Physicochemical properties of apple juice during sequential steps of the industrial processing and functional properties of pectin fractions from the generated pomace

Accepted Manuscript Physicochemical properties of apple juice during sequential steps of the industrial processing and functional properties of pectin fractions from the generated pomace Ana L. Ramos-Aguilar, Claudia I. Victoria-Campos, Emilio Ochoa-Reyes, José de Jesús Ornelas-Paz, Paul B. Zamudio-Flores, Claudio Rios-Velasco, Jaime ReyesHernández, Jaime D. Pérez-Martínez, Vrani Ibarra-Junquera PII:

S0023-6438(17)30600-X

DOI:

10.1016/j.lwt.2017.08.030

Reference:

YFSTL 6452

To appear in:

LWT - Food Science and Technology

Received Date: 16 March 2017 Revised Date:

9 August 2017

Accepted Date: 10 August 2017

Please cite this article as: Ramos-Aguilar, A.L., Victoria-Campos, C.I., Ochoa-Reyes, E., de Jesús Ornelas-Paz, José., Zamudio-Flores, P.B., Rios-Velasco, C., Reyes-Hernández, J., Pérez-Martínez, J.D., Ibarra-Junquera, V., Physicochemical properties of apple juice during sequential steps of the industrial processing and functional properties of pectin fractions from the generated pomace, LWT Food Science and Technology (2017), doi: 10.1016/j.lwt.2017.08.030. 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

1

Physicochemical properties of apple juice during sequential steps of the

2

industrial processing and functional properties of pectin fractions from the

3

generated pomace

RI PT

4 5

Ana L. Ramos-Aguilar a, Claudia I. Victoria-Campos a, Emilio Ochoa-Reyes a,

6

José de Jesús Ornelas-Paz

7

Velasco a, Jaime Reyes-Hernández b, Jaime D. Pérez-Martínez c, Vrani Ibarra-

8

Junquera d

9

a

*, Paul B. Zamudio-Flores a, Claudio Rios-

M AN U

SC

a,

Centro de Investigación en Alimentación y Desarrollo A.C.-Unidad Cuauhtémoc,

10

Av. Río Conchos S/N, Parque Industrial, C.P. 31570, Cd. Cuauhtémoc,

11

Chihuahua, México.

12

b

13

artillero No. 138, Zona Universitaria, C.P. 78210, San Luis Potosí, México.

14

c

15

Manuel Nava 6 Zona Universitaria, C.P. 78210, San Luis Potosí, México.

16

d

17

Colima, C.P. 28400, Coquimatlán, Colima, México.

TE D

EP

Universidad Autónoma de San Luis Potosí, Facultad de Ciencias Químicas,

Universidad de Colima, Bioengineering Laboratory, Km. 9 carretera Coquimatlán-

AC C

18

Universidad Autónoma de San Luis Potosí, Facultad de Enfermería, Av. Niño

19

Short running head: Quality of apple juice and pectin

20

*Corresponding

21

[email protected] (J. J. Ornelas-Paz).

author.

Tel/Fax:

+52-625-5812920.

E-mail

address:

1

ACCEPTED MANUSCRIPT

Abstract

23

The impact of processing at industrial scale on properties of apple juice and pectin

24

fractions extracted from the generated pomace is scarcely known. In this study,

25

apple juice was collected at selected steps of the industrial production process and

26

evaluated for physical and chemical properties. The generated pomace was

27

recovered and subjected to extraction of pectins according to their solubility. They

28

were evaluated for their physicochemical and functional properties. The

29

transmittance for color and clearness of juice increased during clarification step but

30

decreased during concentration. The sugar content was not altered by any step.

31

The content of individual acids showed some changes during the pasteurization.

32

The levels of total and individual phenols tended to increase during the production

33

process. Low levels of 5-HMF (4.0 mg/L) were detected only in the concentrated

34

juice while patulin was absent. The pomace contained only chelator and alkali

35

soluble pectin. In comparison to commercial apple pectin, extracted pectins were of

36

low degree of esterification (67% vs 22.8– 44.6%), GalA (654.1 vs 393.5–436.6

37

mg/g) and Gal (228.6 vs 71.3–115.0 mg/g) content but rich in Ara (27.5 vs 103.4–

38

166.3 mg/g) and of high molecular weight (644.5 vs 1559.6-2360.6 kDa). Their

39

viscosity was lower than that of commercial apple pectin (k values of 0.03–0.05 vs

40

0.14 Pa·sn, respectively). They showed lower thermal stability than commercial

41

apple pectin. The production steps involving high temperature or enzymes effected

42

the properties of apple juice and extracted pectins.

43

Keywords: Industrial

44

Functional properties

45

AC C

EP

TE D

M AN U

SC

RI PT

22

processing;

Nutrients;

Antioxidants; Polysaccharides;

2

ACCEPTED MANUSCRIPT

1. Introduction

47

Clarified apple juice is one of the most popular processed foods in the world.

48

Several chemical components (i.e. sugars, acids, phenolic compounds, pigments,

49

safety-related compounds, etc.) are involved in juice quality; however, the levels of

50

these compounds are highly influenced by the production process (Kadakal & Nas,

51

2003; Suárez-Jacobo et al., 2011). The apple pulp and juice are exposed to

52

exogenous enzymes, adsorbent materials, high temperatures, and high osmotic

53

pressure during the production process (Eisele & Drake, 2005). These conditions

54

favor the release, degradation, transformation, and generation of compounds

55

involved in juice quality (Markowski, Baron, Le Quéré, & Płocharski, 2015;

56

Onsekizoglu, Bahceci, & Acar, 2010). The production process determines the color

57

and turbidity as well as the levels of phenols, sugars, acids, the mycotoxin patulin

58

and 5-hydroxymethylfurfural (5-HMF) of clarified juice, among other compounds

59

(He, Ji, & Li, 2007; Onsekizoglu et al., 2010; Markowski et al., 2015; Gökmen et al.,

60

2001; Welke, Hoeltz, Dottori, & Noll, 2009). However, these processing effects

61

have mainly been determined under laboratory or pilot plant conditions (Gökmen,

62

Artık, Acar, Kahraman, & Poyrazoğlu, 2001; Kadakal & Nas, 2003; Onsekizoglu et

63

al., 2010; Spanos, Wrolstad, & Heatherbell, 1990; Suárez-Jacobo et al., 2011).

64

The effect of apple juice processing at industrial scale has been scarcely studied

65

and limited to a few processing steps and juice attributes (Welke et al., 2009).

66

Further information in this regard is required to be used as a basis for the

67

improvement of the quality of clarified apple juice.

AC C

EP

TE D

M AN U

SC

RI PT

46

3

ACCEPTED MANUSCRIPT

The pomace is the main waste product generated by the apple juice industry. It

69

represents 25% of fruit weight and its disposal involves costs and environmental

70

pollution (O’Shea et al., 2015). However, it also is an important source of

71

commercial pectin, which is a heteropolysaccharide widely used in many industrial

72

sectors for its functional properties (gelling, stabilizing, emulsifying and thickening

73

agent) (Wang, Chen, & Lü, 2014). The functionality of this type of apple pectin

74

depends of its physicochemical characteristics, which are highly influenced by

75

apple variety and ripening stage as well as the using of enzymes during juice

76

production (Canteri-Schemin, Fertonani, Waszczynskyj, & Wosiacki, 2005; Fischer,

77

Arrigoni, & Amadò, 1994). The physicochemical properties of total pectin from

78

apple pomace have extensively been studied (Kammerer, Kammerer, Valet, &

79

Carle, 2014; Kosmala et al., 2010; Min et al., 2010; O’Shea et al., 2015; Wang et

80

al., 2014). However, the physicochemical and functional properties of pectin

81

fractions from apple pomace have received scarce attention (Kosmala et al., 2010).

82

The objective of this work was to determinate the impact of selected steps of the

83

production process at industrial scale in apple juice quality as well as to evaluate

84

the physicochemical and functional properties of different pectin fractions obtained

85

from the generated pomace.

SC

M AN U

TE D

EP

AC C

86

RI PT

68

87

2. Materials and methods

88

2.1. Apple juice and pomace

89

The samples of juice and pomace from ‘Golden Delicious’ apples were obtained

90

under industrial scale conditions. Triplicate samples of juice (1L) and pomace (3 4

ACCEPTED MANUSCRIPT

Kg) were weekly collected during four weeks. Juice was collected at the pressing,

92

pasteurization, clarification, and concentration step while pomace only at pressing

93

step. The first step consisted in pressing apple cubes (2 mm) previously treated

94

with pectin lyase for 60 min at 25 °C. The pomace was collected at this step and

95

stored at -70 °C until pectin extraction. The juice was pasteurized at 90 °C for 60 s

96

and then quickly cooled (50°C). The clarification step involved the treatment with a

97

mixture of polygalacturonases, pectinase and glucoamylase for 100 min at 55 °C

98

followed by ultrafiltration (UF) with tubular membranes of molecular weight cut-off

99

(MWCO) of 100 kDa. The concentration consisted of two heating steps, one at 55

100

°C (23 min) and other at 70 °C (60 min). The average production flow was 3000

101

L/h. The juice samples were immediately stored at -70 °C until analysis. The juice

102

samples were analyzed for total soluble solids content (TSS %) to determine the

103

dehydration level during the production process and then adjusted to 11.2 °Brix

104

before analysis of the physicochemical attributes. Several pectins were

105

sequentially obtained from the apple pomace according to their solubility and

106

evaluated for physicochemical and functional properties.

SC

M AN U

TE D

EP

107

RI PT

91

2.2. Miscellaneous evaluations in juice

109

The content of TSS (%) was determined in three subsamples (0.5 mL) of each

110

juice using an automatic digital refractometer RX 5000 (ATAGO, Tokyo, Japan).

111

The titratable acidity was determined in tree subsamples (5 mL) of each juice

112

according to Eisele and Drake (2005). The total phenolic content was determined

113

three times in each juice by the Folin Ciocalteu method at 750 nm, according to

AC C

108

5

ACCEPTED MANUSCRIPT

Spanos et al. (1990), and expressed as mg of chlorogenic acid equivalent per L of

115

juice. Three subsamples (8 mL) of each juice were evaluated for color and

116

clearness using a Thermo Spectronic 20D+ spectrophotometer (Thermo Scientific,

117

Wisconsin, USA) at 440 and 625 nm (transmittance), respectively. These

118

measurements were performed according to Kadakal and Nas (2003).

119

RI PT

114

2.3. Individual sugars and organic acids

121

The evaluation of sugars and organic acids was performed according to Ornelas-

122

Paz et al. (2017), with slight modifications. For sugars, three subsamples of each

123

juice were centrifuged (24652 g/ 20 min/ 4 °C) and then diluted ten times with

124

water. The pH of diluted juice was adjusted at 7 with 10% NaOH, filtered and

125

manually injected (20 µL) into a HPLC system (Varian Inc., CA, USA), which was

126

equipped with a refractive index detector (Star Model 9040). The sugars were

127

separated in a SUGAR SC 1821 (8.0 x 300 mm) (SHODEX, Tokyo, Japan) ion

128

exchange column at 70 °C. The mobile phase was HPLC grade water at a flow rate

129

of 0.6 mL/min. For organic acids, three subsamples of each juice were filtered and

130

directly injected (20 µL) into the HPLC system described above but fitted to a UV-

131

Vis detector (Model 9050). Acids were separated in an Aminex HPX-87H (7.78 x

132

300 mm) ion exchange column (Bio-Rad Laboratories., CA, USA). The separation

133

was performed at 60 °C using 5mM H2SO4 as mobile phase at a flow rate of 0.4

134

mL/min. The organic acids were monitored at λ=210 nm. The identification and

135

quantification of sugars and acids were performed using standard compounds.

AC C

EP

TE D

M AN U

SC

120

136

6

ACCEPTED MANUSCRIPT

2.4. Analysis of phenolic compounds

138

The extraction and chromatographic separation of phenolic compounds were

139

based in the methodology described by Ornelas-Paz et al. (2017). Three

140

subsamples of each juice were centrifuged (24652 g/ 20 min/ 4 °C), filtered and

141

manually injected (20 µL) into an Agilent 1200 series HPLC system (Agilent Inc.,

142

CA, USA) equipped with a diode array detector. The separation of phenolic

143

compounds was performed in a ZORBAX XDB-C18 column (4.6 x 150 mm)

144

(Agilent Inc., CA, USA) at 30 °C. The phenolic compounds were monitored at λ=

145

280, 320, 350 and 520 nm. The mobile phase consisted of 2% acetic acid (A) and

146

acetonitrile (B), according to the following gradient: 100% A at 0 min, 93% A at 12

147

min, 89% A at 20 min, 86%A at 35 min, 84% A at 36 min, 82% A at 41 min, 76% A

148

at 48 min, 70% A at 54 min, and 65% A at 59 min. The flow rate was 1 mL/min.

149

Reference compounds were used for identification and quantification purposes.

SC

M AN U

TE D

150

RI PT

137

2.5. Analysis of patulin and 5-HMF

152

Three subsamples (10 mL) of each juice were individually placed in a separatory

153

funnel, vigorously mixed with ethyl acetate (20 mL), and kept in repose until

154

separation of phases. The organic phase was recovered while the aqueous phase

155

was extracted two times more with ethyl acetate (20 mL each time), recovering the

156

organic phase after each washing. The organic phases were pooled and washed

157

two times with 1.5% Na2CO3 (10 mL). The organic phase was recovered and

158

evaporated at reduced pressure at 40 °C. The residue was dissolved in 1 mL of

159

water at pH 4 (adjusted with acetic acid), filtered, and manually injected (100 µL) in

AC C

EP

151

7

ACCEPTED MANUSCRIPT

the Agilent 1200 series HPLC system described above. The separation of patulin

161

and 5-HMF was performed according to Gökmen and Acar (1999), using a

162

ZORBAX XDB-C18 (4.6 x 150 mm) (Agilent Inc., CA, USA) column at 30 °C. The

163

patulin and 5-HMF were monitored at 276 and 284 nm, respectively. The mobile

164

phase consisted of water (pH 4, A) and acetonitrile (B) according to the following

165

gradient: 99% A at 0 min, 95% A at 10 min, 90% A at 15 min, 50% A at 20 min,

166

100% B from 25 to 35 min. The flow rate was 1 mL/min. The qualitative and

167

quantitative analyses were performed using reference compounds.

168

M AN U

SC

RI PT

160

2.6. Extraction of pectin

170

Triplicate samples of apple pomace (500 g each) were used as pectin source. The

171

extraction of alcohol-insoluble residues (AIR) and pectins was performed according

172

to Ramos-Aguilar et al. (2015). The AIR was subjected to sequential extraction of

173

pectin with hot water (95 °C) for 5 min, 0.05M CDTA in 0.1 M potassium acetate at

174

pH 6.5 for 6 h at 28 °C, and 0.05 M Na2CO3 containing 0.02 M NaBH4 for 16 h at 4

175

°C and then for 6 h at 28 °C. The ratio of AIR to extraction solution was 1:100 (w/v)

176

in all cases. The pectin was precipitated with ethanol and recovered by

177

centrifugation (12, 000 g/5 min/ 4 °C) and filtration (Whatman paper No. 541). The

178

recovered pectins were named as water, chelator, and alkali soluble pectin (WSP,

179

CSP, and NSP, respectively). Triplicate samples of these pectins as well as of

180

commercial apple pectin (CAP) were evaluate for physicochemical and rheological

181

properties.

AC C

EP

TE D

169

182

8

ACCEPTED MANUSCRIPT

2.7. Degree of esterification of pectin

184

The GalA was liberated from pectins and colorimetrically quantified at 525 nm

185

according to Ramos-Aguilar et al. (2015). The methanol was liberated from pectins

186

according to Voragen, Schols, & Pilnik (1986). The extract was filtered and

187

manually injected (20 µL) to the Varian HPLC system described above (refractive

188

index detection). The methanol was separated in a TSKGel SCX H+ (7.8 x 300

189

mm) ion exchange column (Tosoh Bioscience LLC, Tokyo, Japan) at 30 °C.

190

Deionized water was used as mobile phase at a flow rate of 1 mL/min. The degree

191

of esterification (DE) was calculated according to Voragen et al. (1986).

M AN U

SC

RI PT

183

192

2.8. Monosaccharide composition of pectin

194

Pectin was sequentially subjected to acid and enzymatic hydrolysis according to

195

Ramos-Aguilar et al. (2015). Each hydrolysate was analyzed under two different

196

HPLC conditions using the Varian system described above (refractive index

197

detection). The Glu, Ara, Rha, Gal, and Fuc were separated at 58 °C in a Metacarb

198

H+ (7.8 x 300 mm; Varian Inc., CA, USA) ion-exchange column, using 0.0085N

199

H2SO4 as mobile phase at a flow rate of 0.4 mL/min. Xyl and Man were separated

200

in a Supelcogel Pb (7.8 x 300 mm; Sigma-Aldrich., MO, USA) ion exchange

201

column at 70°C using water as mobile phase at a flow rate of 0.5 mL/min. The

202

qualitative and quantitative analyses were performed using reference compounds.

AC C

EP

TE D

193

203 204

2.9. Molecular weight distribution of pectin

9

ACCEPTED MANUSCRIPT

The molecular weight (MW) distribution was determined by high performance size-

206

exclusion chromatographic, according to Pérez-Martínez et al. (2013). Pectins

207

were solubilized in water (5 g/L). The solutions were filtered through a polyethylene

208

membrane of 0.45 µm of pore size (Millipore Corp., MA, USA) and manually

209

injected (20 µL) into the Varian HPLC system described above (refractive index

210

detection). Separation was performed using a series of TSK-GEL columns

211

(GMPW XL, G5000PW XL, and G4000PW XL; 7.8 x 300 mm each) (Tosoh Bioscience;

212

Tokyo, Japan) at 40° C. Phosphate buffer (0.2 M; pH= 6.9) was used as mobile

213

phase at a flow rate of 0.4 mL/min. The MW was determined relative to dextrans of

214

known MW (1-670 kDa) (Sigma-Aldrich., MO, USA).

215

M AN U

SC

RI PT

205

2.10. Other evaluations of pectin

217

The FT-IR spectra (4000 to 450 cm−1) were collected using a Spectrum Two FT-IR

218

spectrometer (Perkin Elmer Inc., MA, USA). Triplicate samples of each pectin were

219

analyzed and applied on the Attenuated Total Reflection crystal as powder. For

220

each sample, 34 scans were averaged with a spectral resolution of 4 cm−1. Spectra

221

analyses and baseline corrections were performed using the Spectra software ver.

222

10.4.4.449 (Perkin Elmer Inc., MA, USA). The viscosity of pectin solutions (1%)

223

was determined according to Ramos-Aguilar et al. (2015) but using a stainless

224

steel cone geometry (4°, 40 mm diameter) and a gap size of 90 µm. Pectin thermal

225

stability was analyzed with a Q500 thermogravimetric analyzer (TA Instruments.,

226

DE, USA). Ten milligrams of pectin powder were weighted in aluminum pans and

227

heated from 30 to 600 °C at 10 °C/min under nitrogen flow (15 mL/min). The

AC C

EP

TE D

216

10

ACCEPTED MANUSCRIPT

thermogravimetric curves were analyzed with Universal Analysis 2000 software

229

(ver. 4.5A). The tristimulus color (L*, a* and b*) was determined on pectin samples

230

(powder) using Minolta colorimeter (CR-300 model, Minolta Co. Ltd, Osaka,

231

Japan).

RI PT

228

232

2.11. Statistical analysis

234

The values obtained weekly for each measurement were averaged and considered

235

as a single value. Data were analyzed by an analysis of variance under a

236

completely randomized design with a limit of significance of 0.05. The comparison

237

of means was performed using the Tukey-Kramer post hoc test. The analysis data

238

was performed using JMP statistical software (SAS Institute, Inc., NC, USA).

M AN U

SC

233

239

3. Results and discussion

241

3.1. Juice color and clearness

242

The pasteurization and clarification steps sequentially increased the transmittance

243

of the freshly pressed juice at both wavelengths (Table 1). He et al. (2007) also

244

observed that the transmittance of apple juice at 440 nm increased from 53.7% to

245

66.7% after pasteurization. Vladisavljević, Vukosavljević, & Bukvić (2003) observed

246

increases of transmittance after juice clarification that varied between 30 and 21%,

247

depending of the clarification method. The increase of transmittance during the first

248

steps of juice processing might be attributed to the hydrolysis of pectin and starch

249

by added enzymes as well as by removal of these polysaccharides by UF

250

membranes (He et al., 2007). In our study, the transmittance of juice at both

AC C

EP

TE D

240

11

ACCEPTED MANUSCRIPT

wavelengths was reduced 6-8.7% after concentration (Table 1). Others are also

252

observed a slight decrease (4.4-7.7%) in the transmittance of apple juice at both

253

wavelengths after concentration (Kadakal & Nas, 2003; Garza, Giner, Martín,

254

Costa, & Ibarz, 1996). The decrease of transmittance at the concentration step

255

might be consequence of phenol oxidation, the suspension of particles formed by

256

residues of depolymerized pectin and denaturalized proteins that crossed the UF

257

membrane at the clarification step, and formation of Maillard’s reaction products

258

(Garza et al., 1996; He et al., 2007; Kadakal & Nas, 2003; Onsekizoglu et al.,

259

2010; Vladisavljević et al., 2003). The 5-HMF might contribute to juice color

260

because it was detected in concentrated juice at a concentration of 4.0 ± 0.6 mg/L.

261

Similar contents of 5-HMF (up to 7.9 mg/L) have been reported in apple juices after

262

thermal concentration in a rotavapor

263

patulin was not detected.

SC

M AN U

(Spanos et al., 1990). In this study, the

TE D

264

RI PT

251

3.2. Effect of processing on sugars and acids

266

The TSS content in freshly pressed juice did not changed during pasteurization

267

and clarification steps (Table 1). Others have reported similar findings at laboratory

268

and pilot plant scale (Gökmen et al., 2001; Suárez-Jacobo et al., 2011;

269

Vladisavljević et al., 2003). In our study, the juice was concentrated 5.2 times

270

during the evaporation step. Similar changes of concentration (4.8 times) have

271

been reported for apple juice under industrial-scale conditions (Welke et al., 2009).

272

Fructose was the main sugar in tested juices, followed by sucrose and glucose

273

(Table 2). The content of individual sugars was not significantly altered by any

AC C

EP

265

12

ACCEPTED MANUSCRIPT

processing step, as reported in some studies at laboratory and pilot plant scale

275

(Onsekizoglu et al., 2010; Suárez-Jacobo et al., 2011).

276

The TA of juice was similar during the first three steps of the production process

277

(Table 1). Previous studies at laboratory scale with juice from ‘Golden Delicious’

278

apples demonstrated that the TA was not significantly altered by both thermal or

279

non-thermal pasteurization and clarification by UF (Aguilar-Rosas, Ballinas-

280

Casarrubias, Nevarez-Moorillon, Martin-Belloso, & Ortega-Rivas, 2007; Youn,

281

Hong, Bae, Kim, & Kim, 2004). In this study, a slight decrease (11%) of TA was

282

observed after evaporation. Onsekizoglu et al. (2010) observed under laboratory

283

conditions a decrease of 4.7% in acid concentration of juice from the clarification to

284

the evaporation step. The malic and ascorbic acids were the most abundant in

285

tested juices (Table 2). The concentration of each acid in freshly pressed juice was

286

statistically similar to that of the concentrated juice (final juice). However,

287

temporary alterations in acid content were observed at pasteurization step.

288

Interestingly, the highest or lowest concentration of each one of tested acids were

289

observed after pasteurization (Table 2). The effect of this step on acids has not

290

been clearly studied; however, the high temperatures used for pasteurization could

291

improve the release of organic acids from apple particles and the dehydration of

292

some of them, altering consequently their concentrations (Gökmen & Acar, 1998).

294

SC

M AN U

TE D

EP

AC C

293

RI PT

274

3.3. Phenolic compounds in apple juice

13

ACCEPTED MANUSCRIPT

Tested juices were rich in phenolic compounds, as judged by the total phenolic

296

content (Table 1). Interestingly, such content increased (100%) during the

297

production process. This might be a consequence of the thermal and enzymatic

298

release of phenols from fibers during the production process (Kammerer et al.,

299

2014). Eleven phenolic compounds were identified and quantified in tested juices,

300

with chlorogenic acid, procyanidin B2, phloridzin and epicatechin being the most

301

abundant (Table 3). The concentration of these compounds was into the range

302

reported for raw and processed juices (chlorogenic acid, 3.4-113.7 mg/L; caffeic

303

acid, 0.7-8.7 mg/L; p-coumaric acid, 0.1-3.0 mg/L; epichatequin, 0-71.8 mg/L;

304

procyanidin B2, 0-121 mg/L; quercertin-3-galactoside, 0-14.4 mg/L; quercetin-3-

305

glucoside, 0-9.1 mg/L; phloridzin, 1.3-56.0 mg/L) (Gliszczynska-Swiglo &

306

Tyrakowska, 2003; Oszmiański, Wojdyło, & Kolniak, 2011; Spanos et al., 1990).

307

In general, the content of individual phenolic compounds continuously increased

308

from the pressing to the clarification step (Table 3). Some studies at laboratory

309

scale demonstrated the reduction of the phenolic content (4-45%) in apple juice

310

during juice clarification with membranes of MWCO from 10 to 30 kDa (Gökmen et

311

al., 2001; Onsekizoglu, 2013). The membrane used in this study was of a MWCO

312

considerably higher (100 kDa), explaining the low impact of such step on phenols.

313

The concentration step did not alter the levels of phenolic compounds.

314

Substantial increases (34-109%) in chlorogenic, caffeic and p-coumaric acids were

315

observed during the pasteurization and clarification steps. Spanos et al. (1990)

316

demonstrated at pilot plant scale that the juice production at elevated temperatures

317

(55-73 °C) improved the extractability (3-222%) of cinnamic acids and decreased

AC C

EP

TE D

M AN U

SC

RI PT

295

14

ACCEPTED MANUSCRIPT

their degradation due to the inactivation of polyphenol oxidase. Oszmiański et al.

319

(2011) also observed at laboratory scale increases (4-14%) in the content of

320

chlorogenic acid in apple juice after treatment with pectolytic and cellulolytic

321

enzymes. The changes in the content of flavan-3-ols during pasteurization and

322

clarification were higher than those for cinnamic acids (Table 3). The increase in

323

the content of epicatechin and procyanidin B2 ranged from 52 to 163% during

324

pasteurization and clarification steps. Similar increases (109-286%) have been

325

observed for these flavan-3-ols during such steps at laboratory scale (Spanos et

326

al., 1990). The high temperature could induce the hydrolysis of inter-flavonoid

327

linkages in procyanidins, increasing the concentration of their monomeric and

328

dimeric structures, epicatechin and procyanidin B2, while the enzymatic treatment

329

might favor the liberation of procyanidins linked to cell-wall polysaccharides,

330

especially pectin (De Paepe et al., 2014; Oszmiański et al., 2011). The content of

331

quercetin glycosides did not show significant changes during the apple juice

332

processing (Table 3). However, the quercetin aglycone was detected in clarified

333

and evaporated juices, probably as a consequence of the hydrolysis of glycosidic

334

forms during the enzymatic clarification. The effect of processing steps in free and

335

glycosylated quercetin of apple juice has been previously reported under laboratory

336

and pilot scale conditions with contradictory results (increases of up to 300% and

337

reductions of up to 75%), depending of the apple variety, enzymatic treatment, and

338

clarification and concentration methods (Gökmen et al., 2001; Markowski et al,

339

2015; Onsekizoglu et al., 2010; Spanos et al., 1990).

AC C

EP

TE D

M AN U

SC

RI PT

318

340 15

ACCEPTED MANUSCRIPT

3.4. Properties of pectin

342

The total pectin content of apple pomace was 9% (Table 4). Similar content of

343

pectin (7.8%) in apple pomace was recently reported by O’Shea et al. (2015). The

344

FT-IR spectra showed the two characteristic absorption bands for polysaccharides,

345

a strong and wide absorption band at 3100-3700 cm-1 for O-H stretching vibrations

346

and a weak absorption peak of about 2800-3000 cm-1 for C-H stretching vibrations

347

(Fig. 1) (Qiao et al., 2009). It also showed the peaks at 882 and 1273 cm-1, which

348

are characteristic of the pyranose ring of pectin and the C-O dilatation and vibration

349

(Kačuráková, Capek, Sasinkova, Wellner, & Ebringerova, 2000; Wang et al.,

350

2014). In this study, the pomace contained only CSP and NSP, while the WSP was

351

missing. The yield for CSP was 30% higher than that of NSP. These findings differ

352

from those of Kosmala et al. (2010), who observed that content of pectin types in

353

apple pomace followed the order of NSP˃CSP˃WSP (49.9–107.3, 18.9–26.0, 9.4–

354

20.0 mg/g, respectively). This difference might be a consequence of the high

355

activity of the pectin lyase at the pressing step and the using of overripe fruit in the

356

present study. The pectinolytic activity of this enzyme is particularly high for highly

357

esterified pectins and WSP use to show the highest DE (Yadav, Yadav, Yadav, &

358

Yadav, 2009). Laboratory-scale studies conducted in our laboratory with the same

359

apples demonstrated the existence of WSP in apple pomace obtained without the

360

using of enzymes (data not shown). Thus, pectin lyase might hydrolysate all of the

361

WSP as well as high amounts of CSP and NSP. The using of overripe fruit might

362

exacerbate this effect. Recently, Ramos-Aguilar et al. (2015) demonstrated that

AC C

EP

TE D

M AN U

SC

RI PT

341

16

ACCEPTED MANUSCRIPT

the yield of pectin from ripe green peppers follow the order of NSP˃CSP˃WSP but

364

this order was inverted with red peppers (WSP˃ CSP ˃NSP).

365

The extracted pectins were of low DE (Table 4). The DE decreased 49% from CSP

366

to NSP. CAP showed the highest DE (67%), which was virtually identical to that

367

provided by the supplier (70-75%). CSP and NSP from other plant sources have

368

also been described as lowly esterified pectins (DE= 11.2-14.8%) (Sila et al., 2006;

369

Sila et al., 2009). The FT-IR spectra confirmed the high difference of DE for tested

370

pectins (Fig. 1). The absorption peaks at 1750-1730 cm-1 indicated the presence of

371

carbonyl esters (C=O) (Wang et al., 2014) and their intensity and area agreed with

372

the obtained DE values, especially with those of CAP and CSP (Fig. 1, Table 4).

373

The composition of neutral sugars was different for tested pectins (Table 4). The

374

content of GalA was 10% higher in CSP than in NSP; however, CAP showed the

375

highest GalA content, which was up to 40% higher than that of the other pectins.

376

Similarly, Kosmala et al. (2010) observed that the GalA content was 49-74% higher

377

in chelator soluble pectin from apple pomace than in alkali soluble pectin. The

378

content of Man and Ara in CSP and NSP was 68-83% higher than in CAP while

379

CAP was especially rich in Gal and Glu, showing a content of these sugars 50–

380

71% higher than that of the other pectins. Interestingly, the content of almost all of

381

the tested sugars was higher (17-40%) in NSP than in CSP. This finding indicates

382

that NSP was more branched and intact than CSP (Sila et al., 2009). This was

383

supported by the MW distribution of tested pectins. They contained up to three

384

fractions but the first of them was substantially abundant. The peak MW of such

385

fraction is shown in Table 4. First fraction showed the highest MW in NSP, followed

AC C

EP

TE D

M AN U

SC

RI PT

363

17

ACCEPTED MANUSCRIPT

by CSP and CAP. In general, the MW found in this study for the most abundant

387

fractions was into the range reported for apple pectin (100-10000 kDa) (Fischer et

388

al., 1994).

389

The pectin solutions showed a pseudoplastic behavior, especially whiting the range

390

from 50 to 100 s-1 (Fig. 2). The flow behavior showed high correlation values (R2=

391

0.989–0.999) with the power law model. The k values of tested pectins were

392

significantly different, with viscosity of CSP (k= 0.053 Pa.sn) being almost twice

393

than that NSP (k= 0.027 Pa.sn). CAP showed the highest viscosity (k= 0.143

394

Pa.sn). The viscosity of tested pectins showed an inverse relationship with MW of

395

the main fractions (Table 4). The viscosity of pectin solutions is highly influenced

396

by sugar composition, DE and MW. In this study, the content of GalA and Glu and

397

the DE were directly related with viscosity. Wang et al. (2014) demonstrated that

398

the viscosity of apple pectin increased with the GalA content. Liu, Cao, Huang, Cai,

399

and Yao (2010) demonstrated that the gellification and viscosity of pectin increased

400

with the content of GalA and DE.

401

The thermogravimetric analysis showed that the tested pectins released moisture

402

(∼6%) with a maximum derivative of weight loss peak (Pk) at 80°C. The CSP

403

showed a low thermal stability fraction (Pk ≈ 200 °C). The highest degradation

404

amount for all pectins was observed at Pk between 238 and 245 °C (Fig. 3), which

405

has been associated to the pyrolytic decomposition of homogalacturonans (Combo

406

et al., 2013). Accordingly, the pectin with the higher GalA content and viscosity

407

(CAP) had the highest degradation in this region (Table 4; Figs. 2 and 3). At

408

temperatures above 350 °C, the CSP and the NSP showed degradation, which

AC C

EP

TE D

M AN U

SC

RI PT

386

18

ACCEPTED MANUSCRIPT

probably was associated to lignocellulosic materials (Yang et al., 2006). Thus, we

410

could assume that CSP contained a relatively higher content of cellulosic material

411

than NSP. NSP probably contained more lignin-associated polysaccharides

412

because it showed a higher stability at temperature above 350 °C. These

413

compositional changes were probably responsible for the absence of the direct

414

relationship between MW and viscosity and thermogravimetric properties of tested

415

pectins.

416

The color of pectins is not usually reported; however, it is an important property of

417

pectin because it may determine its use in food formulations. In this study, the CSP

418

and CAP showed similar color values, although their L* values were statistically

419

different (Table 4). The color values of these pectins (L*= 72.7- 74.1, a*= 4.1-3.9,

420

b*= 12.9-12.3) were within the ranges reported for pectin from other sources, such

421

as mango, citrus, beet and peppers (L*= 67.5–93.5, a*= -0.6–3.5 and b*= 9.38–

422

20.2) (Berardini, Knödler, Schieber, & Carle, 2005; Mesbahi, Jamalian, &

423

Farahnaky, 2005; Ramos-Aguilar et al., 2015). The NSP was darker than CSP and

424

CAP, according to its significantly lower values of L* and b*, and the higher value

425

of a* (Table 4). The darkness of pectin is highly related with their phenol content

426

(Ramos-Aguilar et al., 2015), thus, the oxidation of phenols during the extraction of

427

NSP might explain the color of this pectin type. However, the extraction conditions

428

can also influence the structure of pectin and these alterations can influence the

429

color of pectin (Pérez-Martínez et al., 2013; Einhorn-Stoll, Kastner, & Drusch,

430

2014).

AC C

EP

TE D

M AN U

SC

RI PT

409

431 19

ACCEPTED MANUSCRIPT

4. Conclusions

433

The steps of the juice production process involving high temperature and enzymes

434

had a significant effect on key components of apple juice (organic acids, phenolics,

435

and 5-HMF). Some effects of the processing coincided with those observed

436

previously at laboratory or pilot plant scale. Tested pomace contained CSP and

437

NSP but not WSP. These pectins differed from CAP in terms of their high MW,

438

Man and Ara content as well as for their low DE, GalA and Gal content. This

439

resulted in lower viscosity and different thermal stability, as compared with the

440

functional properties observed for CAP. The ripening, type of fruit and use of

441

enzymes during the process of juice production were probably involved in the

442

physical and chemical properties of extracted pectins, compromising their

443

functionality.

M AN U

SC

RI PT

432

TE D

444

Acknowledgements

446

This research was funded by the Fondo Mixto CONACYT- Gobierno del Estado de

447

Chihuahua (Project Clave: CHIH-2012-C03-194579).

448

References

449

Aguilar-Rosas, S. F., Ballinas-Casarrubias, M. L., Nevarez-Moorillon, G. V., Martin-

450

Belloso, O., & Ortega-Rivas, E. (2007). Thermal and pulsed electric fields

452

AC C

451

EP

445

pasteurization of apple juice: effects on physicochemical properties and flavour compounds. Journal of Food Engineering, 83, 41–46.

20

ACCEPTED MANUSCRIPT

Berardini, N., Knödler, M., Schieber, A., & Carle, R. (2005). Utilization of mango

454

peels as a source of pectin and polyphenolics. Innovative Food Science &

455

Emerging Technologies, 6, 442-452.

RI PT

453

Canteri-Schemin, M. H., Fertonani, H. C. R., Waszczynskyj, N., & Wosiacki, G.

457

(2005). Extraction of pectin from apple pomace. Brazilian Archives of

458

Biology and Technology, 48, 259–266.

SC

456

Combo, A. M. M., Aguedo, M., Quiévyb, N., Danthine, S., Goffina, D., Jacqueta,

460

N., Blecker C., et al. (2013). Characterization of sugar beet pectic-derived

461

oligosaccharides obtained by enzymatic hydrolysis. International Journal of

462

Biological Macromolecules, 52, 148-156.

M AN U

459

De Paepe, D., Valkenborg, D., Coudijzer, K., Noten, B., Servaes, K., De Loose, M.,

464

Voorspoels, S., et al. (2014). Thermal degradation of cloudy apple juice

465

phenolic constituents. Food chemistry, 162, 176-185.

TE D

463

Eisele, T. A., & Drake, S. R., (2005). The partial compositional characteristics of

467

apple juice from 175 apple varieties. Journal of Food Composition and

468

Analysis, 18, 213–221.

470 471

Einhorn-Stoll, U., Kastner, H., & Drusch, S. (2014). Thermally induced degradation

AC C

469

EP

466

of citrus pectins during storage-Alterations in molecular structure, colour and thermal analysis. Food Hydrocolloids, 35, 565–575.

472

Fischer, M., Arrigoni, E., & Amadò, R. (1994). Changes in the pectic substances of

473

apples during development and postharvest ripening. Part 2: Analysis of the

474

pectic fractions. Carbohydrate Polymers, 25, 167–175. 21

ACCEPTED MANUSCRIPT

Garza, S., Giner, J., Martin, O., Costa, E., & Ibarz, A. (1996). Evolución del color,

476

azúcares y HMF en el tratamiento térmico de zumo de manzana/Colour,

477

sugars and HMF evolution during thermal treatment of apple juice. Food

478

Science and Technology International, 2, 101–110.

RI PT

475

Gökmen, V., & Acar, J. (1998). An investigation on the relationship between patulin

480

and fumaric acid in apple juice concentrates. LWT-Food Science and

481

Technology, 31, 480–483.

SC

479

Gliszczynska‐Swiglo, A. A. G. A., & Tyrakowska, B. (2003). Quality of commercial

483

apple juices evaluated on the basis of the polyphenol content and the TEAC

484

antioxidant activity. Journal of Food Science, 68, 1844–1849.

485

Gökmen

V.,

&

Acar,

J.

M AN U

482

(1999).

Simultaneous

determination

of

5-

hydroxymethylfurfural and patulin in apple juice by reversed-phase liquid

487

chromatography. Journal of Chromatography A, 84, 69-74.

TE D

486

Gökmen, V., Artık, N., Acar, J., Kahraman, N., & Poyrazoğlu, E. (2001). Effects of

489

various clarification treatments on patulin, phenolic compound and organic

490

acid compositions of apple juice. European Food Research and Technology,

491

213, 194–199.

AC C

EP

488

492

He, Y., Ji, Z., & Li, S. (2007). Effective clarification of apple juice using membrane

493

filtration without enzyme and pasteurization pretreatment. Separation and

494

Purification Technology, 57, 366–373.

495

Kačuráková, M., Capek, P., Sasinkova, V., Wellner, N. & Ebringerova, A. (2000).

496

FT-IR study of plant cell wall model compounds: pectic polysaccharides and

497

hemicelluloses. Carbohydrate Polymers, 43, 195–203. 22

ACCEPTED MANUSCRIPT

498

Kadakal, Ç., & Nas, S. (2003). Effect of heat treatment and evaporation on patulin

499

and some other properties of apple juice. Journal of the Science of Food

500

and Agriculture, 83, 987–990. Kammerer, D. R., Kammerer, J., Valet, R., & Carle, R. (2014). Recovery of

502

polyphenols from the by-products of plant food processing and application

503

as valuable food ingredients. Food Research International, 65, 2–12.

504

Kosmala, M., Kołodziejczyk, K., Markowski, J., Mieszczakowska, M., Ginies, C., &

505

Renard, C. M. (2010). Co-products of black-currant and apple juice

506

production: hydration properties and polysaccharide composition. LWT-

507

Food Science and Technology, 43, 173–180.

M AN U

SC

RI PT

501

Liu, L., Cao, J., Huang, J., Cai, Y., & Yao, J. (2010). Extraction of pectins with

509

different degrees of esterification from mulberry branch bark. Bioresource

510

Technology, 101, 3268–3273.

TE D

508

Markowski, J., Baron, A., Le Quéré, J. M., & Płocharski, W. (2015). Composition of

512

clear and cloudy juices from French and Polish apples in relation to

513

processing technology. LWT-Food Science and Technology, 62, 813–820.

515 516

Mesbahi, G., Jamalian, J., & Farahnaky, A. (2005). A comparative study on

AC C

514

EP

511

functionalproperties of beet and citrus pectins in food systems. Food Hydrocolloids, 19,731–738.

517

Min, B., Bae, I. Y., Lee, H. G., Yoo, S. H., & Lee, S. (2010). Utilization of pectin-

518

enriched materials from apple pomace as a fat replacer in a model food

519

system. Bioresource Technology, 101, 5414–5418.

23

ACCEPTED MANUSCRIPT

O’Shea, N., Ktenioudaki, A., Smyth, T. P., McLoughlin, P., Doran, L., Auty, M. A.

521

E., Arendt, E. et al. (2015). Physicochemical assessment of two fruit by-

522

products as functional ingredients: apple and orange pomace. Journal of

523

Food Engineering, 153, 89–95.

RI PT

520

Onsekizoglu, P., Bahceci, K. S., & Acar, M. J. (2010). Clarification and the

525

concentration of apple juice using membrane processes: a comparative

526

quality assessment. Journal of Membrane Science, 352, 160–165.

SC

524

Onsekizoglu, P. (2013). Production of high quality clarified pomegranate juice

528

concentrate by membrane processes. Journal of Membrane Science, 442,

529

264–271.

M AN U

527

530

Ornelas-Paz, J.J., Meza, M.B., Obenland, D., Rodríguez (Friscia), K., Jain, A.,

531

Thornton, S., & Prakash, A. (2017). Effect of phytosanitary irradiation on the

532

postharvest

533

mukakukishu). Food Chemistry, 230, 712-720.

of

Seedless

Kishu

mandarins

(Citrus

kinokuni

TE D

quality

Oszmiański, J., Wojdyło, A., & Kolniak, J. (2011). Effect of pectinase treatment on

535

extraction of antioxidant phenols from pomace, for the production of puree-

536

enriched cloudy apple juices. Food Chemistry, 127, 623–631.

AC C

EP

534

537

Pérez-Martínez, J.D., Sánchez-Becerril, M., Ornelas-Paz, J.J., González-Chávez,

538

M.M., Ibarra-Junquera, V., & Escalante-Minakata, P. (2013). The Effect of

539

extraction conditions on the chemical characteristics of pectin from Opuntia

540

ficus indica cladode flour. Journal of Polymers and the Environment, 21,

541

1040-1051.

24

ACCEPTED MANUSCRIPT

Qiao, D., Hu, B., Gan, D., Sun, Y., Ye, H., & Zeng, X. (2009). Extraction optimized

543

by using response surface methodology, purification and preliminary

544

characterization of polysaccharides from Hyriopsis cumingii. Carbohydrate

545

Polymers, 76, 422–429.

RI PT

542

Ramos-Aguilar, O. P., Ornelas-Paz, J. J., Ruiz-Cruz, S., Zamudio-Flores, P. B.,

547

Cervantes-Paz, B., Gardea-Béjar, A. A., Pérez-Martínez, J. D., et al. (2015).

548

Effect of ripening and heat processing on the physicochemical and

549

rheological properties of pepper pectins. Carbohydrate Polymers, 115, 112–

550

121.

M AN U

SC

546

Sila, D. N., Van Buggenhout, S., Duvetter, T., Fraeye, I., De Roeck, A., Van Loey,

552

A., & Hendrickx, M. (2009). Pectins in processed fruits and vegetables: Part

553

II—structure–function relationships. Comprehensive Reviews in Food

554

Science and Food Safety, 8, 86–104.

555

TE D

551

Sila, D. N., Smout, C., Elliot, F., Loey, A. V., & Hendrickx, M. (2006). Non‐enzymatic

depolymerization

of

carrot

pectin:

toward

a

better

557

understanding of carrot texture during thermal orocessing. Journal of Food

558

Science, 71, E1 – E9.

EP

556

Spanos, G. A., Wrolstad, R. E., & Heatherbell, D. A. (1990). Influence of

560

processing and storage on the phenolic composition of apple juice. Journal

561

AC C

559

of Agricultural and Food Chemistry, 38, 1572–1579.

562

Suárez-Jacobo, Á., Rüfer, C. E., Gervilla, R., Guamis, B., Roig-Sagués, A. X., &

563

Saldo, J. (2011). Influence of ultra-high pressure homogenisation on

25

ACCEPTED MANUSCRIPT

564

antioxidant capacity, polyphenol and vitamin content of clear apple juice.

565

Food Chemistry, 127, 447–454. Vladisavljević, G. T., Vukosavljević, P., & Bukvić, B. (2003). Permeate flux and

567

fouling resistance in ultrafiltration of depectinized apple juice using ceramic

568

membranes. Journal of Food Engineering, 60, 241–247.

RI PT

566

Voragen, A. G. J., Schols, H. A., & Pilnik, W. (1986). Determination of the degree

570

of methylation and acetylation of pectins by HPLC. Food Hydrocolloids, 1,

571

65–70.

573

Wang, X., Chen, Q., & Lü, X. (2014). Pectin extracted from apple pomace and

M AN U

572

SC

569

citrus peel by subcritical water. Food Hydrocolloids, 38, 129–137. Welke, J. E., Hoeltz, M., Dottori, H. A., & Noll, I. B. (2009). Effect of processing

575

stages of apple juice concentrate on patulin levels. Food Control, 20, 48–52.

576

Yadav, S., Yadav, P. K., Yadav, D., & Yadav, K. D. S., 2009. Pectin lyase: A

577 578

TE D

574

review. Process Biochemistry, 44, 1–10. Yang, H., Yan, R., Chen, H., Zheng, C., Lee, D. H., & Liang, D. T. (2006). In-Depth investigation

of

biomass

pyrolysis

based

on

three

major

580

components: hemicellulose, cellulose and lignin. Energy & Fuels, 20, 398-

581

393.

AC C

EP

579

582

Youn, K. S., Hong, J. H., Bae, D. H., Kim, S. J., & Kim, S. D. (2004). Effective

583

clarifying process of reconstituted apple juice using membrane filtration with

584

filter-aid pretreatment. Journal of Membrane Science, 228, 179-186.

585 586 26

ACCEPTED MANUSCRIPT

Figure captions

588

Fig. 1. FT-IR spectra of commercial apple pectin (CAP, ─) and chelator- and alkali-

589

soluble pectin fractions (CSP, ─; NSP, ─) from apple pomace.

590

Fig. 2. Flow curves and parameters of power law model obtained from solutions of

591

commercial apple pectin (CAP, ⚫) and chelator- and alkali-soluble pectin fractions

592

(CSP, ⚪; NSP, ▼) from apple pomace. n, flow behavior. k, consistency index.

593

Fig. 3. Thermogravimetric analysis (A) and derivative thermogravimetric (B) curves

594

for commercial apple pectin (CAP, ─) and chelator- and alkali-soluble pectin

595

fractions (CSP, ─; NSP, ─) from apple pomace.

M AN U

SC

RI PT

587

596

597

Table captions

599

Table 1. Effect of industrial processing on color, clearness, total soluble solids,

600

titratable acidity, and total phenolic content in apple juice

601

Table 2. Concentration of sugars and organic acids in apple juice at selected steps

602

of the industrial production process.

603

Table 3. The effect of the industrial processing on the content of phenolic

604

compounds in of apple juice.

605

Table 4. Yield and physicochemical characteristics of commercial apple pectin

606

(CAP) and chelator- and alkali-soluble pectin fraction (CSP and NSP) from

607

enzyme-treated apple pomace.

AC C

EP

TE D

598

27

ACCEPTED MANUSCRIPT

Table 1.

0.1 ± 0.0 c

0.8 ± 0.2 c

4.9 ± 0.46 d

10.7 ± 1.5 c

11.2 ± 0.4 b

7.0 ± 0.1 ab

Color a

(440 nm)

Clearness

10.7 ± 0.7 b

11.6 ± 0.5 b

69.0 ± 0.6 a

7.8 ± 0.4 a

7.3 ± 0.2 ab

6.5 ± 0.2 b

0.35 ± 0.0 ab

0.4 ± 0.0 a

M AN U

(%)

AC C

equivalents /L)

0.3 ± 0.0 b

EP

0.2 ± 0.0 c

TE D

Titratable acidity

(g chlorogenic acid

47.6 ± 0.3 b

90.1 ± 0.5 b

Total soluble solids

Total phenols

53.8 ± 2.4 a

Concentration

98.8 ± 2.6 a

(625 nm )a

(g malic acid/ L)

Clarification

RI PT

Pasteurization

SC

Pressing

The juice was adjusted at 11.2 °Brix before color, clearness, acidity, and total phenolic content measurements. Values represent the mean of 12 individual measurements ± the standard error. Data in the same row connected by different letters are significant different (p ≤ 0.05). a Values are given % transmittance.

ACCEPTED MANUSCRIPT

Table 2. Pasteurization

Fructose

56.8 ± 2.2 a

64.0 ± 3.5 a

Sucrose

32.0 ± 1.8 a

35.9 ± 1.0 a

Glucose

16.1 ± 0.8 a

16.5 ± 0.8 a

Malic

2732.4 ± 185.7 b

Ascorbic

Clarification

RI PT

Pressing Sugars (g/L)

Concentration

58.6 ± 1.4 a

33.0 ± 1.6 a

31.4 ± 1.0 a

16.0 ± 0.6 a

17.2 ± 0.6 a

3653.8 ± 387.2 a

3137.7 ± 98.7 ab

2953.4 ± 95.3 ab

935.6 ± 121.3 a

722.7 ± 64.6 a

820.6 ± 80.5 a

808.7 ± 170.4 a

Succinic

555.0 ± 52.7 b

707.3 ± 57.2 a

526.1 ± 48.3 b

465.9 ± 27.5 b

Oxalic

196.7 ± 40.0 a

95.3 ± 16.4 b

151.6 ± 13.7 ab

201.0 ± 18.8 a

3.6 ± 0.5 c

5.3 ± 0.4 bc

8.3 ± 0.3 a

7.1 ± 2.9 a

1.9 ± 0.3 a

3.1 ± 0.3 a

Fumaric

4.2 ± 0.5 a

M AN U

AC C

EP

6.8 ± 1.3 ab

TE D

Organic acids (mg/L)

Tartaric

SC

57.7 ± 1.7 a

The juice was adjusted at 11.2 °Brix before analysis. Values represent the mean of 12 individual measurements ± the standard error. Data in the same row connected by different letters are significant different (p ≤ 0.05).

ACCEPTED MANUSCRIPT

Table 3. Compound (mg/L)

Pressing

Pasteurization

Clarification

Concentration

Chlorogenic acid

18.5 ± 1.3 c

31.7 ± 2.4 b

38.8 ± 2.0 a

37.2 ± 1.1 ab

Caffeic acid

0.9 ± 0.0 b

1.0 ± 0.1 b

1.2 ± 0.0 a

1.2 ± 0.0 a

ND

ND

0.04 ± 0.00

ND

0.5 ± 0.0 b

0.5 ± 0.0 b

0.9 ± 0.1 a

0.8 ± 0.1 a

0.2 ± 0.0 a

0.2 ± 0.0 ab

0.1 ± 0.0 b

0.1 ± 0.0 b

6.6 ± 0.8 a

8.4 ± 0.6 a

8.9 ± 0.8 a

15.4 ± 1.5 a

16.9 ± 2.0 a

15.1 ± 0.2 ab

ND

ND

0.3 ± 0.1 a

0.3 ± 0.1 a

Quercetin 3-D-galactoside

1.5 ± 0.1 a

1.8 ± 0.2 a

1.5 ± 0.1 a

1.6 ± 0.1 a

Quercetin 3-β-glucoside

0.5 ± 0.0 a

0.5 ± 0.0 a

0.6 ± 0.0 a

0.6 ± 0.0 a

4.8 ± 0.5 b

6.3 ± 0.9 b

10.7 ± 0.9 a

9.7 ± 0.6 a

p-Coumaric acid

SC

Cinnamic acid

Flavan-3-ols Epicatechin

3.2 ± 0.4 b

Procyanidin B2

10.1 ± 0.8 b

Phloridzin

EP

Dihydrochalcones

AC C

Quercetin

TE D

Flavonols

M AN U

Hydroxybenzoic acid Protocatechuic acid

RI PT

Hydroxycinnamic acids

The juice was adjusted at 11.2 °Brix before analysis. Values represent the mean of 12 individual measurements ± the standard error. Data in the same row connected by different letters are significant different (p ≤ 0.05). ND, not detected.

ACCEPTED MANUSCRIPT

Table 4. Pectin type CSP

NSP

-

5.3 ± 0.1 a

3.7 ± 0.1 b

L*

72.7 ± 0.1 b

74.1 ± 0.4 a

54.6 ± 0.1 c

a*

4.1 ± 0.0 b

3.9 ± 0.1 b

5.3 ± 0.1 a

b*

12.9 ±0.1 a

12.3 ± 0.1 b

11.0 ± 0.1 c

436.6 ± 8.0 b

393.5 ± 5.2 c

Galacturonic acid (mg/g)

654.1 ± 5.7 a

Monosaccharides (mg/g)

M AN U

Tristimulus color

SC

Yield (%)

RI PT

CAP

Mannose Arabinose Galactose

Xylose Rhamnose Fucose

EP

Degree of esterification (%)

132.9 ± 3.0 b

198.3 ± 5.0 a

27.5 ± 0.3 c

103.4 ± 1.9 b

166.3 ± 1.9 a

228.6 ± 2.2 a

71.3 ± 1.8 c

115.0 ± 1.8 b

45.5 ± 0.3 a

16.1 ± 0.2 b

13.4 ± 0.1 c

13.0 ± 0.3 a

11.7 ± 0.5 b

14.0 ± 0.2 a

9.0 ± 0.1 a

4.8 ± 0.2 c

7.9 ± 0.3 b

2.8 ± 0.1 a

2.5 ± 0.1 b

ND

67.4 ± 3.4 a

44.6 ± 2.9 b

22.8 ± 2.4 c

644.5 ± 8.9 c

1559.6 ± 49.6 b

2360.6 ± 38.3 a

TE D

Glucose

42.1 ± 0.5 c

AC C

Peak molecular weight (kDa)

Values represent the mean of at least three individual measurements ± the standard error. Values in the same row with different letters are significantly different (p < 0.05). ND, not detected.

SC

RI PT

ACCEPTED MANUSCRIPT

3000-2800

100

96 3700-3100

92 90

86

Fig. 1.

1273-882

EP

88

84 4000

1750-1730

TE D

94

3500

AC C

Transmittance (%)

98

M AN U

102

3000

2500 2000 Wavenumber (cm-1)

1500

1000

500

ACCEPTED MANUSCRIPT

0.10

M AN U

SC

0.08

n

k (Pa·s ) a 0.143 ± 0.003 b 0.053 ± 0.001 c 0.027 ± 0.001

RI PT

CAP CSP NSP

0.06

0.04

0.00 100

200

EP

0

TE D

0.02

AC C

Apparent viscosity (Pa.s)

0.12

n a 0.919 ± 0.004 b 0.891 ± 0.004 c 0.852 ± 0.006

Fig. 2.

300

Shear rate (1/s)

400

500

600

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 3.

ACCEPTED MANUSCRIPT

-The impact of industrial processing on properties of apple juice is scarcely known -Few pectin fractions from apple pomace have been characterized

RI PT

-The sugar content in apple juice was not altered by any processing step

-The pasteurization step altered the levels of individual phenols and acids -Concentration step favored the formation of 5-HMF

AC C

EP

TE D

M AN U

SC

-Pomace pectins showed less functionality than commercial apple pectin