Catherine Teixeira de CARVALHO a, Wildson Bernardino de Brito LIMA a, Fábio Gonçalves Macêdo de MEDEIROS b, Julia Maria de Medeiros DANTAS a, Carlos Eduardo de Araújo Padilhaa , Everaldo Silvino dos SANTOS a, Gorete Ribeiro de MACÊDOa, Francisco Canindé
de SOUSA JÚNIOR* a,c
a Laboratory of Biochemical Engineering, Chemical Engineering Department, Federal
University of Rio Grande do Norte (UFRN), Natal, Brazil
b Bioprocess Laboratory, Chemical Engineering Department, Federal University of Rio Grande
do Norte (UFRN), Natal, Brazil
c Laboratory of Bromatology, Pharmaceutical Department, Health Sciences Centre, Federal
University of Rio Grande do Norte (UFRN), Natal, Brazil
Corresponding author
*Francisco Canindé de Sousa Júnior
Laboratory of Bromatology, Department of Pharmacy, Health Sciences Center, Federal University of Rio Grande do Norte (UFRN), 59012-970, Natal, Brazil. E-mail: fcfarma@yahoo.com.br
Abstract
The present study aimed to evaluate the hydrolysis conditions of lactose from "coalho" cheese whey using β-galactosidase (β-gal) produced by Kluyveromyces lactis immobilized with sodium alginate. Three sodium alginate-based immobilization systems were evaluated (0.5, 0.7 and 1% w/v) for maximizing the immobilization yield (Y), efficiency (EM) and recovered activity (ar). The stability of the immobilized β-gal was investigated against different pH values,
temperatures and in the presence of metallic ions. The lactose hydrolysis capacity of the immobilized form of β-gal was determined and simulated environments were used in order to assess the preservation of the immobilized enzyme in the gastrointestinal tract. The results showed that β-gal immobilization with 1% (w/v) sodium alginate presented the best results (EM of 66%, Y of 41% and ar of 65%). The immobilization system sustained the highest pH stability
for the range between 5.0 – 7.0, and temperature stability was also favored by immobilization. In 6 h of hydrolysis, the immobilized β-gal was able to hidrolyze 46% of the initial lactose content. For the gastrointestinal simulations, around 40% of the activity was preserved after 2 h of exposure. Overall, the results described here are promising for the industrial applications of β-galactosidase from K. lactis.
1. Introduction
The use of cheese whey by the dairy industry is limited by its high content of lactose, which reduces its solubility and impacts on its low sweetness capacity and digestibility, when incorporated into food products. In addition, lactose is less fermentable when compared to other sugars [1,2].
Lactose is a disaccharide formed by glucose and galactose, a type of carbohydrate found in milk and dairy products. In humans, lactose intolerance occurs in 75% of the population, being caused by the insufficiency of β-galactosidase (lactase) in the body, which compromises the activity of this enzyme in the membrane of the mucosa edge in the small intestine of adults. Lactase enzyme preparations is used in dairy products for reducing the lactose content and promote a beneficial effect for lactose intolerants [3,4]
The β-galactosidase enzymes are widely found in nature, being present in animals, plants and microorganisms. Kluyveromyces spp. yeast are capable of fermenting lactose by producing an intracellular form of β-galactosidase. The lactase obtained from Kluyveromyces spp. yeasts presents optimum activity in pH range around 6.0 – 7.0 [5,6]. As a drug and for the food industry, these biomolecules are of great interest to industry due to their high activity, selectivity and the possibility of manipulating their properties. However, the use of enzymes in pharmaceutical grade administered orally is a challenging aspect due to the potential of inactivation of this molecule in a hostile gastrointestinal environment [7].
Enzyme immobilization is an efficient biotechnological strategy for increasing enzyme stability and enhancing industrial applications of such labile biomolecules, which allows cost reduction [8]. However, a strict criteria selection is required for efficient enzyme immobilization, in order to achieve a simple and economical immobilization strategy that results in high enzyme activity retention and high operation stability [9,10].
Therefore, the present study aimed to evaluate the hydrolysis conditions of the cheese whey lactose using β-galactosidase (β-Gal) of Kluyveromyces lactis immobilized with sodium alginate with potential for industrial application. The viability of this process would help avoiding the inappropriate disposal of the "coalho" cheese whey, a regional industrial by- product, through a value-adding strategy.
2. Materials and methods
2.1. Reagents and enzymes
The synthetic substrate o-nitrophenyl-β-D-galactopyranosid (ONPG) was acquired from Sigma-Aldrich (St. Louis, MO, USA). Pepsin, pancreatin, sodium chloride, lactose, sodium alginate, calcium chloride and all other reagents were of analytical grade.
2.2. Microorganism and inoculum production
Kluyveromyces lactis NRRL Y-8279 used in this study was provided by the collection
of cultures. The strain was maintained in a 30% (v/v) solution of glycerol at -20 °C. For pré- inoculum preparation, K. lactis was cultivated in YEPD medium containing 10 g/L of yeast extract, 20 g/L of dextrose, 20 g/L of peptone and 20 g/L of agar for 24 h, at 30 °C. Then, three isolated colonies (inoculum) were transferred into 50 mL of the culture medium containing 10 g/L of lactose, 5.0 g/L of potassium phosphate, 1.2 g/L of ammonium sulfate, 1.0 g/L of yeast extract and 0.4 g/L of magnesium sulfate, prepared in potassium phosphate buffer (0.2 M, pH 5.5) according to (Lima et al., 2013). The Lactose used in the growth medium was exclusively from the cheese whey partially deproteinized (CWD). The inoculums were cultivated in Erlenmeyers under agitation of 180 rpm, at 30 °C, for 16 h in a rotating incubator.
The enzyme β-galactosidase (β-Gal) was produced through submerged cultivation por
Kluyveromyces lactis NRRL Y-8279 using rotary incubators (Tecnal, model TE-241), with 250
mL Erlenmeyers flasks containing 50 mL of culture medium (20 g/L of QWD, 1.3 g/L of (NH4)2SO4, 12 g/L of yeast extract, 5.0 g/L of KH2PO4 and 0.4 g/L MgSO4.7H2O, in 0.2 M
potassium phosphate buffer at pH 5.5, according to the adapted methodology of (Braga et al., 2014). The fermentation was performed with 10% of inoculum (v/v) prepared as described in section 2.1. The culture medium was previously sterilized and the lactose from the QWD was filtered in a 0.22 μm membrane. The cultivations in rotary incubators were performed at 30 °C, 180 rpm. Samples were removed after 20 h of fermentation.
The enzymatic extract was obtained from the yeast cell disruption after the fermentation, according to (Braga et al., 2014). The procedure was performed in centrifuge tubes of 50 mL, containing 25 mL of the cell suspension and 27.5 g of glass pearls (diameter ranging from 0.95 to 1.05 mm) under vortex agitation for 5 min, followed by 1 minute in an ice bath. This procedure was repeated 4 times. Then, the suspension was centrifuged (Cientec, CT-5000R model, Brazil) at 5200 × g during 10 min at 4 ºC. The enzyme extract was collected and stored at -20 °C.
2.4. β-galactosidase immobilization with sodium alginate
For the immobilization of β-gal, three concentrations of sodium alginate (0.5%, 0.7% and 1% w/v) were tested. The appropriate amount of sodium alginate was mixed into 10 mL of potassium phosphate buffer 50 mM (pH 6.6) and heated under magnetic agitation until complete solubilization. The sodium alginate solution were cooled to room temperature and 0.2 g of freeze-dried β-gal (3 U/mL) were added and homogenized for 10 minutes under magnetic stirring. The enzyme-sodium alginate dispersion was added drop-wise to a solution of calcium chloride 0.25 M, with the aid of a peristaltic pump (TE-BP01 dosing system, TECNAL, Brazil), for the formation of the hydrogel granules. The enzyme-sodium alginate granules were kept
immersed in the calcium chloride solution for 10 minutes under constant agitation. After that, the granules were stored for 10 minutes at 4 °C and subsequently washed with distilled water for removal of excess calcium chloride. The granules were stored at 4 °C in distilled water until further use, following the method adapted from [11,12].
2.4.1. Evaluation of immobilization parameters
In order to evaluate the enzyme immobilization process, the immobilization yield, the recovered enzymatic activity and the immobilization efficiency were assessed. The immobilization yield (Yi) was calculated from the enzymatic activity prior (a0) and after to immobilization (af),
according to Equation 1 [13,14].
Yi (%) = 𝑎0−𝑎𝑓
𝑎0 x 100 (1)
The recovered enzymatic activity (ar) is defined as the ratio between the theoretical enzymatic
activity (at) that the immobilized enzyme should present after the process, and the real
enzymatic activity (ad) of the immobilized system. This parameter evaluates if the active sites
of the enzyme immobilized form are available equations 2 and 2.1:[16,18]
𝑎𝑡= 𝑎0− 𝑎𝑓 (2)
𝑎𝑟(%) = 𝑎𝑑
𝑎𝑡 x 100 (2.1)
The immobilization efficiency (EI) is determined as the ratio between the enzymatic activity of the immobilized system and the enzymatic activity of the free β-gal enzyme (al), according to
Equation 3 [13].
𝐸𝐼 (%) = 𝑎𝑑
The best immobilization condition was selected for the stability, lactose hydrolysis and gastrointestinal simulation tests. All experiments were performed in triplicates.
2.5. Enzymatic stability of immobilized β-galactosidase and operational stability
The β-Gal stability was assessed at different pHs, in the presence of metal ions and at different temperatures based on the remaining activity after exposure according to [19]. For pH stability, the immobilized β-gal was exposed to pH 5.0 to 10.0, at 30 °C, for 30, 60 and 120 minutes. The following buffer solutions were used: sodium acetate (0.2 M, pH 5.0), sodium phosphate (0.2 M, pH 6.0-8.0), glycine-NaOH (0.2 M, pH 9.0-10.0). For thermal stability, the enzymatic extract was exposed to temperatures of 40 °C, 45 °C and 50 °C, at pH 6.6, for 15, 30 and 60 minutes [20].
The effect of metal ions on the β-gal activity was assessed as described by [21], with minor modification. The immobilized β-gal was exposed to solutions containing ions Mg2+
(MgSO4·7H2O, 5 mM), Cu2+ (CuSO4 ·5H2O, 5 mM), Zn2+ (ZnSO4 ·7H2O, 5 mM), Mn2+
(MnCl2, 5 mM), Fe2+ (FeSO4, 5 mM) e Co2+ (CoCl2, 5 mM), at 25 °C, for 30, 60 and 120
minutes. After each treatment, the β-gal activity was determined and the results were expressed as the enzymatic activity retention [22].
The enzyme β-galactosidase immobilized in alginate according to item 1.2 was further submitted to the operational storage stability study, being stored in potassium phosphate buffer pH 6.6 and temperature of 4 °C for a period of 62 days. Residual activity was determined by the initial rates method according to [4]. Activity was calculated relative to initial activity [23].
2.6. Scanning Electron microscopy (SEM)
For evaluating the microstructure of the immobilization system, SEM images of immobilized enzyme and the enzyme-free biopolymer (sodium alginate 1%) were acquired with a scanning electron microscope (Hitachi Tabletop Microscope TM-3000, USA). Samples were freeze dried
prior to the analysis. Images were acquired accelerating voltage of 5kV and 15kV, and the electron micrographs were taken at 150x and 1000x magnification [16,19].
2.7. Fourier transformer infrared spectroscopy analysis (FT-IR)
For the study of polymers, the FT-IR technique allows analyzing the compounds present in the structure and the interactions between them. Biopolymer (sodium alginate 1 %) and free recovered enzyme were initially freeze dried for 24 h. Then, the immobilized enzyme complex, and the biopolymer without enzyme were analyzed by Fourier transform infrared spectroscopy (model IRTracer-100, Shimadzu, USA). Measurements were performed by ATR (Total Attenuated Reflectance) with zinc selenide crystal. FT-IR spectra were obtained in the range of 400 to 4000 cm-1 [24]
2.8. Analytical methods
The enzymatic activity of β-gal was determined using the synthetic substrate o- nitrophenyl-β-D-galactopyranosid (ONPG), as described by [4]. The enzymatic activity of β- gal was expressed in U/mL. The enzymatic activity unit (U) was defined as the enzyme necessary to produce 1.0 μmol of o-nitrophenol per minute at the assay’s conditions (37 °C, pH 6.6). The total protein was assessed by Bradford method [25]. Bovine Serum Albumin (BSA) was used as standard and reads were performed at 595 nm. The protein concentration was expressed in mg/mL and used to calculate the specific activity of the enzyme β-Gal (U/mg).
2.9. Application of β-galactosidase enzyme 2.9.1. Hydrolysis of cheese whey lactose
Lactose from the "coalho" cheese whey (10 g/L) was used for the hydrolysis experiments. The hydrolysis of lactose was performed in order to compare the immobilized enzyme and crude enzyme extract. Cheese whey aliquots (1 mL) were added to test tubes with
either 0.1 g of alginate-immobilized β-Gal or 1 mL of crude enzyme extract, at pH 6.0, 40 °C and 100 rpm. Samples were collected in triplicate after 0, 120, 240 and 360 minutes of hydrolysis. For inactivation of the β-Gal enzyme, the samples were subjected to heating in a water bath at 100 °C for 5 minutes [11]. The lactose and glucose contents after hydrolysis were evaluated by High-performance Liquid Chromatography (HPLC; model Ultimate 3000, Thermo Fisher Scientific, USA) using a Shim-Pack column (model SCR-101H, Shimadzu, Japan) with refraction index detector (RID), at 40 °C. Sulfuric acid 0.005 M was used as a mobile phase, under flow of 1.0 mL/min. All samples were filtered through 0.22 µm membrane before analysis [11,18].
2.9.2. Stability of β-galactosidase in simulated gastrointestinal conditions
The stability of the immobilized enzyme under simulated gastric and intestinal conditions was performed using the methodology described by [26]. For simulation of gastric conditions, 0.2 g of immobilized enzyme was incubated with 2 mL of simulated gastric fluid (SGF, pH 2.0; 2.0 g sodium chloride, 3.2 g pepsin, 7.0 mL HCl 37%, for 1000 mL) at 37 °C, 100 rpm. For intestinal conditions, 0.2 g of immobilized enzyme was incubated with 2 mL of 2 mL of simulated intestinal fluid (SIF, pH 7.5; 6.8 g monobasic potassium phosphate, 10 g pancreatin, 190 mL NaOH 0.2 N, for 1000 mL), at the same previous conditions. Samples were taken after 5, 10, 30, 45, 60 and 120 minutes and enzymatic activity was evaluated [27].
2.10. Statistical analysis
All experiments were performed as three independent replicates (n = 3). Statistical analysis was performed using the software Statistica v. 8.0 (TIBCO Statistica, Palo Alto, CA, USA). One-way ANOVA combined with Tukey HSD post hoc test was applied to establish statistical significance (p < 0.05).
3. Results and discussion
The results regarding the immobilization conditions of the β-gal enzyme in relation to the different concentrations of sodium alginate are shown in Table 1.
Table 1: Immobilization parameters of β-gal enzyme for the different concentrations of sodium
alginate Sodium alginate (% w/v) Y (%) rar (%) EI (%) 0,5 66.95 ± 1.48 a 17.40 ± 0.51 c 39.54 ± 0.02 c 0,7 17.98 ± 0.26 c 73.28 ± 0.11 a 45.71 ± 0.09 b 1,0 41.77 ± 0.23 b 65.02 ± 0.10 b 66.03 ± 0.10 a
Where Y – Yield, ar – recovered activity and EI -immobilization efficiency, respectively. Mean values ± Standard deviation. Different letters (a, b, c) in the same column indicate statistical differences (p < 0.05) according to Tukey’s test.
The enzyme concentration for alginate ratio was established at 2.6 U/g of granules. For the three concentrations of sodium alginate tested, the granules obtained at 0.7% sodium alginate (w/v) presented the highest recovered activity (73.28 ± 0.11%). On the other hand, when the three key parameters (Y, EI, ar) are taken all together, the granules obtained at 1.0%
sodium alginate (w/v) presented better higher EM (66.03 ± 0.10%) with low reduction of Y (41.77 ± 0.23%) and ar (65.02 ± 0.10%).
Shen et al. [28] developed a hybrid matrix of sodium alginate-gelatin-calcium phosphate for immobilization of the β-gal enzyme of K. lactis. The authors observed a lower relative activity of the enzyme immobilized with the hybrid matrix (58.6%) when compared to the control matrix with only sodium alginate (62.3%). These results were attributed by the authors
to problems of mass transfer caused by the calcium phosphate layer and gelatin in the immobilization matrix, which indicates the correct choice of support for this research.
Freitas et al. [29] investigated the development of a biocatalysts system using β-gal of
K. lactis immobilized in sodium alginate-gelatin-cross-linking with glutaraldehyde, and
observed that the immobilized enzyme retained 80% of its initial activity after 25 cycles of use. In addition, the hydrolysis of lactose using 10 and 100 g/L of substrate did not present inhibition by the product for the immobilized enzyme.
In a study conducted by Souza et al. [12] using a polysaccharide complexed with sodium alginate for immobilization of β-gal, the authors obtained 69.4% of recovered activity, very similar to the results presented here. Souza et al. [15] observed in their study that the formation of the alginate complex and β-gal promoted changes in the enzyme structure, however the conformational modification was reversible after the dissociation of the complex which allowed the enzyme to recover its activity. This fact may contribute to expanding the functional applications of enzymatic immobilization.
3.1. Effect of pH value, temperature and metallic ions on stability of immobilized β-galactosidase
The immobilization of β-gal in sodium alginate was performed with the objective of increasing enzymatic activity, enzyme stability and reduction of operation costs by allowing the recycle use [13]. Some factors such as temperature, pH, presence and concentration of metal ions may influence the activity of the enzyme β-Gal [30]. In the present study, the stability of the β-Gal enzyme immobilized with alginate was evaluated in different pH values, temperature and in the presence of several metal ions. 1 shows that the complex enzyme/support showed higher stability in the pH range between 5.0-7.0, with the highest relative activity obtained at pH 5.0 remaining stable for 30 and 60 minutes, conditions in which the enzyme maintained an
average of 105.50 % ± 0.25 of the relative activity. In addition, the immobilized form of the enzyme presented higher values for relative activity when compared to the same pH range in the free enzyme.
Albuquerque et al. [31] analyzed the stability of the enzymatic activity of β- galactosidase immobilized with agarose at pH 4.5, 7.0 and 9.0. In the immobilized enzyme the pH 9.0 was less stable when compared to the free enzyme. On the other hand, when the immobilized form of the enzyme in solutions with pH 5.0 or 7.0 was clearly more stable than the free enzyme.
Figure 1: Stability of the enzymatic residual activity of immobilized β-galactosidase with
sodium alginate at different pH values. Mean values ± Standard deviation. Different low case letters (a,b,c) indicate statistical difference (p < 0.05) for different pH values at the same incubation time according to Tukey’s test. Different capital letters (A, B, C) indicate statistical difference (p < 0.05) for different incubation times at the same pH according to Tukey’s test.
It was observed that of higher pH values (8-10), there was a significant reduction of the enzymatic activity. Depending on the nature and charges on the surface of the immobilization matrix, and the source of the enzyme, the optimum pH is changed between free and immobilized forms [32]. According to Vasileva et al. [33], the optimum immobilization pH for one enzyme depends on the chosen immobilization method and the type of matrix used. The optimum pH can be maintained or displaced to a more basic or more acidic region in relation to the free enzyme, this change in the profile of the pH curve occurs by the unequal distribution of charges in the microenvironment of the immobilized enzyme form. This modification may occur by the introduction of negative charges, such as amine groups, promoting displacement to the acidic pH range, or carboxyl groups that promote this displacement to the basic range.
When it comes to the effect of temperature on the stability of the immobilized enzyme, it was observed that the highest stability was obtained at 50 ºC for 30 minutes, with a relative activity of 180.0 ± 1.37% (Figure 2). According to Sousa Junior et al. [34] there is a strong dependence on the stability of the immobilized enzyme with the temperature. For temperatures of 55, 57 and 60 ºC, the authors reported the results similar to this work. Therefore, it is clear that the immobilization strategy also favors the increase of enzyme stability against temperature.
According to Souza et al. [18] the denaturation of the immobilized enzyme is less observed because of the protective effect credited to the increased structural stiffness of the enzyme, thus promoting the protection of amino acids in the active site, as well as on the surface.
In a study produced by Freitas et al. [35] on the immobilization of the β-gal enzyme of
Aspergillus oryzae in sodium alginate-reticulated gelatin with glutaraldehyde, it was observed
that the optimal enzymatic activity of the immobilized enzyme increased from 55 ºC to 60 ºC, and the immobilized form also presented higher tolerance to high temperatures. In addition, in
studies conducted by El-Masry et al. [36], the authors observed that the change of stability to higher temperatures indicates that the immobilization process provides a higher resistance against thermal inactivation, preserving the structure of the enzyme when compared with the free form, which was also observed in this study.
Figure 2: Stability of the enzymatic residual activity of immobilized β-galactosidase with
sodium alginate at different temperatures. Mean values ± Standard deviation. Different low case letters (a, b, c) indicate statistical difference (p < 0.05) for different temperatures at the same incubation time according to Tukey’s test. Different capital letters (A,B,C) indicate statistical difference (p < 0.05) for different incubation times at the same temperature according to Tukey’s test.
As for the effect of metallic ions on the stability of the immobilized β-Gal, Figure 3 shows that during the incubation time of 60 minutes the enzyme was stable for most of the ions tested. A pronounced reduction was observed only when the enzyme/support complex was in contact with the Co2+ and Cu2+ ions with a relative activity of 59.82 ± 0.93% and 52.29 ± 0.13%.In contrast, in the presence of Ag+, Mg2+ and Fe2+ there was a significant increase in the
relative activity in the immobilized enzyme exceeding the percentages between 144.75 ± 1.53%, 154.59 ± 0.60% and 158.90 ± 1.25% in 2 hours of incubation.
Figure 3: Stability of the enzymatic residual activity of immobilized β-galactosidase with
sodium alginate at different metallic ions. Mean values ± Standard deviation. Different low case