Accepted Manuscript

Immobilization of pectinases into calcium alginate microspheres for fruit juice

application

Ziyu Deng, Fang Wang, Bin Zhou, Jing Li, Bin Li, Hongshan Liang

PII: S0268-005X(18)31045-2
DOI: https://doi.org/10.1016/j.foodhyd.2018.11.031
Reference: FOOHYD 4772

To appear in: Food Hydrocolloids

Received Date: 7 June 2018

Revised Date: 13 November 2018
Accepted Date: 13 November 2018

Please cite this article as: Deng, Z., Wang, F., Zhou, B., Li, J., Li, B., Liang, H., Immobilization of
pectinases into calcium alginate microspheres for fruit juice application, Food Hydrocolloids (2018), doi:
https://doi.org/10.1016/j.foodhyd.2018.11.031.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.
 ACCEPTED MANUSCRIPT
Graphical Abstract

PT
RI
U SC
AN
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT

1 Immobilization of pectinases into calcium alginate microspheres

2 for fruit juice application

3 Ziyu Deng1 a,b, Fang Wang 1 a,b, Bin Zhou d, Jing Li a,b, Bin Li a,b,c, Hongshan Liang a,b*

a
4 College of Food Science and Technology, Huazhong Agricultural University,

PT
5 Wuhan 430070, China

RI
6 Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural

7 University), Ministry of Education, China

SC
c
8 Functional Food Engineering & Technology Research Center of Hubei Province,

U
9 China
AN
d
10 School of Food and Biological Engineering, Hubei University of Technology,

11 Wuhan 430068, China

M

1
12 Contributed equally to this work as co-first authors.
D

13 *Corresponding author: Hongshan Liang

TE

14 E-mail address: lianghongshan@mail.hzau.edu.cn

15
EP

16
C

17
AC

18

19

20

21

1
 ACCEPTED MANUSCRIPT

22 Abstract:

23 In this study, polygalacturonase (PG) was immobilized in calcium alginate

24 microspheres prepared using endogenous emulsification method and its application in

25 apple juice clarification was evaluated. Preparation conditions of calcium alginate

PT
26 microspheres prepared by endogenous emulsification were optimized, additionally,

RI
27 the formed microspheres were characterized. Under optimal conditions，microspheres

28 with the particle size of 38.0 ± 0.2 µm showed good sphericity, storage stability and a

SC
29 narrow particle size distribution. Meanwhile, single factor experiment and response

U
30 surface experiment were combined to get the optimum conditions of immobilizing PG.
AN
31 The immobilized enzyme presented the optimum temperature at 60.0 °C, which was

32 almost 10.0 °C higher than the free PG and the activity was less dependent upon pH
M

33 within the interval of 3.0-4.5 than free enzyme, supporting the immobilized enzyme
D

34 more stable in apple juice clarification (pH=4.3±0.1). The immobilized enzyme also
TE

35 exhibited good operational stability and 63.0% of its initial activity retained after

36 three batch reactions. In the present of the immobilized PG, the turbidity of apple
EP

37 juice decreased, as well as apple juice had good thermal and freezing ability.
C

38 Keywords: Calcium alginate microspheres, endogenous emulsification,

AC

39 polygalacturonase, immobilization.

40

41

42

2
 ACCEPTED MANUSCRIPT

43 1. Introduction

44 One of the main problems encountered in the preparation of industrially processed

45 fruit-based beverages is sometimes undesirable turbidity of the final product (Siebert,

46 2006). Immediate turbidity in freshly produced juice is led by suspension of

PT
47 polysaccharide particulates (pectin, cellulose, hemicellulose, starch and lignin) arising

RI
48 from the primary and inner cell wall (Sorrivas, Genovese, & Lozano, 2006).

49 Particularly, pectin causes turbidity and undesired solid suspensions in fruit juices,

SC
50 and commercial production of fruit juices often utilizes pectinases to degrade and

U
51 remove pectin (DiCosimo, McAuliffe, Poulose, & Bohlmann, 2013; Sharma, Patel, &
AN
52 Sugandha, 2017; Tapre & Jain, 2014). Hence, commercial pectinases enzymes play an

53 important role in fruit juice technology and exert a favorable effect in improving the
M

54 yield, clarity and filterability of juice (Khandare, Walia, Singh, & Kaur, 2011; Sandri,
D

55 Lorenzoni, Fontana, & da Silveira, 2013; Uzuner & Cekmecelioglu, 2015).

TE

56 In spite of high catalytic properties of pectinase, free enzymes always present some

57 drawbacks such as poor stability under operational conditions, impossibility of

EP

58 multiple reuses in an industrial process, the presence of compounds arising from

C

59 enzyme preparation in the final food product (Garcia-Galan, Berenguer-Murcia,

AC

60 Fernandez-Lafuente, & Rodrigues, 2011). Therefore pectinases immobilization arises

61 as a solution to the necessity of reusing the expensive enzymes in industrial processes

62 (Fang, Chen, Zhang, & Chen, 2016; Mateo, Palomo, Fernandez-Lorente, Guisan, &

63 Fernandez-Lafuente, 2007; Rajdeo, Harini, Lavanya, & Fadnavis, 2016; Rehman, et

3
 ACCEPTED MANUSCRIPT

64 al., 2013). Moreover, immobilization has been revealed itself as a powerful tool to

65 improve many enzyme properties, like stability, activity, selectivity or specificity,

66 resistance to inhibitors, etc (O. Barbosa, et al., 2015; O. Barbosa, et al., 2013;

67 Cipolatti, et al., 2016; Mateo, et al., 2007; Rodrigues, Ortiz, Berenguer-Murcia,

PT
68 Torres, & Fernandez-Lafuente, 2013).

RI
69 The immobilization of enzymes is usually carried out by three main methods (i)

70 covalent binding to supports (carrier), (ii)adsorption of enzyme molecules on a

SC
71 support material and (iii)entrapment or encapsulation of enzyme in polymers

U
72 (Oveimar Barbosa, et al., 2015; Cantone, et al., 2013; Fernandez-Lafuente, 2009;
AN
73 Mohamad, Marzuki, Buang, Huyop, & Wahab, 2015; Vaghari, et al., 2016). To date,

74 pectinase has been immobilized on various supports, like nylon, ion-exchange resins
M

75 and silk (DiCosimo, et al., 2013). However, sufficient improvement of pectinase

D

76 immobilization efficiency and stability has not been achieved (Li, Li, Wang, & Tain,
TE

77 2008).

78 Entrapment within insoluble calcium alginate microspheres has shown to be a most

EP

79 effective approach due to the biocompatibility, low cost, availability and resistance to
C

80 microbial contamination as compared to other methods (Rehman, et al., 2013;

AC

81 Sheldon & van Pelt, 2013; Zhao, et al., 2017). Water-in-oil (W/O) emulsion

82 cross-linking method has been used to prepare Ca-alginate microspheres (F. J. Zhang,

83 Cheng, Gao, & Li, 2006), which also can be divided into external emulsification and

84 endogenous emulsification (Sosnik, 2014). As for the external emulsification method,

4
 ACCEPTED MANUSCRIPT

85 microspheres generated were with poor performance (Chan, Lee, & Heng, 2002;

86 Chuah, Kuroiwa, Kobayashi, Zhang, & Nakajima, 2009; Rastogi, et al., 2007).

87 Compared with the external emulsification method, the endogenous emulsification

88 method uses a poorly soluble calcium salt, which can avoid clustering aggregation of

PT
89 microspheres and make the size of microspheres easy to control (Reis, Neufeld, Vilela,

RI
90 Ribeiro, & Veiga, 2008). However, current preparation methods for calcium alginate

91 microspheres still have a number of flaws (Hariyadi, et al., 2014; Martín, et al., 2015;

SC
92 Shi, Zheng, Zhang, & Tang, 2016; Silva, Ribeiro, Ferreira, & Veiga, 2006; Zou, et al.,

U
93 2011), including for example, poor range of particle size and uneven distribution
AN
94 (Chan, et al., 2002; Chuah, et al., 2009; Hwang & Gu, 2013; Rastogi, et al., 2007).

95 Up to now, using the calcium alginate microspheres are prepared by endogenous

M

96 emulsification method for polygalacturonase (PG) immobilizing has not been reported
D

97 to our best knowledge. In this study, firstly, the optimum conditions of preparing
TE

98 calcium alginate microspheres through the combination of single factor experiment

99 and orthogonal experiment were performed. The effect of different drying methods on
EP

100 the morphology of microspheres was investigated. Additionally, the formed

C

101 microspheres were characterized. Following this, response surface methodology was
AC

102 used to establish the optimum conditions for immobilizing PG on calcium alginate

103 microspheres to acquire the maximum recovery of enzyme activity. Finally, the

104 properties of immobilized enzymes, such as kinetic parameters, pH and temperature

5
 ACCEPTED MANUSCRIPT

105 profiles and operational stability, were investigated and compared with those of free

106 PG.

107 2. Materials and methods

108 2.1. Materials

PT
109 Calcium carbonate purchased from the United States Aladdin company Co., Ltd.

RI
110 Alginate was obtained from the Sinopharm Chemical Reagents Co., Ltd. (Shanghai,

111 China). Polygalacturonase from Aspergillus niger (1 U/mg, EC 3.2.1.15), pectin from

SC
112 citrus peel (Galacturonic acid > 74 % and 50-70 % esterification, P9135) and pectin

U
113 from apple (50-75 % esterification, 93854) were the product of Sigma (St. Louis, MI,
AN
114 USA). Other chemicals were all analytical grade and used without purification.

115 Aqueous solutions were prepared using ultrapure water through a Millipore (Millipore,
M

116 Milford, MA, USA) Milli-Q water purification system.

D

117 2.2. Preparation of alginate microspheres

TE

118 Alginate was dissolved in distilled water to a final concentration of 2.0%, 3.0%, 4.0%,

119 4.5%, 5.0% (mass percentage), gently stirred at room temperature for 30.0 min at
EP

120 4.0 °C refrigerator overnight degassed spare. Calcium carbonate suspension (5%, w/v)
C

121 was obtained by dissolving calcium carbonate powder in deionized water and
AC

122 ultrasonically dispersing for 15 min.

123 Take a certain concentration of sodium alginate solution and calcium carbonate was

124 mechanically stirred evenly according to a certain proportion. It was dispersed into a

125 paraffin oil phase containing Span 80, controlling the speed and stirring time to form

6
 ACCEPTED MANUSCRIPT

126 a W/O emulsion. A certain amount of liquid paraffin containing glacial acetic acid

127 was added to the emulsion to dissociate Ca2 + in CaCO3. Ca2+ interacted with sodium

128 alginate to form calcium alginate microspheres under a certain speed stirring

129 (Oveimar Barbosa, et al., 2014). 1.0% Tween 80 solution was added to the emulsion

PT
130 to demulsify, the emulsion was left overnight, and microspheres were separated from

RI
131 it. The oil phase was removed with ethanol at 50.0 °C and ethanol was removed by

132 water washing. Microspheres were then dried and stored for subsequent use (see

SC
133 below).

U
134 2.3. Drying of calcium alginate microspheres
AN
135 Freeze-drying method: Microspheres were quick-frozen with liquid nitrogen and then

136 freeze-dried in a freeze-dryer for 48.0 h.

M

137 Ethanol gradient dehydration method (Wei, et al., 2008): 20.0%, 50.0%, 75.0%,
D

138 85.0%, 95.0% concentrations of ethanol were prepared and sequentially added to the
TE

139 wet sample microspheres were eluted in 20.0 min. Finally using ethanol elution,

140 ethanol was removed after the treatment. The sample was placed in a vacuum drying
EP

141 box dried at 50.0 °C to achieve constant weight.

C

142 2.4. Characterizations of calcium alginate microspheres

AC

143 2.4.1. Particle size distribution and SPAN factor

144 The particle distribution and volume-average particle size (d4, 3) of calcium alginate

145 microspheres was measured using a laser diffraction device (Mastersizer 2000,

146 Malvern Instruments, MA). The volume size distribution was calculated from the

7
 ACCEPTED MANUSCRIPT

147 intensity of light diffracted at each angle using Mie theory. The analysis required a

148 parameter referred to as the presentation value, a combination of the absorbance of the

149 dispersed phase, the ratio of the relative refractive indices of the dispersed phase and

150 water. The presentation code used was Alg-Ca, corresponding to a relative refractive

PT
151 index of the particles of 1.52, sample absorption of 0.10, and a refractive index of the

RI
152 solvent of 1.33. Each sample was tested three times. Polydispersity was determined

153 by the SPAN factor expressed as

SC
154 SPAN = [D (v, 90) - D (v, 10)] / D (v, 50) （1）

U
155 where D (v, 90), D (v, 10) and D (v, 50) are volume size diameters at 90.0%, 10.0%
AN
156 and 50.0% of the cumulative volume, respectively (Y. Zhang, Wei, Lv, Wang, & Ma,

157 2011).
M

158 2.4.2. Morphology observations

D

159 An optical microscope installed with picture capturer was used to observe the overall
TE

160 morphology of microspheres. The surface morphology of the dried samples was

161 observed by a scanning electron microscopy (SEM, SU8000, Japan). Images were
EP

162 recorded by using FESEM with an acceleration voltage of 10.0 kV at different

C

163 magnifications.
AC

164 2.4.3. Differential scanning calorimetry (DSC) analysis

165 In Nitrogen gas atmosphere, accurate weighing 3.0 ± 0.2 mg samples were put into

166 sample cell. Heated from 30.0 °C to 300.0 °C at a rate of 10.0 °C/min, with 204 F1

167 differential scanning calorimeter. A sealed empty cell was used as reference. All

8
 ACCEPTED MANUSCRIPT

168 experiments were conducted in triplicate.

169 2.4.4. X-ray diffraction (XRD)

170 Dry sample X-ray diffraction patterns acquired at 20.0 °C on a Bruker D8-Advance

171 Diffractometer (Bruker AXS Inc., Madison, WI, USA) with backgroundless sample

PT
172 holders. The data were collected over an angular range from 5.0° to 85.0° 2θ in

RI
173 continuous mode using a step size of 0.02° 2θ and step time of 5.0 seconds.

174 2.5. Immobilization process

SC
175 0.5 g of calcium alginate microspheres were soaked in citric acid-sodium citrate

U
176 buffer at pH = 3.5. After suction filtration, 15.0 mL of 5.0 mg/mL PG was added and
AN
177 shook at 40.0 °C for 6.0 h. Then, samples were placed at 4.0 °C overnight. After

178 immobilization, the calcium alginate microspheres were washed several times with
M

179 distilled water until the absorbance of the washings at 280 nm was less than 0.01. The
D

180 immobilized PG was stored at 4.0 °C.

TE

181 2.6. Polygalacturonase activity assay

182 The PG activity was determined by measuring the reducing sugar (galacturonicacid)
EP

183 produced as a result of the reaction between the PG and the pectin. The concentration
C

184 of reducing sugar in the supernatant was estimated using DNS method (Li, et al., 2008;
AC

185 Li, et al., 2007). The standard compound used for the calibration curve for

186 determining PG activity was D-(+)- galacturonic acid monohydrate and the

187 absorbance was spectrometrically measured at 540 nm. One unit was defined as

188 liberating 1.0 mg of galacturonic acid from pectin per minute. To promote accuracy,

9
 ACCEPTED MANUSCRIPT

189 all the experiments were done in triplicates and the mean was calculated.

190 2.7. Optimum conditions of immobilization by response surface methodology

191 The Design Expert Software (version 6.0) was used for the statistical design of

192 experiments and data analysis. Response surface methodology was used to determine

PT
193 optimum conditions of immobilization to acquire the maximum activity recovery, and

RI
194 a 3-level-3-factor Box-Behnken design with 17 experiments was performed. The

195 experimental design chosen for the study was a Box–Behnken design (Hari, 2000;

SC
196 Zhou, Wang, Wu, Tang, & Pan, 2013), that helped in investigating linear, quadratic,

U
197 and cross-product effected of three factors, each varied at three levels and also
AN
198 included three center points for replication. The three factors were studied on

199 immobilization of PG into calcium alginate microspheres and their levels were:
M

200 enzyme amount (35.0 mg-45.0 mg), time (5.0-7.0 h) and pH (3.5-4.5).
D

201 2.8. Characterization of free and immobilized enzymes

TE

202 The effect of pH on the activity and stability of free and immobilized PG were

203 assayed by using 0.1 M phosphate buffers (pH 3.0-6.0) for 30.0 min. The effect of
EP

204 temperature on the activity and stability of free and immobilized PG were examined
C

205 by measuring PG activity from 20.0 °C to 80.0 °C for 30.0 min.

AC

206 Kinetic parameters Km and Vmax for both free and immobilized enzymes were

207 calculated from the Lineweaver–Burk equation using computed linear regression

208 calculations, under conditions given earlier at different substrate concentrations

10
 ACCEPTED MANUSCRIPT

209 between 0.2% and 1% (w/v) (Busto, Garcia-Tramontin, Ortega, & Perez-Mateos,

210 2006).

211 2.9. Operational stability of the immobilized polygalacturonase

212 The operational stability of the immobilized enzyme in 0.25% (w/v) apple pectin

PT
213 (Sigma) solution was determined by quantifying its activity at 40 °C, with stirring rate

RI
214 of 150 rpm, in consecutive cycles of repeated use of the enzyme. After each cycle of

215 30 min, immobilized particles were washed with Milli-Q water. The hydrolysis

SC
216 reaction was initiated by adding 0.5% (w/v) free enzyme or the same concentration of

U
217 immobilized enzyme (0.1 g/mL).
AN
218 2.10. Clarified the apple juice before and after characterization

219 2.10.1. Clarification of apple juice

M

220 The transmittance of apple juice before and after clarification was spectrometrically
D

221 measured at 650 nm, with distilled water as a reference. All experiments were
TE

222 repeated at least three times.

223 2.10.2 Color value of apple juice

EP

224 Apple juice before and after clarification added to equal volume of ethanol, mixed for
C

225 30.0 min. After sample filtration, the absorbance of samples was spectrometrically
AC

226 measured at 430 nm with distilled water as a reference. All experiments were repeated

227 at least three times.

228 2.10.3. Determination of soluble solids

11
 ACCEPTED MANUSCRIPT

229 Soluble solids content of apple juice before and after clarification were determined by

230 refractometer (B20T, Guangzhou Suwei electronic instrument Co., Ltd). Meanwhile,

231 the temperature was corrected according to the method of the previous report (Sugiura,

232 Kataoka, & Tomana, 1983).

PT
233 2.10.4. Determination of total acid and total sugar

RI
234 With reference to the previous report (Canan, et al., 2016), the sodium hydroxide

235 solution was used to titrate the total acid content of samples. The content of total

SC
236 sugar in samples was determined by direct titration. All experiments were repeated at

U
237 least three times.
AN
238 2.10.5. Pectin test

239 Referred to the previous report, apple juice before and after clarification added to
M

240 twice the volume of acidified ethanol. Then samples were mixed upside down three
D

241 times, stood 15.0 min. If there was a gel, the juice contained pectin (Liew Abdullah,
TE

242 Sulaiman, Aroua, & Megat Mohd Noor, 2007).

243 2.11. Apple juice stability determination

EP

244 Apple juice before and after clarification heated at 30.0 °C, 40.0 °C, 50.0 °C, 60.0 °C,
C

245 70.0 °C, 80.0 °C for 30.0 min and froze at -18.0 °C for 24.0 h, respectively. The
AC

246 transmittance of apple juice before and after clarification was compared after

247 processing to judge the stability of juice.

248 2.12. Statistics analysis

249 Data were presented as the mean ± standard deviation. Statistical comparisons were

12
 ACCEPTED MANUSCRIPT

250 analyzed by ANOVA analysis. A value of p < 0.05 was considered to be significant.

251 3. Results and discussion

252 3.1. Optimization experiment of preparing calcium alginate microspheres

253 The conditions including mass concentration of sodium alginate, mass percentage of

PT
254 span80, stirring speed, etc, for calcium alginate microspheres preparation were

RI
255 optimized, based on the single factor experiment and nine well-planned orthogonal

256 experiments (Fig.S1, S2, S3, S4, S5, S6, Table S1, S2, S3). Eventually, the optimum

SC
257 preparation conditions of microspheres were found to be aqueous volume fraction of

U
258 30.0%, mass concentration of sodium alginate of 4.5%, mass percentage of span 80 of
AN
259 3.0%, stirring speed of 400 r/min, W (Ca): W (Alg) of 13.0% and n (HAC): n (CaCO3)

260 of 3:1. Under optimal conditions, microspheres with the particle size of 38.0 ± 0.2 µm
M

261 showed good sphericity, good storage stability and a narrow particle size distribution
D

262 (Fig.S7).
TE

263 3.2. Characterizations of calcium alginate microspheres

264 The SEM images (Fig.1a, b) clearly demonstrated that different drying methods had a
EP

265 great impact on the morphology of microspheres under the optimal preparation
C

266 conditions. In Fig.1a, microspheres sample was freeze-dried after being frozen in
AC

267 liquid nitrogen. Due to the freezing effect, the microspheres had different degrees of

268 contraction, resulting in the majority of microspheres were elliptical. In Fig.1b,

269 microspheres were dried by ethanol gradient dehydration, which maintained good

270 sphericity of microspheres. According to Fig.1c, the major characteristic peaks of the

13
 ACCEPTED MANUSCRIPT

271 sodium alginate powder appeared at 13.0° and 20.0° (Liu, Chen, Zhong, & Wu, 2009).

272 However, the calcium alginate microspheres peaks at 13° disappeared and peaks

273 intensity at 20.0° significantly decreased. Thereby, it proved that sodium alginate did

274 crosslink with Ca2+. In Fig.1d, we could see the thermogram of the sodium alginate

PT
275 original powder which showed initial endothermic peaks at 73.3 °C and higher

RI
276 exothermic peaks at 245.8 °C. However thermogram of calcium alginate

277 microspheres did not show characteristic peak. The reason was that in the

SC
278 microsphere formation process, sodium alginate and Ca2+ was cross-linked to form a

U
279 network structure (Sarmento, Ferreira, Veiga, & Ribeiro, 2006).
AN
280 3.3. Optimization of enzyme activity recovery using response surface

281 methodology (RSM)

M

282 The most important parameters, which affected the recovery of enzyme activity, were
D

283 found to be enzyme amount, immobilization time and immobilization pH (Das, Roy,
TE

284 Bhattacharjee, Chakraborty, & Bhattacharjee, 2015; Fernandez-Lopez, et al., 2017;

285 Zaak, et al., 2017). Preliminary experiments were carried out to determine the initial
EP

286 values of the factors: enzyme amount (35.0 mg-45.0 mg), time (5.0 h-7.0 h) and pH
C

287 (3.5-4.5) (Fig.S8). Based on these experiments, the levels of the significant
AC

288 parameters and the interaction effects between various factors were studied. The

289 levels of each factor were illustrated in Table 1. The range and center point values of

290 three independent variables presented in Table 1 were based on the results of

291 preliminary experiments.

14
 ACCEPTED MANUSCRIPT

292 Using polynomial multivariate regression technique, Eq. (1) had been obtained after

293 the analysis of variance (ANOVA), which provided an estimation of enzyme activity

294 recovery as a function of enzyme amount, time and pH.

295 Y=-121.9121+7.9206X1+5.7308X2+3.7766X3+0.1629X2X3-0.1019X12-0.5166X22-0.5

PT
296 461X32 (1)

RI
297 where Y = enzyme activity recovery (%), X1 = enzyme amount (mL), X2 = time (h),

298 X3 = pH.

SC
299 Relatively high value of R2 = 0.9984 indicated that the full quadratic model equation

U
300 was capable of representing the system under the given experimental domain. Table 2
AN
301 showed the ANOVA results indicated that all the quadratic models obtained were

302 significant, since the probability values (Prob> F< 0.0001) were low. Values of P less
M

303 than 0.05 indicated that the model terms were significant. ANOVA revealed that the
D

304 most significant factor effecting enzyme activity recovery was X1 (p < 0.0001), X2 (p
TE

305 = 0.0004) followed by X3 (p = 0.0001).

306 The response surface plots or contour plots generated by response surface
EP

307 methodology could be used to study the effect of interaction of variables on enzyme
C

308 activity recovery and also to predict the optimal values of those variables. The 3D
AC

309 surface plots were generated by plotting the response (enzyme activity recovery) on

310 Z-axis against any two independent variables while keeping the third independent

311 variable at its constant level.

312 Fig.2a stated the effect of enzyme amount and pH on enzyme activity recovery. When

15
 ACCEPTED MANUSCRIPT

313 the pH value remains unchanged, with the increase of enzyme dosage, a substantial

314 increase in enzyme activity recovery was observed, while at higher enzyme amount

315 beyond 39.0 mg, enzyme activity recovery was found to decrease. Due to excessive

316 amount of enzyme, it could lead to the enzyme reaction required space was blocked

PT
317 and the recovery rate of enzyme activity declined. Fig.2b stated the effect of time and

RI
318 pH on enzyme activity recovery. When time remained unchanged, with the increase of

319 pH, increase in enzyme activity recovery was observed, while at higher pH beyond

SC
320 4.0, enzyme activity recovery was found to significantly decrease. When the pH value

U
321 was 4.0, the enzyme activity recovery reached to the highest level. Fig.2c showed the
AN
322 effect of enzyme amount and time on enzyme activity recovery. From Fig.2c, it was

323 clear that enzyme activity recovery was peaked at around 39.0 mg and 6.5 h,
M

324 respectively.
D

325 3.3.1. Validation of the model

TE

326 According to the response surface analysis, optimum enzyme activity recovery was

327 estimated to be 58.0% at pH value of 4.0, time of 6.5 h and enzyme amount of 39.0
EP

328 mg. To validate the optimum conditions, an experiment for enzyme activity recovery
C

329 was performed with the specified conditions. Under these conditions enzyme activity
AC

330 recovery was found to be 57.8% which was similar to the predicted value of 58.0%.

331 Thus, the model developed was accurate and reliable for predicting the optimized

332 conditions, as well as for maximum enzyme activity recovery.

333 3.4. Characterization of free and immobilized enzymes

16
 ACCEPTED MANUSCRIPT

334 3.4.1 Optimal pH and temperature for free and immobilized polygalacturonase

335 As shown in Fig.3a, the optimum temperature of immobilized enzymes was about

336 60.0 °C, which was almost 10.0 °C higher than the free PG. After immobilization, the

337 optimum temperature shifted to high temperatures, indicating that the immobilized

PT
338 PG had a stronger rigidity than free enzyme and may better prevent its unfolding at

RI
339 high temperatures (Tardioli, Zanin, & de Moraes, 2006). Meanwhile, the temperature

340 range over which the adsorbed enzyme retained more than 80% of its initial activity

SC
341 (20-60 °C) broader than that for free enzyme (20-50 °C). This was due to

U
342 immobilization improved enzyme stability at extreme temperatures (Cipolatti, et al.,
AN
343 2016; Mateo, et al., 2007). According to the Fig.3b, the optimum pH value of the free

344 PG was around 4.0 and the activity of immobilized enzyme was less dependent upon
M

345 pH within the interval of 3.0-4.5 than free enzyme. We speculated that the
D

346 immobilized enzyme more stable in apple juice clarification (pH=4.3±0.1) than free
TE

347 enzyme (Özoğlu & Bayındırlı, 2002). Ascribe to immobilization improved enzyme

348 stability in acidic solution (pH=3.0-4.5) (Garcia-Galan, et al., 2011), the immobilized
EP

349 PG was more suitable for the apple juice clarification.

C

350 3.4.2 Enzyme kinetics

AC

351 The Michealis-Menten constant (Km value) was obtained from Lineweaver-Burk plots

352 (Fig.4). The kinetic behavior for PG was changed via immobilization. Km value of the

353 immobilized PG (5.041 mg/mL) was higher than free PG (4.472 mg/mL). Due to the

354 effect of calcium alginate microspheres on PG diffusion in three-dimensional space,

17
 ACCEPTED MANUSCRIPT

355 the substrate concentration required for reaction of immobilization enzyme was

356 slightly higher than free enzyme (Arslan, Kıralp, Toppare, & Bozkurt, 2006; Lei & Bi,

357 2007). There was similarly report in other immobilization studies (Haupt, Neumann,

358 Wittemann, & Ballauff, 2005; Roy, Sardar, & Gupta, 2003).

PT
359 As usual, the maximum rate of reaction catalysed by the immobilized PG (0.112 U)

RI
360 was lower than that of the free enzyme (0.214 U). This decrease may be due to steric

361 hindrances imposed by the support on the macromolecular substrate (Busto, et al.,

SC
362 2006). Additionally, the accumulation of reaction products in the enzyme

U
363 microenvironment as a consequence of slow diffusion and interactions with H+ could
AN
364 have triggered competitive inhibition, resulting in Vmax value decreased (Rodrigues, et

365 al., 2013).

M

366 3.4.3. Operational stability of the immobilized polygalacturonase

D

367 Operational stability of a batch reactor with apple pectin solution at 40 °C was shown
TE

368 in Fig.5. The reduction of activity with both free and immobilized enzymes after 30

369 min treatment was about 7.0% (Fig.5a). This may ascribe to the viscosity of the apple
EP

370 pectin solution could produce some negative effects on the diffusion and enzyme
C

371 activity (Rodrigues, et al., 2013). As can be seen from Fig.5b, the activity of
AC

372 immobilized PG decreased considerably after the first five batch reactions, followed

373 by a decrease of 14.0%, 23.0%, 37.0% and 58.0%, respectively. After that, the activity

374 of enzyme decreased slightly. The immobilized enzyme retained about 20.0% of their

375 initial activity after repeating ten times. This was ascribe to the Ca2+ in the alginate

18
 ACCEPTED MANUSCRIPT

376 beads was exchanged with Na+ ions in solution, resulting in swelling, whereafter,

377 enzyme leakage and a subsequent inhibition on recycling of enzyme in the reaction

378 media (Hwang, et al., 2013).

379 3.5. Clarified the apple juice before and after characterization

PT
380 The immobilized PG clarification of apple juice conditions were optimized by

RI
381 response surface methodology. A 3-level-3-factor Box-Behnken design with 17

382 experiments was performed and the results were shown in Fig.S9. Eventually, the

SC
383 light transmittance of apple juice was improved to 96.8% at the following conditions:

U
384 9.0 mL (0.1 g/mL) of immobilized PG was added to 20.0 mL of apple juice at 45.0 °C
AN
385 for 90.0 min. The subsequent experiments were conducted with the apple juice

386 clarified by the optimized conditions.

M

387 According to Table.3, apple juice was treated by adding 0.5% (w/v) free enzyme or
D

388 the same concentration of immobilized enzyme (0.1 g/mL), the total sugar, acid value,
TE

389 soluble solids, light transmittance and color value had varying degrees of change. The

390 pectin tests result showed that both treatments were all no pectin. It was proved that
EP

391 the free and immobilized PG could effectively decompose the pectin in apple juice.
C

392 Compared with the free PG, the immobilized PG treated apple juice had higher light
AC

393 transmittance and smaller color value, smaller soluble solids content (Table.3).

394 Photograph of apple juice before and after clarification by immobilized PG was

395 showed in Fig.6a.

396 3.5.1. Thermal stability of apple juice

19
 ACCEPTED MANUSCRIPT

397 Thermal stability of apple juice before and after clarification was showed in Fig.6b.

398 The light transmittance of the untreated apple juice increased as the temperature rose.

399 High temperature caused pectin in apple juice aggregation and precipitation, so the

400 light transmittance of apple juice increased. Meanwhile, the effect of temperature rise

PT
401 on the light transmittance of the treated apple juice was not significant, and the

RI
402 transmittance of the treated apple juice was stable in a certain temperature range.

403 3.5.2. Freeze-thaw stability of apple juice

SC
404 Immobilized PG treated and untreated apple juice was frozen-thawed, and the

U
405 freeze-thaw stability was judged by measuring the change of light transmittance.
AN
406 Table.4 showed the light transmittance of treated apple juice was significantly (P

407 <0.05) higher than untreated after freeze-thaw treatment. Meanwhile, the light
M

408 transmittance of treated apple juice remained nearly constant after freeze-thaw
D

409 treatment. Therefore, the treated apple juice showed better freeze-thaw stability.
TE

410 4. Conclusions

411 In the present study, commercial PG were immobilized into calcium alginate
EP

412 microspheres and its application in apple juice clarification was evaluated. The
C

413 conditions of calcium alginate microspheres prepared by endogenous emulsification

AC

414 were optimized. Eventually, the optimum preparation conditions of microspheres

415 were found to be aqueous volume fraction of 30.0%, mass concentration of sodium

416 alginate of 4.5%, mass percentage of span 80 of 3.0%, stirring speed of 400.0 r/min,

417 W(Ca):W(Alg) of 13.0% and n(HAC): n(CaCO3) of 3:1. Meanwhile microspheres

20
 ACCEPTED MANUSCRIPT

418 were dried by ethanol gradient dehydration-vacuum drying and showed good

419 sphericity, storage stability, and narrow particle size distribution. Under the optimized

420 conditions, the enzyme activity recovery of immobilized PG prepared was 57.8% and

421 the light transmittance of apple juice was improved to 96.8%. Moreover, the

PT
422 immobilized enzyme had more temperature tolerance and wider pH adaptation range.

RI
423 Through the analysis of physico-chemical indicators of apple juice, apple juice

424 clarified by the immobilized PG proved transparent. Meanwhile, immobilized PG

SC
425 treated apple juice had good thermal stability and freeze-thaw stability. This study

U
426 provided a suitable strategy in PG immobilization for applying in the industrial
AN
427 production of apple juice.

428
M

429 Acknowledgments
D

430 This work was financially supported by the National Natural Science Foundation of
TE

431 China (Grant No. 31801586), Hubei Provincial Natural Science Foundation of China

432 (Grant No. 2018CFB233) and the Fundamental Research Funds for the Central
EP

433 Universities (Grant No. 2662018QD016). The authors greatly thank colleagues of Key
C

434 Laboratory of Environment Correlative Dietology of Huazhong Agricultural

AC

435 University for offering many conveniences.

436

437 References

21
 ACCEPTED MANUSCRIPT

438 Arslan, A., Kıralp, S., Toppare, L., & Bozkurt, A. (2006). Novel conducting polymer

439 electrolyte biosensor based on poly (1-vinyl imidazole) and poly (acrylic acid)

440 networks. Langmuir, 22(6), 2912-2915.

441 Barbosa, O., Ortiz, C., Berenguer-Murcia, A., Torres, R., Rodrigues, R. C., &

PT
442 Fernandez-Lafuente, R. (2015). Strategies for the one-step

RI
443 immobilization-purification of enzymes as industrial biocatalysts.

444 Biotechnology Advances, 33(5), 435-456.

SC
445 Barbosa, O., Ortiz, C., Berenguer-Murcia, Á., Torres, R., Rodrigues, R. C., &

U
446 Fernandez-Lafuente, R. (2014). Glutaraldehyde in bio-catalysts design: a
AN
447 useful crosslinker and a versatile tool in enzyme immobilization. RSC

448 Advances, 4(4), 1583-1600.

M

449 Barbosa, O., Ortiz, C., Berenguer-Murcia, Á., Torres, R., Rodrigues, R. C., &
D

450 Fernandez-Lafuente, R. (2015). Strategies for the one-step

TE

451 immobilization–purification of enzymes as industrial biocatalysts.

452 Biotechnology Advances, 33(5), 435-456.

EP

453 Barbosa, O., Torres, R., Ortiz, C., Berenguer-Murcia, A., Rodrigues, R. C., &
C

454 Fernandez-Lafuente, R. (2013). Heterofunctional supports in enzyme

AC

455 immobilization: from traditional immobilization protocols to opportunities in

456 tuning enzyme properties. Biomacromolecules, 14(8), 2433-2462.

22
 ACCEPTED MANUSCRIPT

457 Busto, M. D., Garcia-Tramontin, K. E., Ortega, N., & Perez-Mateos, M. (2006).

458 Preparation and properties of an immobilized pectinlyase for the treatment of

459 fruit juices. Bioresource Technology, 97(13), 1477-1483.

460 Canan, İ., Gündoğdu, M., Seday, U., Oluk, C. A., Karaşahin, Z., Eroğlu, E. Ç., Yazici,

PT
461 E., & Ünlü, M. (2016). Determination of antioxidant, total phenolic, total

RI
462 carotenoid, lycopene, ascorbic acid, and sugar contents of citrus species and

463 mandarin hybrids. Turkish Journal of Agriculture and Forestry, 40(6),

SC
464 894-899.

U
465 Cantone, S., Ferrario, V., Corici, L., Ebert, C., Fattor, D., Spizzo, P., & Gardossi, L.
AN
466 (2013). Efficient immobilisation of industrial biocatalysts: criteria and

467 constraints for the selection of organic polymeric carriers and immobilisation
M

468 methods. Chemical Society Reviews, 42(15), 6262-6276.

D

469 Chan, L., Lee, H., & Heng, P. (2002). Production of alginate microspheres by internal
TE

470 gelation using an emulsification method. International Journal of

471 Pharmaceutics, 242(1), 259-262.

EP

472 Chuah, A. M., Kuroiwa, T., Kobayashi, I., Zhang, X., & Nakajima, M. (2009).
C

473 Preparation of uniformly sized alginate microspheres using the novel

AC

474 combined methods of microchannel emulsification and external gelation.

475 Colloids and Surfaces A: Physicochemical and Engineering Aspects, 351(1-3),

476 9-17.

23
 ACCEPTED MANUSCRIPT

477 Cipolatti, E. P., Valério, A., Henriques, R. O., Moritz, D. E., Ninow, J. L., Freire, D.

478 M. G., Manoel, E. A., Fernandez-Lafuente, R., & de Oliveira, D. (2016).

479 Nanomaterials for biocatalyst immobilization-state of the art and future trends.

480 RSC Advances, 6(106), 104675-104692.

PT
481 Das, B., Roy, A. P., Bhattacharjee, S., Chakraborty, S., & Bhattacharjee, C. (2015).

RI
482 Lactose hydrolysis by beta-galactosidase enzyme: optimization using response

483 surface methodology. Ecotoxicology and Environmental Safety, 121, 244-252.

SC
484 DiCosimo, R., McAuliffe, J., Poulose, A. J., & Bohlmann, G. (2013). Industrial use of

U
485 immobilized enzymes. Chemical Society Reviews, 42(15), 6437-6474.
AN
486 Fang, G., Chen, H., Zhang, Y., & Chen, A. (2016). Immobilization of pectinase onto

487 Fe3O4@ SiO2–NH2 and its activity and stability. International Journal
M

488 Biological Macromolecules, 88, 189-195.

D

489 Fernandez-Lafuente, R. (2009). Stabilization of multimeric enzymes: Strategies to

TE

490 prevent subunit dissociation. Enzyme and Microbial Technology, 45(6-7),

491 405-418.
EP

492 Fernandez-Lopez, L., Pedrero, S. G., Lopez-Carrobles, N., Gorines, B. C.,

C

493 Virgen-Ortiz, J. J., & Fernandez-Lafuente, R. (2017). Effect of protein load on

AC

494 stability of immobilized enzymes. Enzyme and Microbial Technology, 98,

495 18-25.

24
 ACCEPTED MANUSCRIPT

496 Garcia-Galan, C., Berenguer-Murcia, Á., Fernandez-Lafuente, R., & Rodrigues, R. C.

497 (2011). Potential of Different Enzyme Immobilization Strategies to Improve

498 Enzyme Performance. Advanced Synthesis and Catalysis, 353(16), 2885-2904.

499 Hari, K. S. (2000). Optimization of isoamyl acetate production by using immobilized

PT
500 lipase from Mucor miehei by response surface methodology. Enzyme and

RI
501 Microbial Technology, 26(2), 131-136.

502 Hariyadi, D. M., Ma, Y., Wang, Y., Bostrom, T., Malouf, J., Turner, M. S., Bhandari,

SC
503 B., & Coombes, A. G. A. (2014). The potential for production of freeze-dried

U
504 oral vaccines using alginate hydrogel microspheres as protein carriers. Journal
AN
505 of Drug Delivery Science and Technology, 24(2), 178-184.

506 Haupt, B., Neumann, T., Wittemann, A., & Ballauff, M. (2005). Activity of enzymes
M

507 immobilized in colloidal spherical polyelectrolyte brushes.

D

508 Biomacromolecules, 6(2), 948-955.

TE

509 Hwang, E. T., & Gu, M. B. (2013). Enzyme stabilization by nano/microsized hybrid

510 materials. Engineering in Life Sciences, 13(1), 49-61.

EP

511 Khandare, V., Walia, S., Singh, M., & Kaur, C. (2011). Black carrot (Daucus carota
C

512 ssp. sativus) juice: processing effects on antioxidant composition and color.
AC

513 Food and Bioproducts Processing, 89(4), 482-486.

514 Lei, Z., & Bi, S. (2007). The silica-coated chitosan particle from a layer-by-layer

515 approach for pectinase immobilization. Enzyme and Microbial Technology,

516 40(5), 1442-1447.

25
 ACCEPTED MANUSCRIPT

517 Li, T., Li, S., Wang, N., & Tain, L. (2008). Immobilization and stabilization of

518 pectinase by multipoint attachment onto an activated agar-gel support. Food

519 Chemistry, 109(4), 703-708.

520 Li, T., Wang, N., Li, S., Zhao, Q., Guo, M., & Zhang, C. (2007). Optimization of

PT
521 covalent immobilization of pectinase on sodium alginate support.

RI
522 Biotechnology Letters, 29(9), 1413-1416.

523 Liew Abdullah, A. G., Sulaiman, N. M., Aroua, M. K., & Megat Mohd Noor, M. J.

SC
524 (2007). Response surface optimization of conditions for clarification of

U
525 carambola fruit juice using a commercial enzyme. Journal of Food
AN
526 Engineering, 81(1), 65-71.

527 Liu, Y., Chen, S., Zhong, L., & Wu, G. (2009). Preparation of high-stable silver
M

528 nanoparticle dispersion by using sodium alginate as a stabilizer under gamma

D

529 radiation. Radiation Physics and Chemistry, 78(4), 251-255.

TE

530 Martín, M. J., Calpena, A. C., Fernández, F., Mallandrich, M., Gálvez, P., & Clares, B.

531 (2015). Development of alginate microspheres as nystatin carriers for oral

EP

532 mucosa drug delivery. Carbohydrate Polymers, 117, 140-149.

C

533 Mateo, C., Palomo, J. M., Fernandez-Lorente, G., Guisan, J. M., &
AC

534 Fernandez-Lafuente, R. (2007). Improvement of enzyme activity, stability and

535 selectivity via immobilization techniques. Enzyme and Microbial Technology,

536 40(6), 1451-1463.

26
 ACCEPTED MANUSCRIPT

537 Mohamad, N. R., Marzuki, N. H. C., Buang, N. A., Huyop, F., & Wahab, R. A.

538 (2015). An overview of technologies for immobilization of enzymes and

539 surface analysis techniques for immobilized enzymes. Biotechnology and

540 Biotechnological Equipment, 29(2), 205-220.

PT
541 Özoğlu, H., & Bayındırlı, A. (2002). Inhibition of enzymic browning in cloudy apple

RI
542 juice with selected antibrowning agents. Food Control, 13(4), 213-221.

543 Rajdeo, K., Harini, T., Lavanya, K., & Fadnavis, N. W. (2016). Immobilization of

SC
544 pectinase on reusable polymer support for clarification of apple juice. Food

U
545 and Bioproducts Processing, 99, 12-19.
AN
546 Rastogi, R., Sultana, Y., Aqil, M., Ali, A., Kumar, S., Chuttani, K., & Mishra, A.

547 (2007). Alginate microspheres of isoniazid for oral sustained drug delivery.
M

548 International Journal of Pharmaceutics, 334(1), 71-77.

D

549 Rehman, H. U., Aman, A., Silipo, A., Qader, S. A. U., Molinaro, A., & Ansari, A.
TE

550 (2013). Degradation of complex carbohydrate: immobilization of pectinase

551 from Bacillus licheniformis KIBGE-IB21 using calcium alginate as a support.

EP

552 Food Chemistry, 139(1), 1081-1086.

C

553 Reis, C. P., Neufeld, R. J., Vilela, S., Ribeiro, A. J., & Veiga, F. (2008). Review and
AC

554 current status of emulsion/dispersion technology using an internal gelation

555 process for the design of alginate particles. Journal of Microencapsul, 23(3),

556 245-257.

27
 ACCEPTED MANUSCRIPT

557 Rodrigues, R. C., Ortiz, C., Berenguer-Murcia, A., Torres, R., & Fernandez-Lafuente,

558 R. (2013). Modifying enzyme activity and selectivity by immobilization.

559 Chemical Society Reviews, 42(15), 6290-6307.

560 Roy, I., Sardar, M., & Gupta, M. N. (2003). Evaluation of a smart bioconjugate of

PT
561 pectinase for chitin hydrolysis. Biochemical Engineering Journal, 16(3),

RI
562 329-335.

563 Sandri, I. G., Lorenzoni, C. M. T., Fontana, R. C., & da Silveira, M. M. (2013). Use

SC
564 of pectinases produced by a new strain of Aspergillus niger for the enzymatic

U
565 treatment of apple and blueberry juice. LWT-Food Science and Technology,
AN
566 51(2), 469-475.

567 Sarmento, B., Ferreira, D., Veiga, F., & Ribeiro, A. (2006). Characterization of
M

568 insulin-loaded alginate nanoparticles produced by ionotropic pre-gelation

D

569 through DSC and FTIR studies. Carbohydrate Polymers, 66(1), 1-7.
TE

570 Sharma, H. P., Patel, H., & Sugandha. (2017). Enzymatic added extraction and

571 clarification of fruit juices–A review. Critical Reviews in Food Science and
EP

572 Mutrition, 57(6), 1215-1227.

C

573 Sheldon, R. A., & van Pelt, S. (2013). Enzyme immobilisation in biocatalysis: why,
AC

574 what and how. Chemical Society Reviews, 42(15), 6223-6235.

575 Shi, L.-E., Zheng, W., Zhang, Y., & Tang, Z.-X. (2016). Milk-alginate microspheres:

576 Protection and delivery of Enterococcus faecalis HZNU P2. LWT - Food

577 Science and Technology, 65, 840-844.

28
 ACCEPTED MANUSCRIPT

578 Siebert, K. J. (2006). Haze formation in beverages. LWT-Food Science and

579 Technology, 39(9), 987-994.

580 Silva, C. M., Ribeiro, A. J., Ferreira, D., & Veiga, F. (2006). Insulin encapsulation in

581 reinforced alginate microspheres prepared by internal gelation. European

PT
582 Journal of Pharmaceutical Sciences, 29(2), 148-159.

RI
583 Sorrivas, V., Genovese, D., & Lozano, J. (2006). Effect of pectinolytic and amylolytic

584 enzymes on apple juice turbidity. Journal of Food Processing and

SC
585 Preservation, 30(2), 118-133.

U
586 Sosnik, A. (2014). Alginate Particles as Platform for Drug Delivery by the Oral Route:
AN
587 State-of-the-Art. ISRN Pharmaceutics, 2014, 926157.

588 Sugiura, A., Kataoka, I., & Tomana, T. (1983). Use of refractometer to determine
M

589 soluble solids of astringent fruits of Japanese persimmon (Diospyros kaki L.).
D

590 Journal of Pomology and Horticultural Science, 58(2), 241-246.

TE

591 Tapre, A., & Jain, R. (2014). Pectinases: Enzymes for fruit processing industry.

592 International Food Research Journal, 21(2).

EP

593 Tardioli, P. W., Zanin, G. M., & de Moraes, F. F. (2006). Characterization of

C

594 Thermoanaerobacter cyclomaltodextrin glucanotransferase immobilized on

AC

595 glyoxyl-agarose. Enzyme and Microbial Technology, 39(6), 1270-1278.

596 Uzuner, S., & Cekmecelioglu, D. (2015). Optimising clarification of carrot juice by

597 bacterial crude pectinase. International Journal of Food Science and

598 Technology, 50(12), 2707-2712.

29
 ACCEPTED MANUSCRIPT

599 Vaghari, H., Jafarizadeh-Malmiri, H., Mohammadlou, M., Berenjian, A., Anarjan, N.,

600 Jafari, N., & Nasiri, S. (2016). Application of magnetic nanoparticles in smart

601 enzyme immobilization. Biotechnology Letters, 38(2), 223-233.

602 Wei, W., Wang, L., Yuan, L., Yang, X., Su, Z., & Ma, G. (2008). Bioprocess of

PT
603 uniform-sized crosslinked chitosan microspheres in rats following oral

RI
604 administration. European Journal of Pharmaceutics and Biopharmaceutics,

605 69(3), 878-886.

SC
606 Zaak, H., Siar, E.-H., Kornecki, J. F., Fernandez-Lopez, L., Pedrero, S. G.,

U
607 Virgen-Ortíz, J. J., & Fernandez-Lafuente, R. (2017). Effect of immobilization
AN
608 rate and enzyme crowding on enzyme stability under different conditions. The

609 case of lipase from Thermomyces lanuginosus immobilized on octyl agarose

M

610 beads. Process Biochemistry, 56, 117-123.

D

611 Zhang, F. J., Cheng, G. X., Gao, Z., & Li, C. P. (2006). Preparation of Porous
TE

612 Calcium Alginate Membranes/Microspheres via an Emulsion Templating

613 Method. Macromolecular Materials and Engineering, 291(5), 485-492.

EP

614 Zhang, Y., Wei, W., Lv, P., Wang, L., & Ma, G. (2011). Preparation and evaluation of
C

615 alginate–chitosan microspheres for oral delivery of insulin. European Journal

AC

616 of Pharmaceutics and Biopharmaceutics, 77(1), 11-19.

617 Zhao, F., Wang, Q., Dong, J., Xian, M., Yu, J., Yin, H., Chang, Z., Mu, X., Hou, T.,

618 & Wang, J. (2017). Enzyme-inorganic nanoflowers/alginate microbeads: An

30
 ACCEPTED MANUSCRIPT

619 enzyme immobilization system and its potential application. Process

620 Biochemistry, 57, 87-94.

621 Zhou, Y., Wang, L., Wu, T., Tang, X., & Pan, S. (2013). Optimal immobilization of

622 B-glucosidase into chitosan beads using response surface methodology.

PT
623 Electronic Journal of Biotechnology, 16(6), 6-6.

RI
624 Zou, Q., Zhao, J., Liu, X., Tian, F., Zhang, H.-p., Zhang, H., & Chen, W. (2011).

625 Microencapsulation of Bifidobacterium bifidum F-35 in reinforced alginate

SC
626 microspheres prepared by emulsification/internal gelation. International

U
627 Journal of Food Science and Technology, 46(8), 1672-1678.
AN
628
M

629
D

630
TE

631
EP

632
C

633
AC

634

635

636

31
 ACCEPTED MANUSCRIPT

637 Figure captions:

638 Fig.1. The morphology of microspheres under different drying methods by SEM (a, b).

639 X-ray diffractograms of pure sodium alginate and calcium alginate microspheres (c).

640 DSC thermographs for pure sodium alginate and calcium alginate microsphere (d).

PT
641

RI
642 Fig.2 Three-dimensional response surface of enzyme amount and immobilization pH

643 on the recovery of enzyme (a). Three-dimensional response surface of immobilization

SC
644 pH and immobilization time on the recovery of enzyme (b). Three-dimensional

U
645 response surface of enzyme amount and immobilization time on the recovery of
AN
646 enzyme (c).

647
M

648 Fig.3. The optimum temperature of immobilized and free PG were examined by
D

649 measuring PG activity from 20.0 °C to 80.0 °C for 30.0 min. (a). The optimum pH of
TE

650 immobilized and free PG were assayed by using 0.1 M phosphate buffers (pH 3.0-6.0)

651 for 30.0 min (b).

EP

652
C

653 Fig.4. Lineweaver-Burk curve of polygalacturonase.

AC

654

655 Fig.5. Operational stability of immobilized PG in 0.25% (w/v) pectin solution at 30

656 min (a). Utilization frequency of immobilized PG (b).

657

32
 ACCEPTED MANUSCRIPT

658 Fig.6. Photograph of apple juice before (1) and after (2) clarification by immobilized

659 PG (a). Thermal stability of apple juice before and after clarification (b).

660

661

PT
662

RI
663

664

SC
665

U
666
AN
667

668
M

669
D

670
TE

671

672
EP

673
C

674
AC

675

676

677

678

33
 ACCEPTED MANUSCRIPT

679 Fig.1.

PT
RI
U SC
AN
M

680

681
D
TE

682
EP

683

684
C
AC

685

686

687

688
34
 ACCEPTED MANUSCRIPT

689 Fig.2.

PT
RI
U SC
AN
M
D
TE
EP

690
C

691
AC

692

693

694

35
 ACCEPTED MANUSCRIPT

695 Fig.3.

PT
RI
SC
696

U
697
AN
698
M

699
D

700
TE

701
EP

702
C

703
AC

704

705

706

36
 ACCEPTED MANUSCRIPT

707 Fig.4.

PT
RI
U SC
AN
M

708
D

709
TE

710
EP

711
C

712
AC

713

714

715

37
 ACCEPTED MANUSCRIPT

716 Fig.5.

PT
RI
U SC
AN
M
D
TE
C EP
AC

717

718

719

38
 ACCEPTED MANUSCRIPT

720 Fig.6.

PT
RI
SC
721

722

U
AN
723
M

724
D

725
TE

726
EP

727
C

728
AC

729

730

731

39
 ACCEPTED MANUSCRIPT

732 Table.1 Factor levels table of response surface experiment.

733
Levels
Independent variables 734
-1 0 1
735

PT
Enzyme amount（mg） 35 40 45
736
Time（h） 5 6 7

RI
pH 3.5 4 4.5 737

738

SC
739

740

U
AN
741
M

742
D

743
TE

744
EP

745
C

746
AC

747

748

749

40
 ACCEPTED MANUSCRIPT

750 Table.2 Variance analysis of enzyme recovery of immobilized polygalacturonase.

Source Sum of square dF Mean square F value P value (Prob>F）

Model 40.70 7 5.81 787.59 <0.0001

PT
X1 10.03 1 10.03 1358.91 <0.0001

X2 0.22 1 0.22 29.48 0.0004

RI
X3 0.30 1 0.30 40.26 0.0001

SC
X2X3 0.027 1 0.027 3.06 0.0904

X12 26.83 1 26.83 3634.32 <0.0001

X22
U
AN
1.00 1 1.00 135.39 <0.0001

X32 0.075 1 0.075 10.17 0.0110

M

Lack of fit 0.051 5 0.010 2.69 0.1795

D

Pure error 0.015 4 0.003807

TE

751

752
EP

753
C

754
AC

755

756

757

41
 ACCEPTED MANUSCRIPT

758 Table.3 The physical and chemical indexes of apple juice before and after clarification

759
Physicochemical Pectinase Immobilized
Fruit juice
760
indexes treatment enzyme treatment

PT
Total sugar (g/L) 142.54±1.75a 140.37±2.63a a
139.57±3.11761

Acid value (g/L) 1.45±0.16a 1.36±0.08b 1.42±0.15a762

RI
pH value 3.89±0.02a 3.74±0.01b 3.81±0.02b763

SC
Soluble solids 10.9±0.1a 11.9±0.2b 11.5±0.1b 764

Light transmittance 765

U
a b b
37.6±0.2 94.8±0.2 97.1±0.3
766
AN
（670nm）

Colour (420nm) 0.784±0.009a 0.232±0.007b b

0.197±0.016767
M

Pectin Have None None 768

D

769
TE

770
EP

771
C

772
AC

773

774

775

42
 ACCEPTED MANUSCRIPT

776 Table.4 The stability of apple juice after freeze-thaw treatment

Apple juice T（%） Freeze-thaw T% Sediment

Untreated 37.6±0.2 46.3±0.3 Have

Treated 97.1±0.3* 96.5±0.3* No

PT
777

RI
778

SC
779

U
AN
M
D
TE
C EP
AC

43
 ACCEPTED MANUSCRIPT
Hight lights

1. Conditions of calcium alginate microspheres prepared were optimized.

2. Calcium alginate microspheres showed arrow particle size distribution.

3. The enzyme activity recovery of immobilized pectinase prepared was 57.83%.

4. The light transmittance of apple juice was improved to 96.8%.

PT
5. The immobilized enzyme had more temperature tolerance, wider pH adaptation
range.

RI
U SC
AN
M
D
TE
C EP
AC