Scholarly article on topic 'Soybean β-Glucosidase immobilisated on chitosan beads and its application in soy drink increase the aglycones'

Soybean β-Glucosidase immobilisated on chitosan beads and its application in soy drink increase the aglycones Academic research paper on "Industrial Biotechnology"

0
0
Share paper
OECD Field of science

Academic research paper on topic "Soybean β-Glucosidase immobilisated on chitosan beads and its application in soy drink increase the aglycones"

Vol.57, n.5: pp. 766-773, September-October 2014 BRAZILIAN ARCHIVES OF

http://dx.doi.org/10.1590/S1516-8913201402331 ^«w « n.^. ^«w

p xSSN 1516-8913 Printed in Brazil BIOLOGY AND TECHNOLOGY

AN INTERNATIONAL JOURNAL

Soybean P-Glucosidase Immobilisated on Chitosan Beads and its Application in Soy Drink Increase the Aglycones

Luciana Carvalho Grade1, Amanda Aleixo Moreira1, Geni da Silva Varea1, José Marcos

Gontijo Mandarino , Josemeyre Bonifácio da Silva , Elza Iouko Ida and Mara Lúcia Luiz

Ribeiro

'Departamento de Bioquímica e Biotecnologia; Universidade Estadual de Londrina. 2Departamento de Ciencia e Tecnologia de Alimentos; Universidade Estadual de Londrina; Londrina - Paraná - Brasil. 3Embrapa Soja; Londrina - Paraná - Brasil

ABSTRACT

The objective of this study was to investigate the immobilisation efficiency of soybean fi-glucosidase (181.6 U/mL; 23.8 mg protein/mL) on activated chitosan beads. Central Composite Rotational Design (CCDR) 23 was used and the application of immobilised enzyme in commercial soy drink was evaluated. The activation of chitosan beads was achieved with established 2.5% glutaraldehyde, pH 7.5, 8 h incubation time (6 h with agitation and 2 h without agitation) at 37°C. The highest immobilisation efficiency (%) of soybean fi-glucosidase on chitosan beads obtained was 37.74 U/mL and 18.84 mg protein/4 chitosan beads at pH 7.5 and 20 h coupling time of enzyme-matrix (7 h with agitation and 13 h without agitation) at 4°C. The immobilised enzyme incubated at 50°C, pH 5.5 resulted in 24% increase in the aglycones content in commercial soy drink after 60 min.

Key words: fi-glucosidase soybean, immobilisation, chitosan, aglycones

INTRODUCTION

^-glucosidases (^-D-glucopyranoside

glucohydrolases- E.C. 3.2.1.21) are enzymes that hydrolyze glycosidic bonds to release the non-reducing terminal glucosyl residues from glycosides and oligossaccharides. These enzymes are found widely in nature, can be synthetized by the plants or produced by the animals and microorganisms. Soy contains glucosidic isoflavones (daidzin, genistin and glycitin), which can be hydrolysed by ^-glucosidases and then releases the aglycones (daidzein, genistein and glycitein). Aglycones forms exhibit higher biological activity and are more metabolically that can be absorved faster in higher amounts than glycosides (Izumi et al. 2000). The isoflavones are

*Author for correspondence: maralucia.ribeiro@gmail.com

associated with the risk reduction or prevention of various diseases, such as breast cancer and prostate cancer (Liggins et al. 2000), osteoporosis, menopause symptoms (Levis et al. 2010), cardiovascular disease (Rimbach et al. 2008), improved memory (Lephart et al. 2002), estrogenic and antioxidant activity (Liu et al. 2010; Ma et al. 2010). Soy contains 2% of the aglycones in relation of the total isoflavones (Matsuura and Obata 1993). Thus, the application of endogenous ß-glucosidase and its immobilisation can be an alternative to promote the bioconversion of the glycosidic isoflavones to aglycones with the aim to produce soyfoods with higher aglycones content. The immobilisation of soybean ß-glucosidase and its application has not been described.

The application of immobilised enzymes is a strategy for continuous bioprocesses and can improve the efficiency of biotechnological processes with reduction in production costs. This process increases enzyme stability and facilitates the separation of the reaction product (Chang and Juang 2007; Su et al. 2010). Several chemical and physical methods are used for immobilisation of an enzyme and must be materials of readily available and abudant, inexpensive, easy to operate on large scale, show high retention capacity (Nagashima 1984).

The enzyme immobilisation efficiency depends on various factors such as pH, agitation time and time-out (Cao 2006; Shing 2009; Sheldon 2011). Support activation with glutaraldehyde is a simple and efficient method that improves the stability of the enzyme due to multipontuais linkage or among the subunits of the enzyme (Betancor et al. 2006). Chitosan is a suitable support for the enzymes immobilisation because it is biocompatible, available in various forms (gel, membrane, fiber and film), nontoxic, biodegradable, and resistant to chemical modifications (Yi et al. 2009; Gomathi et al. 2010). Therefore, chitosan has potential for application as biomaterial (Arnaud et al. 2010).

The objective of this study was to investigate the immobilisation of soybean p-glucosidase on chitosan and evaluated its application in commercial soy drink to increase the aglycone.

MATERIAL AND METHODS Material

Soybean cultivar BRS 213, developed at Empresa Brasileira de Pesquisa Agropecuâria, Embrapa/Soja, Londrina, Parana, Brazil, was used. The grains were ground in a knife grinder (100 mesh) (TE 631, Tecnal, Brazil) to obtain a finely granulometric flour. The soymilk was obtained from a local commercial centre.

Extration of P-glucosidase of soy cotyledon flour

The conditions for p-glucosidase extration were as described by Matsuura and Obata (1993) with some modifications. Sixty grams of soy cotyledon flour and 100 mM sodium phosphate buffer, pH 6.6, in a 1:10 proportion (w/v) were mixed and slowly agitated at 4°C for 1 h and then centrifuged at 4,000 xg at 4°C for 15 min. The supernatant was

acidified with 0.1 M HCl to pH 5.0, and the samples were centrifuged again under the same conditions. The supernatant obtained (extract crude) was precipitated by ammonium sulfate at 4°C according to Santos et al. (2013). The crude extract was first precipitated by ammonium sulfate at 4°C and 40% saturation. After centrifugation at 4,000 xg at 4°C for 15 min, ammonium sulfate was added to the supernatant until 85% saturation was achieved and centifuged again under the same conditions. The precipitateds were resuspended in a 50 mM citrate phosphate bufer, pH 5.0 and dialysed with the same buffer at 4°C for 14 h.

P-glucosidase activity

P-glucosidase activity was determined using p-nitrophenyl-p-D-glucopyranoside (p-NPG)

substrate according to the procedure described by Matsuura and Obata (1993). The standard curve of p-nitrophenol (0.04-0.32 ^mol) was prepared. One P-glucosidase activity unit (U) was defined as the amount of enzyme needed to liberate 1 ^mol of p-nitrophenol/min under the assay conditions. The soluble protein content was quantified using the method described by Lowry et al. (1951) with a bovine serum albumin standard solution (40-400 ^g/mL). The specific activity was determined as the relationship between the enzymatic activity and the protein content, which was expressed as U/mg.

Experimental design for the activation of chitosan beads with glutaraldehyde

Chitosan beads in the concentration of 1% glutaraldehyde (p/v) (Sigma-Aldrich) were prepared according to Kumar et al. (2009) and activated by incubation and agitation at 37°C. Central Composite Rotational Design (CCDR) with 23 factorial, six axial points and three replicates at the central point was used, with a total of 17 randomized experiments. Table 1 presents the code and the real levels of independent variables Xi (glutaraldehyde concentration, %), X2 (pH of wash buffer) and X3 (incubation time of activation system at 37°C). The response function Y1 (immobilisation efficiency of P-glucosidase on activated chitosan beads with glutaraldehyde, %) was evaluated according to mathematical model: Y — p0 + p1x1 + P2X2 + P3X3 + P11 X12 + P22 X22 + P33 X32 + P12 X1X2 + p13 x1x3 + p23 x2x3 + e (Equation 1) where: Y = response function, Xi, X2 e X3 — levels of coded variables, P = estimated coefficients on the

response surface and e = pure error. The response function (immobilisation efficiency, %) was calculated as a relationship between the specific activity of immobilised and specific activity of free enzyme multiplied by 100.

Table 1 - Coded and real levels of independents variables used in a Central Composite Rotational Design (CCRD) 23 to activation of chitosan beads with

Variables Levels

-1.68 -1 0 +1 +1.68

X1 = glutaraldehyde 0.0 1.7 2.5 4.2 5.0

concentration (%)

X2 = pH (wash buffer) 5.0 5.5 6.5 7.5 8.0

X3 = incubation time 0/8 2/6 4/4 6/2 8/0

at 37°C*

* agitation time (h)/time without agitation (h).

Experimental design for the immobilization of P-glucosidase on activaded chitosan beads with glutaraldehyde

Soybean p-glucosidase immobilisation was conducted after determining the conditions of activation of the chitosan beads with glutaraldehyde using a new Central Composite Rotational Design (CCDR) with 23 factorial, six axial points and three replicates at the central point with a total of 17 randomized experiments. Table 2 presents the code and the real levels of independent variables X4 (protein content (mg)/4 activated chitosan beads with glutaraldehyde), X5 (pH of wash buffer) and X6 (linkage time between enzyme and activated chitosan at 37°C). The response function Y2 (immobilisation efficiency (%) of p-glucosidase on activated chitosan beads with glutaraldehyde to 2.5%) was evaluated according to mathematical model expressed in the Equation 1. The response function Y2 (immobilisation efficiency, %) also was calculated as a relationship between specific activity of immobilised and specific activity of free enzyme multiplied by 100.

Statistical analysis

After analysing the response functions (Yi and Y2), the analysis of variance (ANOVA) of the regressions and the coefficients of determination (R2) were determined and used to compare the fit of the model by the experimental data. Response surface graphs were generated for each response function evaluated. All the analyses were carried

out and all the graphs were created using Statistica 7.0 software, version 4.0 (Statsoft, Inc., 2004, Tulsa, USA).

Commercial soy drink with immobilised soybean P-glucosidase

Commercial soybean drink (1.0 mL) and two chitosan beads with immobilised soybean p-glucosidase were incubated at 50°C, pH 5.5 for 15, 20, 30, 45 and 60 min, then the commercial soy drink was filtered for the analysis. Isoflavones content were quantified by High Performance Liquid Chromatography (HPLC).

Table 2 - Coded and real levels of independents

variables used in a Central Composite Rotational

Design (CCRD) 23 to soybean p-glucosidase immobilisation.

Variables Levels

-1.68 -1 0 +1 +1.68

X4 = protein content 6.0 10.0 15.0 20.0 24.0

(mg)/ chitosan beads*

X5 = pH (wash buffer) 5.0 5.5 6.5 7.5 8.0

X6 = linkage time at 0/20 7/13 10/10 13/7 20/0

4°C**

*4 activated chitosan beads with 2.5% glutaraldehyde. **agitation time (h)/time without agitation (h).

Determination of isoflavones content by HPLC

The isoflavones were extracted according to Carrao-Panizzi et al. (2002) and quantified by the method of Berhow (2002) by HPLC (Model 2690, Waters, USA) with a reverse phase column ODS C18 (YMC-Pack ODS-AM S-5 ^m, with a diameter of 4.6 mm and length 250 mm) and a diode array detector (model 996, Waters, USA) adjusted to a wavelength of 254 nm. A linear binary gradient system with methanol, trifluoroacetic acid and ultrapure deionized water was used for separation. The initial gradient was 20%, reached 80% at 35 min and returned to 20% at 40 min. The mobile phase flow rate was of 1.0 mL/min, and the temperature during the race was kept constant at 25°C. Quantitation was performed with the external standard calibration curves of daidzin, genistin, glycitin, daidzein, genistein, glycitein, malonyldaidzin, malonylgenistin, malonylglycitin, acetyldaidzin, acetylgenistin and acetylglycitin purchased by Sigma Chemicals Co. (St. Louis, E.U.A.), and the results were expressed as ^g isoflavones/ mL of commercial soy drink.

RESULTS AND DISCUSSION

The extract obtained from the cotyledon soybean flour with p-glucosidase activity of 181.6 U/mL and the protein content of 23.8 mg/mL was used as enzyme source for immobilization. Chitosan beads activation with glutaraldehyde increased significantly (p < 0.05) the immobilisation efficiency of soybean P-glucosidase. The high immobilisation efficiency (Y1 — 41.55%) was observed in seven assays (Table 3) with 1.7% glutaraldehyde, pH 7.5 and 8 h incubation time (6 h with agitation and 2 h without agitation). This result was 11.5 times higher than the efficiency obtained for un-activated immobilised enzyme active. However, at 4.2 and 5.0% glutaraldehyde,

a descrease in the immobilisation efficiency (22 and 44%, respectively) was observed. The increase in the concentration of glutaraldehyde increases the amount of free aldehyde groups on the support surface (Hua et al. 2009), promoting the immobilisation of the enzyme. However, the gradual increase of the concentration of glutaraldehyde can produce excessive crosslinking, which may cause aggregation, precipitation, distortion in the three-dimensional structure of the enzyme and decreased activity. In addition, at high concentrations of glutaraldehyde, the chitosan beads become fragile and brittle and the immobilisation efficiency of the enzyme decreases (Kumar et al. 2009).

Table 3 - Central Composite Rotational Design (CCRD) 2 with independent variables and the response function

Yj and Y2.

Experiments X1 X2 X3 Y1 X4 X5 X6 Y2

1 1.7 5.5 2/6 10.59 10.0 5.5 7/13 32.60

2 4.2 5.5 2/6 22.70 20.0 5.5 7/13 53.48

3 1.7 7.5 2/6 32.66 10.0 7.5 7/13 70.27

4 4.2 7.5 2/6 39.10 20.0 7.5 7/13 73.75

5 1.7 5.5 6/2 12.19 10.0 5.5 13/7 11.46

6 4.2 5.5 6/2 32.56 20.0 5.5 13/7 20.64

7 1.7 7.5 6/2 41.55 10.0 7.5 13/7 22.90

8 4.2 7.5 6/2 32.26 20.0 7.5 13/7 45.37

9 0.0 6.5 4/4 3.62 6.0 6.5 10/10 17.57

10 5.0 6.5 4/4 23.39 24.0 6.5 10/10 60.15

11 2.5 5.0 4/4 11.88 15.0 5.0 10/10 13.65

12 2.5 8.0 4/4 31.64 15.0 8.0 10/10 22.72

13 2.5 6.5 0/8 25.03 15.0 6.5 0/20 46.10

14 2.5 6.5 8/0 33.99 15.0 6.5 20/0 39.41

15 2.5 6.5 4/4 24.92 15.0 6.5 10/10 55.33

16 2.5 6.5 4/4 26.06 15.0 6.5 10/10 57.70

17 2.5 6.5 4/4 26.78 15.0 6.5 10/10 52.65

Y1 (immobilisation efficiency of P-glucosidase on activated chitosan beads with glutaraldehyde, %), Y2 [immobilization efficiency (%) of immobilised P-glucosidase on activated chitosan beads with glutaraldehyde 2.5%], X1 (glutaraldehyde, %), X2 (pH), X3 (incubation time, h at 37°C), X4 (protein content/4 chitosan beads activated with glutaraldehyde 2.5%), X5 (pH) e X6 [coupling time: agitation time (h)/time without agitation (h) at 4°C]

The pH variation also significantly influenced (p < 0.05) the chitosan beads activation with glutaraldehyde. The immobilisation efficiency increased from 10.6 to 41.6% at pH from 5.5 to 7.5 at 1.7% glutaraldehyde. The pH variation can increase the amount of amino groups on the surface of chitosan, favoring the binding of glutaraldehyde and therefore, increasing the immobilisation efficiency (Dwevedi and Kayastha 2009).

The agitation of the immobilization system during the incubation time at 37°C had a significant effect

(p < 0.05) on the chitosan beads activation with glutaraldehyde. The immobilisation efficiency increased from 22.7 to 32.56% with the increase of agitation time 2 to 6 h. Thus, the relationship between the incubation time with and without agitation of the immobilisation system was important in the activation process of the support. The highest agitation time advance the contact of glutaraldehyde with chitosan beads. According to regression parameters, independent variables X1 (glutaraldehyde concentration, %) (P1 — 4.61), X2 (pH) (p2 — 7.38) and X3 (agitation time

of immobilisation system, h) (p3 = 2.09) showed a significant linear positive effect at a 5% significance level on the response function Yj (immobilisation efficiency of p-glucosidase on chitosan beads activated with gluctaraldehyde). The quadratic effect of variable Xj (Pj2 = - 2.92), X3 (P32 = - 2.76) and the interation XjX2 (pap2 = -4.42) had also significant effects on the response function Yj. However, the quadratic effect of variable X2 (p22 = 0.01) and interation XjX3 (PiP3 = - 0.93) and X2X3 (p2p3 = - 1.18) were not significant at a 5% significance level. Thus, considering only the significant variables, the following mathematical model was developed: Y1 = 25.48 + 4.61 x1 - 2.92 x12 + 7.38 x2 + 2.09 x3 +

2.76 x3 - 4.42 x1x2. The lack-of-fit of the model was significant (95%) and 86% (R2) of the experimental data was properly adjusted to the model. The significant lack-of-fit should not be considered relevant when the mean square of the pure error was low (1.759) (Box and Drapper 1987).

The response surface (Fig. 1) showed a region where the chitosan beads activation reflected on immobilisation efficiency. For the interaction between pH and glutaraldehyde, the ranges were 7.5- 8.5 and 1.7-4.0% (Fig. 1A) and for the interaction glutaraldehyde concentration and incubation time, the ranges were 1.7-4.0% and 7-8 h (Fig. 1B).

Figure 1 - Model of response surface for glutaraldehyde (%) and pH (A) and glutaraldehyde (%) and incubation time (h) (B) on the immobilisation efficiency of soybean p-glucosidase on chitosan beads activated with glutaraldehyde (Y1).

After establishing the optimum conditions for chitosan beads with glutaraldehyde (2.5% glutaraldehyde, pH 7.5 and 8 h incubation time with agitation), the variables protein content (mg) per support (X4), pH wash buffer (X5) and coupling time of enzyme on the matrix at 4°C (X6) were investigated.

The higher immobilisation efficiency (%) (Y2) on the chitosan beads activated with 2.5% glutaraldehyde was observed in the 4th assay (73.75%) (Table 3) with 20 mg of protein, pH 7.5 and coupling time of 20 h incubation time (7 h with agitation and 13 h without agitation). Reducing the pH from 7.5 to 5.5 and maintaining the other variables, the immoblisation efficiency

decreased to 27%. The increase of agitation time from 7 to 13 h decreased it to 39%. With 10 mg of protein, pH 5.5 and 13 h of agitation time, 6.5 times reduction in the immobilisation efficiency (%) were achieved. These results suggested the importance of the pH and agitation time on the immobilisation efficiency. The variable X4 (protein content per support, mg) (p4 = 9.34), X5 (pH wash buffer) (p5 = 8.00) and X6 (coupling time enzyme-matrix at 4°C (p6 = 10.32) showed a significant linear positive effect at a 5% significance level on the response function Y2 (immobilisation efficiency (%) on the chitosan beads activated with 2.5% glutaraldehyde). The quadratic effect of variable X4 (p4 = - 3.71) and

X5 (p5 — - 11.01) were significant and negative while the quadratic effect of variable X6 (p6 — -10.32) and of interation (X4, X5, X6) were not significant.

Considering only the significant variables, the following mathematical model was developed: Y2 = 54.62 + 9.34 x4 - 3.71 x42 + 8.00 x5 - 11.01 x52 -10.32 x6. The lack-of-fit was not significant (at 95%) and 77% (R2) of the experimental data was properly adjusted to the model. The response surface (Fig. 2) showed a region where Y2 was maximal. Therefore, the ranges of pH and protein content established were 7.5-8.5 and 20-24 mg (Fig. 2A) and pH and coupling time enzyme-matrix as 7.5-8.5 and 7 h with agitation and 13 h without agitation (Fig. 2B). There were no reports on the immobilization of soybean p-glucosidase. However, an efficiency of 48.2% p-glucosidase immobilisation has been reported from almonds on chitosan activated with

0.5% glutaraldehyde (Chang and Juang 2007). Immobilisation of mature seeds soybean urease and of pea p-galactosidase on chitosan activated with 1.0% glutaraldehyde resulted in 65 and 67% efficiency, respectively (Kumar et al. 2009; Dwevedi and Kayastha 2009). These differences in immobilisation efficiencies could be attributed to the diversity of enzymes used and the type of interactions between the enzyme and matrix. Results showed that the soybean p-glucosidase immobilisation efficiency obtained in this work was higher than the others enzymes and matrix, e.g., a and y-alumina (15%) and cellulose (18%) (Martino et al. 1996), Eupergit C (30%) (Tu et al. 2006), chitosan (25.1%) and alginate (31.4%) (Su et al. 2010). Thus, the the present results could be considered satisfactory, because it was possible to obtain 73.75% immobilisation efficiency. The soybean p-glucosidase immobilisation efficiency increased from 41.6 to 73.8%.

Figure 2 - Model of response surface for pH and protein content (A) and pH and coupling time enzyme-support (B) on the immobilisation efficiency of soybean ß-glucosidase on chitosan beads activated with glutaraldehyde 2.5% (Y2).

The P-glucosidase enzyme immobilised on chitosan beads activated with 2.5% glutaraldehyde was added in the commercial soy drink and isoflavones content were quantified (Table 4). The commercial soy drink without P-glucosidase enzyme immobilised showed 169.40 ^g/mL of total isoflavones content (109.30 ^g/mL P-glucosides, 27.90 ^g/mL malonyl-glucosides and

32.20 ^g/mL aglycones, respectively). After 60 min of incubation, an increase of 24% in the aglycones content was obtained, which was probably due the p-glucoside activity that hydrolyzed the p-glucosides forms in aglyconas. Thus, the addition of p-glucosidase enzyme immobilised in the commercial soy drink increased the aglycones content. No reports about

soybean p-glucosidase immobilisation and on the addition in soy drinks were found. Chen et al. (2013) reported that p-glucosidase of Aspergillus niger immobilised on spent coffee grounds was capable of catalysing the hydrolysis of isoflavones in black soymilk and aglycones content increased from 13.29 to 69.72% after 60 min of application of the enzyme.

Table 4 - Isoflavones content (^g de isoflavonas/mL) in commercial soy drink without and with soybean p-glucosidase immobilised on chitosan beads activated with 2.5% glutaraldehyde*._

Incubation Glycosides Malonyl- Aglycones

time (min) glycosides

0 109.30a 27.90bc 32.20d

15 100.20c 27.50c 31.70d

20 100.50bc 27.90bc 34.00c

30 102.40b 29.30a 35.30bc

45 98.20d 29.20a 36.10b

60 88.70e 26.10d 40.00a

*values obtained are an average of two repeats. Means followed by equal letters in the same column do not differ by Tukey's test at 5% probability.

CONCLUSION

The highest immobilisation efficiency (%) of soybean p-glucosidase on chitosan beads activated with 2.5% glutaraldehyde was obtained with 18.84 mg protein/mL and 37.74 U/mL per 4 chitosan beads at pH 7.5 and coupling time of enzymematrix of 7 h with agitation and 13 h without agitation at 4°C. After incubation with immobilised enzyme, the commercial soy drink showed an increase of 24% in the aglycones contents after 60 min.

ACKNOWLEDGMENTS

This work was partially funded by CNPq/MCT and Funda^ao Araucária/PR - Chamada PRONEX and Grade, L.C would like to thank Funda^ao CAPES/MEC for a graduate scholarship.

REFERENCES

Arnaud TMS, Barros-Neto B, Diniz FB. Chitosan effect on dental enamel de-remineralization: An in vitro evaluation. J Dent. 2010; 38: 848-852.

Berhow MA. Moderm analytical techniques for flavonoid determination. In: Buslig BS, Manthey JA, editor. Flavonoids in the living cell. New York: Klusher Academic; 2002. p. 61-76.

Betancor L, Lopez-Gallego F, Hidalgo A, Alonso-Morales N, Mateo GDC, Fernandez-Lafuente R, et al. Different mechanisms of protein immobilization on glutaraldehyde activated supports: effect of support activation and immobilization conditions. Enzyme Microb Technol. 2006; 39: 877-882.

Box GEP, Draper NR. Empirical Model Building and Response Surfaces. 1. ed. New York: Wiley, 1987, 688p.

Cao L. Immobilized enzymes: past, present and prospects. In: Carrier-bound immobilized enzymes: principles, application and design. Weinheim: Wiley; 2006. p. 1-52.

Carrao-Panizzi MC, Favoni SPG, Kikuchi A. Extraction time for soybean isoflavone determination. Braz Arch Biol Technol. 2002; 45: 515-518.

Chang M, Juang R. Use of chitosan-clay composite as immobilization support for improved activity and stability of ß-glicosidase. Biochem Eng J. 2007; 35: 93-98.

Chen KI, Lo YC, Liu CW, Yu RC, Chou CC, Cheng KC. Enrichment of two isoflavones aglycones in black soymilk by using spent coffee grounds as an immobilizer for beta-glucosidase. Food Chem. 2013; 139: 79-85.

Dwevedi A, Kayastha AM. Optimal immobilization of ß-galactosidase from Pea (PsBGAL) onto Sephadex and chitosan beads using response surface methodology and its applications. Bioresour Technol. 2009; 100: 2667-2675.

Gomathi P, Ragupathy D, Choi JH, Yeum JH, Lee SC, Kim JC, et al. Fabrication of novel chitosan nanofiber/gold nanoparticles composite towards improved performance for a cholesterol sensor. Sensor Actuat B-Chem. 2010; 153: 44-49.

Hua F, Jun H, Liyun D, Mingtian L, Zhao C J. Preparation of magnetic chitosan nanoparticles and immobilization of laccase. J Wuhan Uni Technol. 2009; 24: 42-47.

Izumi T, Piskula MK, Osawa S, Obata A, Tobe K, Saito M, et al. Soy isoflavone aglycones are absorbed faster and in higher amounts than their glucosides in humans. JNutr. 2000; 130: 1695-1699.

Kumar S, Dwevedi A, Kayastha AM. Immobilization of soybean (Glycine max) urease on alginate and chitosan beads showing improved stability: analytical applications. J Mol Catal B:Enzym. 2009; 58: 138145.

Lephart ED, West TW, Weber KS, Rhees RW, Setchell KDR, Adlercreutz H, et al. Neurobehavioral effects of dietary soy phytoestrogens. Neurotoxicol Teratol. 2002; 24(1): 5-16.

Levis S, Strickman-Stein N, Doerge DR, Krischer J. Design and baseline characteristics of the Soy Phytoestrogens As Replacement Estrogen (SPARE) study - A clinical trial of the effects of soy isoflavones in menopausal women. Contemp Clin Trials. 2010; 31: 293-302.

Liggins J, Bluck LJC, Runswick S, Atkinson C, Coward WA, Bingham SA. Daidzein and genistein contents of vegetables. Brit J Nutr. 2000; 84: 717725.

Liu Z, Kanjo Y, Mizutani S. A review of phytoestrogens: Their occurrence and fate in the environment. Water Res. 2010; 44: 567-577.

Lowry OH, Rosenbrough NJ, Farr AL, Randal RJ. Protein measurement with the folin phenol reagent. J Biol Chem. 1951; 193: 265-275.

Ma W, Yuan L, Yu H, Ding B, Xi Y, Feng J, et al. Genistein as a neuroprotective antioxidant attenuates redox imbalance induced by ß-amyloid peptides 2535 in PC12 cells. Int J Dev Neurosci. 2010; 28: 289295.

Martino A, Durante M, Pifferi PG, Spagna G, Bianchi G. Immobilization of ß-glucosidase from a commercial preparation. Part 1. A comparative study of natural supports. Process Biochem. 1996; 31: 281285.

Matsuura M, Obata A. ß-glucosidases from soybeans hydrolyze daidzin and genistin. J Food Sci. 1993; 58: 144-147.

Nagashima M. Continuous ethanol fermentation using immobilized yeast cells. Biotechnol Bioeng. 1984; 21: 49-58.

Rimbach G, Boesch-Saadatmandi C, Frank J, Fuchs D, Wenzel U, Daniel H, et al. Dietary isoflavones in the prevention of cardiovascular disease - A molecular perspective. Food Chem Toxicol. 2008; 46: 13081319.

Santos RF, Oliveira CF, Varea GS, Corradi da Silva MLC, Ida EI, Mandarino JMG, et al. Purification and characterization of soy cotyledons beta-glucosidase. J Food Biochem. 2013; 37: 302-312.

Sheldon RA. Characteristics features and biotechnological applications of cross-linked enzyme aggregates (CLEAs). Appl Microbiol Biotecnol. 2011; 92: 467-477.

Shing BD. Biotechnology expanding horizons. 1ed. India: Kalyani Publishers, 2009.

Su E, Xia T, Gao L, Dai Q, Zhang Z. Immobilization of ß-glucosidase and its aroma-increasing effect on tea beverage. FoodBioprodProcess. 2010: 83-839

Tu M, Zhang X, Kurabi A, Gilkes N, Mabee W, Saddler I. Immobilization of b-glucosidase on Eupergit C for lignocellulose hydrolysis. Biotechnol Lett. 2006; 28: 151-156.

Yi S, Noh J, Lee Y. Amino acid modified chitosan beads: improved polymers supports for immobilization of lipase from Candida rugosa. J Mol CatalB: Enzym. 2009; 57: 123-129.

Received: August 26, 2013; Accepted: April 22, 2014.