of HF (40%) as a catalyst. After drying for 15 days at room temperature, the obtained xerogels were ground until they passed through a Tyler standard sieve 35 mesh series. After washing with distilled water and ethanol, the xerogels were vacuum dried at 90 °C for 2 hours. These samples were designated SiQT10 for 10% of chitosan and SiQT20 for 20% chitosan.

2.3.

Preparation of the organofunctionalized silica The silica matrix was prepared by the sol-gel method. Hydrolysis of the inorganic

precursor occurred by the addition of 5.0 mL of TEOS in a beaker containing 5.0 mL of ethanol, in which 0.3 mL of water and 0.05 mL of HCl (37%) were added. This solution remained under magnetic stirring for 1 hour and was slowly added to a solution containing 1.4 mL of water, 0.8 mL of acetic acid and 5.0 mL of ethanol under constant stirring until homogenization. To this mixture were added 10 drops of HF (40%) as a catalyst. After drying for 15 days at room temperature, the obtained xerogel was ground until it passed through a Tyler standard sieve 35 mesh series. Later, the material was washed with distilled water and ethanol, and vacuum dried at 90 °C for 2 hours. This sample was designated SiO2. The silica surface was modified by a grafting reaction with the addition of 1.0 mmol of APTMS per gram of silica under reflux in toluene at 65 °C for 18 hours with mechanical stirring and an inert atmosphere. The resulting material was washed with portions of toluene, ethanol, water and ether, and then vacuum dried at 90 °C for 2 hours. This material is referred to as SiO2aptms.

2.4.

Activation with glutaraldehyde Activation was performed by adding 1.0 g of the obtained materials in 50 mL of a 5.0%

v/v solution of glutaraldehyde under agitation for three hours. Subsequently, the materials were -1

washed several times with potassium phosphate buffer solution (0.1 mol L at pH 7.0) and dried

6

under vacuum for two hours. The materials are referred to as SiQT10glut for 10% of chitosan, SiQT20glut for 20% of chitosan and SiO2glut for organofunctionalized silica.

2.5.

β-galactosidase immobilization β-galactosidase was immobilized with 0.010 g of each support incubated with 1.0 mL of -1

-1

a 0.1 mg mL enzyme solution prepared with potassium phosphate buffer 0.1 mol L (pH 7.0) -1

containing MgCl2 1.5 mmol L

under gentle stirring for 18 hours at room temperature. After

immobilization, the materials were washed with buffer solution, NaCl (1.0 mol L-1) and ethylene glycol (30% v/v) to remove non-covalently bound enzymes. The materials are referred to as SiQT10enz for 10% of chitosan, SiQT20enz for 20% of chitosan and SiO2enz for organofunctionalized silica.

2.6.

Elemental analysis of C, H and N Elemental analysis was used for quantification of organic groups present in the

silica/chitosan composite materials. The analyses were performed in a CHN Perkim Elmer M CHNS/O Analyzer, model 2400.

2.7.

Thermogravimetric Analysis (TGA) The thermogravimetric analyses of the materials were performed using a Shimadzu

Instrument TGA-50 under an argon flow with a velocity of 50 mL min-1 and a heating rate of 20 -1

°C min from ambient temperature to 800 °C.

2.8.

Fourier Transform Infrared Spectroscopy (FT-IR) The SiQT10, SiQT20 and SiO2 materials were analyzed by infrared spectroscopy using

a quartz cell that allows collection of spectra after heat treatment under vacuum to avoid air exposure. Disks of material samples with a diameter of 2.5 cm, weighing approximately 100 mg, were prepared and accommodated within the cell (Foschiera, Pizzolato, & Benvenutti, 2001).

7

-2

The samples were heated to 140 °C under vacuum (10 torr) for 2 hours. The spectra were obtained using a Shimadzu FTIR instrument, namely, Patronize 21 with a resolution of 4 cm-1 and 100 cumulative scans.

2.9.

N2 adsorption and desorption isotherms N2 adsorption and desorption isotherms were obtained using Tristar II 3020 Krypton

Micromeritics equipment at the liquid nitrogen boiling point (77 K). The pore-size distribution curves of the materials were obtained using the BJH method (Barret, Joyner and Halenda). The materials’ specific surface area was obtained using the BET method (Brunauer, Emmett and Teller) (Gregg & Sing, 1982; Webb & Orr, 1997).

2.10.

Scanning Electron Microscopy (SEM) The morphology of the samples, before and after surface modification, was investigated

using images taken with a scanning electron microscope. The materials were dispersed without coating on double-sided conductive tape on an aluminum support. The equipment used was a Zeiss EVO MA10. Elemental analysis of samples was performed via X-ray spectroscopy with energy dispersion.

2.11.

Activity assays The hydrolytic activity of free and immobilized enzyme was determined by measuring

the release of o-nitrophenol from o-NPG, as determined spectrophotometrically at 415 nm in a thermostatically controlled water bath at 37 °C. A β-galactosidase unit is defined as the amount of enzyme that catalyzes the conversion of 1.0 µmol o-NPG to ο-nitrophenol per minute. Free enzyme analysis was performed in a 0.5-mL assay volume containing 270 µL of -1

-1

potassium phosphate buffer solution 0.1 mol L (pH 7.0) containing MgCl2 1.5 mmol L , 180 µL o-NPG (30 mmol L-1) and 50 µL of enzyme solution. After 2 minutes, the reaction was stopped -1

by adding 1.5 mL of sodium carbonate buffer solution 0.1 mol L (pH 10.0). Using the same procedure, after enzyme immobilization, analyses were performed with aliquots taken from the

8

resulting washing fractions with buffer solution, NaCl and ethylene glycol, as described in item 2.5 (Klein et al., 2012). Assays were performed in triplicate. For hydrolytic activity analysis of the immobilized enzyme, 0.010 g of biocatalyst was mixed with 1.62 mL of potassium phosphate buffer solution 0.1 mol L-1 (pH 7.0) containing -1

-1

MgCl2 1.5 mmol L , under constant stirring. Next, 1.08 mL of o-NPG (30 mmol L ) was added. After 30 seconds, the reaction was stopped by adding 9.0 mL of sodium carbonate buffer -1

solution 0.1 mol L (pH 10.0). Assays were performed in triplicate. The hydrolytic activity results obtained in these tests were used to determine the immobilization parameters (Sheldon & van Pelt, 2013):
 % = 100 ×



   

!"!# % = 100 ×

   

$!%&%# '!(&'# % = 100 ×

2.12.

   

Equation 1 Equation 2 Equation 3

Thermal stability The effect of temperature on the stability of free and immobilized enzyme was evaluated

through incubation of biocatalyst in tubes containing activity buffer solution in a thermostatically controlled water bath at 40, 50, 60 and 70 °C. After defined time intervals, the samples were removed from the bath and placed in an ice bath to stop thermal inactivation. The residual activities were determined by o-NPG hydrolysis at 37 °C, as described by Klein et al. (2012), and the relative activities were calculated as the ratio between the activity at each temperature and the maximum activity obtained. Assays were performed in triplicate to determine the mean square errors.

2.13.

Determination of optimal pH The optimal pH was evaluated for both free and immobilized enzyme with o-NPG

hydrolysis using potassium phosphate buffer solution 0.1 mol L-1 at pH(s) 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0, with a constant temperature of 37 °C. The relative activity was calculated as the ratio

9

between the observed activity at each pH and the maximum activity obtained for each sample. Assays were performed in triplicate to determine the mean square errors.

2.14.

Operational stability of immobilized β-galactosidase The operational stability of immobilized β-galactosidase on the SiQT10 sample was -1

determined by hydrolysis of lactose (50 g L ) with a fixed bed reactor. The reactor consisted of a glass column with a water jacket, which contained 1.5 g of the biocatalyst. The substrate flow rate was controlled by a peristaltic pump at 0.3 mL min-1. The fixed bed column (height, 15.0 cm; inner diameter, 1.0 cm; volume, 28.0 mL) has an entry at the bottom and exit at the top, both equipped with a sintered glass disc to retain support particles within the column. The outside water jacket enabled the circulation of liquid, maintaining the system at 37 °C.

3. Results and Discussion

3.1.

Structural and textural characterization of the materials CHN analysis of the composite materials is shown in Table 1 of the Supplementary

Material. It is possible to quantify the proportion of chitosan incorporated into the composite material. Sample SiQT20 showed a proportionally greater amount of chitosan incorporated in its structure: 5.93% compared to 3.60% in the SiQT10 sample. Even if 10% and 20% of chitosan were added in the syntheses, the amount actually incorporated in the material is lower, as observed in other studies (Ramos et al., 2015). The curves for thermogravimetric analysis obtained for all samples before and after surface modifications are presented as supplementary material (Figure 1). Weight loss was observed between room temperature and 150 °C and is ascribed to the adsorbed water evaporation. The weight loss between 150 and 650 °C can be related to dehydroxylation of silanol groups from silica and to desorption and decomposition of organic matter. The weight loss between 150 and 650 °C increases with increasing chitosan content in the samples: 2.68% for SiO2, 6.29 for SiQT10 and 11.83 for SiQT20, as shown on Table 1. Increasing weight loss

10

between 150 and 650 °C was observed after surface modifications of SiO2 (with APTMS and glutaraldehyde) and SiQT10 (with glutaraldehyde), proving the presence of these components. It was not possible to show immobilization of the enzyme using this technique, possibly due to the small amount of enzyme added.

Table 1. Thermogravimetric and textural analyses of samples.

Samples

0-150 °C (%)

150-650 °C (%)

SiO2

2.18

SiO2aptms

SBET 2

Pore Volume -1

3

-1

(± 10 m g )

(± 0.05 cm g )

2.68

206

1.25

0.85

5.64

184

1.13

SiO2glut

5.94

8.44

194

0.92

SiO2enz

8.51

8.21

190

0.92

SiQT10

4.19

6.29

174

1.11

SiQT10glut

1.86

8.83

180

1.09

SiQT10enz

12.37

8.57

163

1.05

SiQT20

3.13

11.83

107

0.83

SiQT20glut

2.87

11.58

104

0.77

SiQT20enz

3.09

11.32

102

0.79

The SiO2 and SiQT10 sample morphologies were investigated using SEM analysis, which is presented in Figure 1. For the SiO2 sample obtained by the sol-gel method (Figure 1a), a typical morphology of micrometer silica particles was observed. However, images of the SiQT10 sample showed rough regions on its surface (Figure 1b-d). The backscattered electron images (BSE) (Figure 1c and 1d) showed darker micrometric regions related to carbon atoms, indicating that chitosan is dispersed in micrometric domains in the silica matrix, which can be seen more clearly in the BSE images. In Figure 1(e) and 1(f) and Table 2 of the Supplementary Material, EDS analysis shows the mass percentages of the main elements present in certain points of the samples. This analysis was performed to identify the distribution and location of chitosan in silica samples. The

11

points were chosen to show differences between the composition of silica and chitosan moieties. The points SiQT10-2 and SiQT10glut-3, located in lighter areas of the images, showed a higher percentage of silicon and a lower percentage of carbon in their composition, near 75% and 4%, respectively. The points SiQT10-1, SiQT10glut-1 and SiQT10glut-2, located in the darker area, showed a greater amount of nitrogen and carbon, near 10% and 60% respectively, indicating the presence of chitosan, while the silica percentage was near 10%. The infrared spectra obtained at room temperature for, of SiO2, SiQT10, and SiQT20 samples treated at 140 °C under vacuum are shown as Supplementary Material (Figure 2). The bands at 1870 and 1980 cm

-1

correspond to silica overtones (Costa, Gallas, Benvenutti, &

-1

Jornada, 1997). The 1640 cm band is due to silica overtones and NH2 bending from chitosan, -1

since it increases with chitosan amount. The bands above 3200 cm were assigned to the O-H stretching of silanol groups. The band at 3740 cm-1 is due to free silanols (Läufer, 1980; -1

McDonald, 1958). The band with a maximum at 3675 cm was assigned to bridged geminal silanol groups (Parida, Dash, Patel, & Mishra, 2006), while the band with a maximum between -1

3460 and 3525 cm was attributed to bridged vicinal silanol groups (Morrow & McFarlan, 1992). A shift of this last band towards lower frequencies was observed as the organic content increases. This shift was interpreted to be the result of silanol interactions with the organics. The results for the specific surface area and pore volume of the samples are detailed in Table 1. The samples SiO2, SiQT10 and SiQT20 showed surface area values of 206, 174 and 2

-1

3

-1

107 mg g , respectively, and pore volume values of 1.25, 1.11 and 0.83 cm g . The addition of chitosan to the materials resulted in a reduction in N2 adsorbed volume per gram of material and a decrease in surface area values. For the SiO2 sample, there is a small decrease in surface area values with the successive incorporation of the organic moieties. However, for SiQT10 and SiQT20 samples, these values remained almost unaltered after surface modification and enzyme immobilization. Nitrogen adsorption and desorption isotherms of SiO2, SiQT10 and SiQT20 samples are shown in Figure 2(a) and are typical of mesoporous materials, classified as type IV (Gregg & Sing, 1982). The isotherm of the SiQT20 sample shows N2 adsorption at higher relative pressure compared to other samples, which indicates the presence of pores with larger size. The samples showed a mesopore distribution with maxima near 20 nm, as seen in Inset Figure

12

2(a). After surface modifications of the SiO2 sample with APTMS and glutaraldehyde, the closing of pores with larger diameters was observed. However, the addition of enzyme did not cause pore closing, as seen in Figure 2(b). For the samples SiQT10 and SiQT20, the curves of which are presented respectively in Figure 2(c) and 2(d), surface modifications and enzyme immobilization produced no significant change in the pore size distribution and in the isotherms curves, indicating that the modifications do not affect the pore structure. The obtained results indicate that chitosan was incorporated in the silica matrix in micrometric domains. The enzyme was immobilized only on the chitosan moiety, while the pore structure was formed only by the silica moiety. In pure silica material, successive modifications with APTMS and glutaraldehyde occurred on larger pores, but enzyme immobilization did not cause changes in pore volume or the pore size distribution curve. These results can be explained by the fact that glutaraldehyde and enzymes do not react with the silica matrix, which is the component responsible for the pores. As silica and chitosan are present in separate micrometric phases, as observed by SEM analysis, glutaraldehyde activation occurs only in the chitosan phase, which is the non-porous portion of the material.

3.2.

Immobilization parameters Table 2 presents the immobilization process parameters. The enzyme immobilized on

the organofunctionalized silica (SiO2enz) shows the highest immobilization yield values (97%) but a lower efficiency (37%) compared to the supports containing chitosan, indicating that most of the immobilized enzyme on silica lost its activity. The recovered activities of the immobilized enzyme showed approximately the same amounts of composite materials and SiO2enz. The enzyme immobilized on the support containing 10% of chitosan showed the highest efficiency -1

(62%) and higher observed activity per gram of support (851 U g ). These results demonstrate that the presence of chitosan in the composite improves the support potential for enzyme immobilization, reflecting better total activity per gram of support. In this case, chitosan offers a stronger protecting effect on the enzyme than organofunctionalized silica, since it showed better efficiency by reflecting a higher observed activity per gram of support (Kumar, Attri, & Venkatesu, 2012).

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Table 2. Immobilization process parameters of SiQT10enz, SiQT20enz and SiO2enz samples. Immobilized

Efficiency

Activity

Activity (U g )

Yield (%)

(%)

Recovery (%)

SiQT10enz

851(± 65)

53(± 0.1)

62(± 0.2)

33(±0.002)

SiQT20enz

686(± 25)

54(± 0.8)

47(± 1.4)

26(±0.005)

SiO2enz

736(± 8)

97(± 2)

37(± 1)

36(± 0.02)

Sample

Observed -1

When comparing the efficiency and observed activity for both composites, contrary to what was expected, SiQT10enz presented better results. Part of the chitosan moiety of SiQT20enz is occluded in the silica, and this may be why it is not available to bond with enzyme.

3.3.

Determination of optimal pH The pH of the medium was altered between 5.5 and 8.0 for both free and immobilized

enzyme, and the resulting effect on relative activity of the enzyme is shown in Figure 3(a). As observed, the relative activity of the free and immobilized β-galactosidase was clearly dependent on pH. The highest relative activity for the free and immobilized enzyme was observed at pH 7.0, indicating that free and immobilized enzymes maintained their active conformation at this pH. The significant decrease in relative activity in more acidic and alkaline pH could be attributed to inactivation of the enzyme. Similar behaviors were reported by Klein et al. (2012) for the β-galactosidase enzyme and by Schöffer et al. (2013) for the cyclodextrin glycosyltransferase enzyme, both immobilized on chitosan. Therefore, the results obtained here suggest that the supports containing silica/chitosan and organofunctionalized silica could be used as supports for immobilizing β-galactosidase at the pH of milk (approximately 7.0), where the enzyme was more active.

14

3.4.

Thermal stability The thermal stability of free and immobilized β-galactosidase, determined by o-NPG

hydrolysis at 37 °C and pH 7.0 for the 5-min assays, are shown in Figure 3(b). In tests performed at 60 and 70 °C, there was a common tendency for a total loss of relative activity of free and immobilized enzyme after 5 min of heating. The results showed higher relative activity for free and immobilized enzyme at 40 °C. As seen in Figure 3(c), at 40 °C the enzyme immobilized on the support containing 10% chitosan (SiQT10enz) retained 45% of relative activity for up to 350 minutes of immersion, which is the best result among the biocatalysts presented here. After 1440 minutes of incubation, both free and immobilized enzyme decreased in relative activity. Because free enzyme exhibits a thermal stability similar to immobilized enzyme, the immobilization process used for the three materials was adequate. However, this process did not change the basic characteristics of the enzyme, as already observed by other authors when pure chitosan was used as support (Klein et al., 2012; Lorenzoni et al., 2014; Schöffer et al., 2013).

3.5.

Operational stability A primary objective in immobilized enzyme technology is to increase enzyme stability,

since stability directly affects costs (Nie, Xie, Wang, & Tan, 2006). Thus, the immobilized enzyme operational stability was evaluated by hydrolysis of lactose using a fixed bed reactor. Figure 4 shows the results of relative activity as a function of reactor operating time. The enzyme immobilized in SiQT10 was chosen for this step because it presented the best efficiency and activity per gram of support. After 200 hours of continuous reactor operation, the biocatalyst maintained a high relative activity of approximately 90%. The conversion rate for lactose into glucose was 14%. This value can be improved by optimizing operational parameters, such as the increase in substrate residence time in the reactor, either by decreasing substrate flow or using a larger amount of biocatalyst in the column.

15

In our recent work (Klein et al., 2012; Lorenzoni, Aydos, Klein, Rodrigues, & Hertz, 2014; Schöffer, Klein, Rodrigues, & Hertz, 2013), we reported similar high thermal and operational stability for enzymes immobilized on chitosan particles. However, these derivatives lack good mechanical stability, hindering the scale-up process. In generally, the support used should be affordable, safe and stable. Therefore, the biocatalyst obtained in the present work satisfies these requirements, since it was prepared from chitosan (an affordable and non-toxic polysaccharide) and silica, which improves mechanical stability. The results reported in the present work show that it is possible to improve mechanical resistance of this carrier without loss in catalyst performance. From a practical point of view, the obtained particles are easy to handle and more mechanically resistant than particles prepared with chitosan alone. These results indicate that silica/chitosan supports can be an attractive alternative for enzyme immobilization in food processing, particularly β-galactosidase, by improving cost-effectiveness due to the possibility of using the enzyme repeatedly in batch or in a continuous process without losing its relative activity.

4. Conclusions In this work, novel silica-based composite materials were prepared with high amounts of chitosan (10 and 20%) and a silica material, which was organofunctionalized with APTMS for comparison. These materials were applied to the immobilization of β-galactosidase from Kluyveromyces lactis. The presence of chitosan was evidenced by elemental and thermogravimetric analysis, indicating that chitosan content increases with the amount of chitosan added. Scanning electron microscopy of the composite samples indicated that chitosan was dispersed in micrometric regions in the silica. The samples presented surface area 2

-1

with values between 100 and 200 m g and pore distribution curves with maxima near 20 nm. These curves did not change after enzyme immobilization, indicating that the enzyme is only linked in the chitosan regions of the material. For immobilization parameters, enzyme immobilized in the composite materials showed higher efficiency than enzyme immobilized in the organofunctionalized silica. The immobilized

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enzyme on the composite material SiQT10 showed higher activity (U g ). Additionally, the enzyme retained approximately 90% activity for lactose hydrolysis in a fixed bed reactor when used for a continuous period of 200 hours. In conclusion, β-galactosidase immobilized on silica/chitosan supports can be an excellent alternative for the production of food with a low concentration of lactose based on the possibility of reusing the same enzyme repeatedly and maintaining its enzymatic activity. The development of a composite support enabled the incorporation of both widely known characteristics of silica and the positive characteristics of chitosan to the biocatalyst, showing for the first time that this support is a viable, interesting alternative for applications in the food industry.

Acknowledgments This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), which are Brazilian agencies. We would like to express gratitude for scholarships and grants. We would also like to thank the Centro de Microscopia e Microanálise of UFRGS (CMM).

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Figure Captions

Figure 1. SEM images of (a) SiO2 and (b) SiQT10 samples; SEM BS images of (c) SiQT10glut and (d) SiQT10enz samples. Bar size is 100 µm; and SEM BS images with EDS points (e) SiQT10 and (f) SiQT10glut samples.

Figure 2. N2 adsorption-desorption isotherms and pore-size distribution curves (Inset): (a) SiO2, SiQT10 and SiQT20 samples; (b) SiO2 sample before and after modifications; (c) SiQT10 sample before and after modifications; (d) SiQT20 sample before and after modifications.

Figure 3. (a) Effect of pH on relative activity of free and immobilized enzyme. (b) Thermal stability of free and immobilized enzyme at 40 to 70 °C, determined by o-NPG hydrolysis at 37 °C and pH 7.0 for 5-min assay. (c) Thermal stability of free and immobilized enzyme at 40 °C, determined by o-NPG hydrolysis at 37 °C and pH 7.0.

Figure 4. Operational stability of enzyme immobilized onmn support containing 10% of chitosan (SiQT10enz) using 50 g L

−1

buffered lactose solution, pH 7.0 at 37 °C and flow rate of 0.3 mL

min-1.

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Highlights Silica/chitosan biocatalyst for application in dairy technology Novel

silica/chitosan

composite

support

for

β-galactosidase

immobilization Morphological and textural characterization of silica/chitosan composite materials Application of the biocatalysts in lactose hydrolysis reaction in continuous operation system Biocatalyst with high activity for continuous use, showing mechanical stability

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