Accepted Manuscript

A new natural source for obtainment of inulin and fructo-oligosaccharides from

industrial waste of Stevia rebaudiana Bertoni

Sheila Mara Sanches Lopes, Gabriela Krausová, José Walter Pedroza Carneiro,
José Eduardo Gonçalves, Regina Aparecida Correia Gonçalves, Arildo José
Braz de Oliveira

PII: S0308-8146(16)32130-6
DOI: http://dx.doi.org/10.1016/j.foodchem.2016.12.100
Reference: FOCH 20414

To appear in: Food Chemistry

Received Date: 16 August 2016

Revised Date: 28 November 2016
Accepted Date: 31 December 2016

Please cite this article as: Mara Sanches Lopes, S., Krausová, G., Walter Pedroza Carneiro, J., Eduardo Gonçalves,
J., Aparecida Correia Gonçalves, R., José Braz de Oliveira, A., A new natural source for obtainment of inulin and
fructo-oligosaccharides from industrial waste of Stevia rebaudiana Bertoni, Food Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.foodchem.2016.12.100

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 1

1 A new natural source for obtainment of inulin and fructo-oligosaccharides

2 from industrial waste of Stevia rebaudiana Bertoni

3 Sheila Mara Sanches Lopesa, Gabriela Krausováb, José Walter Pedroza Carneiroc, José

4 Eduardo Gonçalvesd, Regina Aparecida Correia Gonçalvesa, Arildo José Braz de

5 Oliveiraa*

a
6 Graduate Program in Pharmaceutical Sciences, Department of Pharmacy, State

7 University of Maringá, Ave. Colombo 5790, 87.020-900, Maringá, Brazil.

b
8 Department of Microbiology and Technology, Dairy Research Institute, Ke Dvoru 12a,

9 160 00 Prague, Czech Republic.

c
10 Department of Agronomy, Iguatemi Research Farm, State University of Maringá, Av.

11 Colombo 5790, 87.020-900, Maringá, Brazil.

d
12 Program of Master in Health Promotion and Program of Master in Clean

13 Technologies, University Center of Maringá, Av. Guedner, 1610, 87.050-900, Maringá,

14 Brazil.

15

16

17 * Corresponding author: Graduate Program in Pharmaceutical Science, Department of

18 Pharmacy, State University of Maringá, Ave. Colombo 5790, 87.020-900, Maringá,

19 Brazil. Tel.: +55 44-3011-4872; e-mail address: ajboliveira@uem.br

20

21

22

23

24
 2

25 ABSTRACT

26 Fructan-type inulin and fructo-oligosaccharides (FOS) are reserve polysaccharides that

27 offer an interesting combination of nutritional and technological properties for food

28 industry. Stevia rebaudiana is used commercially in the sweetener industry due to the

29 high content of steviol glycosides in its leaves. With the proposal of using industrial

30 waste, the objective of the present study was to isolate, characterize and evaluate the

31 prebiotic activity of inulin and FOS from S. rebaudiana stems. The chemical

32 characterization of the samples by GC-MS, NMR and off-line ESI-MS showed that it

33 was possible to obtain inulin molecules from the S. rebaudiana stems with a degree of

34 polymerization (DP) of 12, and FOS with a DP < 6. The in vitro prebiotic assay of these

35 molecules indicates a strain specificity in fermentation capacity of fructans as substrate.

36 FOS molecules with a low DP are preferably fermented by beneficial microbiota than

37 inulin molecules with higher DP.

38

39 Chemical compounds studied in this article

40 Inulin from chicory (PubChem CID: 16219508); Fructose (PubChem CID: 5984);

41 Glucose (PubChem CID: 5793); Myo-inositol (PubChem CID: 892); Ethanol (PubChem

42 CID: 702); Trifluoroacetic Acid (PubChem CID: 6422); Hexamethyldisilazane

43 (PubChem CID: 13838); Chlorotrimethylsilane (PubChem CID: 6397); Pyridine

44 (PubChem CID: 1049).

45

46 Keywords: Industrial waste, Stevia rebaudiana, inulin, fructo-oligosaccharides, prebiotic

47 activity

48

49
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50 1. Introduction

51 Stevia rebaudiana Bertoni is a perennial herb of the Asteraceae family and is

52 native from South America. It has been used commercially in the sweetener industry

53 since the 1970s, when Japanese researchers developed a process for the extraction and

54 refinement of stevioside and rebaudioside from its leaves (Serfaty, Ibdah, Fischer,

55 Chaimovitsh, Saranga & Dudai, 2013; Tavarini & Angelini, 2013). Fructans are another

56 type of metabolite of commercial interest, and have been isolated from the roots of S.

57 rebaudiana (Lopes et al., 2016; Lopes, Krausová, Rada, Gonçalves, Gonçalves &

58 Oliveira, 2015; Oliveira et al., 2011).

59 Inulin and fructo-oligosaccharides (FOS) are fructan-type polysaccharides that

60 consisting of (2→1)-linked β-D-fructofuranosyl (β-D-Fruf) residues with a terminal

61 glucose residue linked by an α-D-(1→2) bond. The general structure of fructans can be

62 described as GFn, where G and F represent glucose and fructose, respectively, and ‘n’ is

63 the number of fructosyl units (Shoaib et al., 2016; Singh; Singh & Kennedy, 2016).

64 The fructans differ in chain length, or degree of polymerization (DP). Fructo-

65 oligosaccharides are smaller molecules with a DP < 9, and inulin has DP ranging from

66 10 to 65 fructose units, but the most common are molecules with DP between 12-15

67 (Singh et al., 2016).

68 These compounds are considered a functional food ingredient that can be used as

69 a dietary fiber and prebiotic (Caleffi et al., 2015; Moreno-Vilet et al., 2014). Regular

70 consumption of such non-digestible polysaccharides (i.e. prebiotics) has positive effects

71 on human health (Shoaib et al., 2016). These compounds can reduce cardiometabolic

72 risk by diminishing levels of triglycerides and cholesterol, modulating hyperglycemia

73 (Mitchell et al., 2015), preventing colorectal cancer (Ambalam, Raman, Purama &

74 Doble, 2016), increasing the intestinal absorption of minerals (Lobo et al., 2011) and
 4

75 improving immune system efficiency (Moreno-Vilet et al., 2014; Peshv & Van den

76 Ende, 2014).

77 Fructans offer a unique combination of nutritional properties and technological

78 applications, they are used as a fat and sugar substitute in low-calorie foods, while

79 improving the taste and mouthfeel of food items such as dairy products (Aravind,

80 Sissons, Fellows, Blazek & Gilbert, 2012; Chi, Zhang, Cao, Liu, Cui & Zhao, 2011;

81 Crispin-Isidio, Lobato-Calleros, Espinosa-Andrews, Alvarez-Ramirez &Vernon-Carter,

82 2015).

83 Sustainable technological development aims to reduce environmental impact

84 using renewable resources such as raw materials from industry (Misselbrooka, Menzi &

85 Cordovil, 2012). Organic wastes generated by agriculture and industry are an abundant

86 natural biomass source, which can be recycled for the production of compounds of

87 commercial interest. Mainly, functional ingredients with health promotion potential and

88 prevention of diseases, that meet consumer requirements for health foods (Castro,

89 Soares, Albernaz & Sato, 2016; Salihu, Alam, AbdulKarima & Salleh, 2012).

90 The objective of the present study was to isolate and characterize fructans (inulin

91 and fructo-oligosaccharides) from S. rebaudiana stems, as well as evaluate the prebiotic

92 activity of these molecules in vitro, with the propose of obtaining products of

93 commercial interest from waste generated by the sweetener industry.

94 2. Materials and methods

95 2.1. Materials

96 The S. rebaudiana stems were obtained from field cultivar of the Iguatemi

97 Research Station (located at 230 20' SL , 52 0 04 'WL and altitude of 506 m) of the State

98 University of Maringá. The S. rebaudiana voucher specimen (14301-HUEM) was

 5

99 deposited at the Herbarium of the State University of Maringá, Brazil. The stems were

100 dried in a drying oven at 48°C for 3 days and milled.

101 Inulin extracted from chicory (Orafti® GR) and fructo-oligosaccharides (Orafti®

102 P95) obtained by the hydrolysis of the chicory inulin used as standard, were purchased

103 from Beneo, Belgium. The fructose, glucose and myo-inositol standards were from

104 Sigma-Aldrich®. All other chemical reagents were analytical grade.

105

106 2.2. Fructan extraction

107 The S. rebaudiana stems (24.0 g) were extracted with deionized water (300 mL)

108 under reflux conditions, at 80 °C. Sequential extraction was carried out at 4, 8 and 12 h.

109 The crude extracts were separately concentrated in a rotary evaporator and were then

110 precipitated with ethanol at a 1:3 (v/v) ratio and maintained at 4°C for 24 h under

111 refrigerated conditions. The crude extracts were centrifuged (6000 x g at 4 °C for 20

112 min).

113 The inulin molecules with high molecular weight were obtained from the

114 precipitate fraction. The fructo-oligosaccharides (FOS) were obtained in a soluble form

115 from the ethanolic supernatant. Both fractions were lyophilized to obtain the dry inulin

116 and FOS extracts.

117

118 2.3. Total sugar determination

119 The total carbohydrate content in the extracts was determined by the phenol-

120 sulfuric acid method with D-fructose as the standard (Dubois, Gilles, Hamilton, Reberes,

121 & Smith, 1956).

122

123
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124 2.4. Monosaccharide composition

125 The samples of inulin and FOS (5.0 mg) from S. rebaudiana stems were

126 hydrolyzed with 0.5 M trifluoroacetic acid (TFA) at 60 °C for 1 h, followed by oxime

127 derivatization with methoxyamine hydrochloric acid in pyridine (20 mg/mL) at 70 °C

128 for 1 hour. Then, the oximes were trimethylsilylated with 200 µL of the following

129 derivatization mixture: HMDS + TMCS + Pyridine, 3:1:9 (Lopes et al., 2015).

130 The silylated oxime derivatives (dissolved in 200 µL hexane) were analyzed by

131 Gas Chromatography Agilent 7890B coupled with Mass Spectrometry Agilent 5977A

132 MSD, using an HP5-MS UI-Agilent with a fused silica capillary column (30 m × 0.25

133 mm × 0.25 µm). Helium was used as the carrier gas, with an oven temperature of 170-

134 210 °C (2 °C/min). The injector and interface were kept at 280 ºC and 260 ºC,

135 respectively. The injections were made in split mode with a split ratio of 1:40. The mass

136 spectrometer was operated in electron impact (EI) mode at 70 eV. The quadrupole and

137 source temperatures were 150 ºC and 230 °C, respectively.

138 The characterization of the compounds was performed using the NIST Mass

139 Spectral Library Database - NIST 11 and through comparison with the mass spectrum

140 and retention time of the standard glucose and fructose analyzed under the same

141 conditions.

142 The monosaccharide concentrations were obtained by analysis of the relative

143 area of the integration of the chromatographic peaks, corrected by internal standard. The

144 degree of polymerization by GC-MS was calculated by the ratio between the fructose

145 and glucose concentrations (Lopes et al., 2015).

146

147 2.5. 1H and 13C NMR analyzes

 7

148 The samples of inulin and FOS (20.0 mg) were dissolved in deuterium oxide

149 (D2O) 99.9%, maintained at 45 °C for 24 h for hydrogen exchange and then lyophilized.

150 The dried waste was solubilized in 700 µL of D2O and then analyzed.

151 One- and two-dimensional NMR spectra were recorded at 298 K using

152 a Bruker Spectrometer (Avance III HD model) operating at 500.0 MHz for the 1 H
13
153 nucleus and 125.0 MHz for the C nucleus, using the standard pulse sequences

154 available in the Bruker software. The chemical shifts (δ) were expressed in parts per

155 million (ppm).

156 The spectra signals were assigned in comparison with the inulin standard

157 (Orafti® GR), FOS standard (Orafti® P95), and according to literature data (Caleffi et

158 al., 2015; Lopes et al., 2015, Oliveira et al., 2011).

159 The DP by 1H NMR was calculated from the mean ratio between the integral

160 proton signal (H3-Fru and H4-Fru) and the integral of the anomeric hydrogen glucose

161 signal (H1’-Glc) (Caleffi et al., 2015). The integrals of the protons were calculated by

162 the Mestre Nova 6.1 software package (Mestrelab Research S. L., 2010).

163

164 2.6. Electrospray ionization mass spectrometric analysis of FOS

165 The FOS molecules were dissolved in Milli-Q water (1 mg/mL), and an aliquot

166 of 250 µL of FOS was added to 250 µL of acetonitrile. One drop of formic acid was

167 added and the samples were centrifuged at 6000 x g for 5 min.

168 The supernatant was introduced into the Quattro MicroTM API (Waters) mass

169 spectrometer using a syringe pump (40 µL/min), for off-line ESI-MS analysis. Mass

170 spectra were obtained in the positive ionization mode, setting the capillary voltage at

171 3500 V, the cone voltage at 70 V, the source temperature at 120 °C and the desolvation
 8

172 temperature at 350 °C. Each spectrum was produced by the accumulation of data over 1

173 min (Lopes et al., 2016; Oliveira et al., 2011).

174

175 2.7. Prebiotic activity testing

176 Prebiotic activity was tested in seven strains of bifidobacteria and lactobacilli

177 originating from various sources including commercially used dairy strains, as well as

178 isolates from infant feces and biopsy samples. The strains Bifidobacterium bifidum

179 CCDM 559, Bifidobacterium breve CCDM 562 and Lactobacillus animalis CCDM 382

180 were obtained from the Culture Collection of Dairy Microorganisms Laktoflora

181 (Prague, Czech Republic). The Lactobacillus casei subsp. paracasei PE1TB-P and

182 Lactobacillus gasseri PHM-7E1 strains were isolated from biopsy samples (Dairy

183 Research Institute, Tábor, Czech Republic) and the Lactobacillus fermentum RL25

184 strain came from infant feces (Czech University of Life Sciences, Prague, Czech

185 Republic). The commercial strain Bifidobacterium animalis subsp. lactis Bb12 was

186 purchased from Chr. Hansen (Hoersholm, Denmark).

187 Bacterial strains were grown in a basal medium containing 10.0 g of peptone, 10.0

188 g of tryptone, 5.0 g of yeast extract, 1 mL of Tween 80® and 2.0 g of carbon source per

189 1 L distilled water. The medium was autoclaved at 21 °C for 15 min. Inulin and fructo-

190 oligosaccharides from S. rebaudiana stems were used as a carbon source. As a negative

191 control, a medium without carbohydrate and an uninoculated medium were used. The

192 Wilkins Chalgren broth (Oxoid, Basingstoke, UK) was used as a control of bacterial

193 growth and viability. The Orafti® P95, a commercially available prebiotic (containing

194 FOS from chicory) was used for comparison with the FOS isolated from S. rebaudiana.

195 All strains were precultivated in Wilkins Chalgren broth (Oxoid, Basingstoke, UK)

196 overnight (37 °C for 16 h).

 9

197 Bacterial suspensions (0.5 mL) of the respective strains in the exponential growth

198 phase were used to inoculate each tested media. Prior to this, they were centrifuged

199 (6000 x g for 7 min) and resuspended in saline (0.9%) at a concentration of 106

200 CFU/mL. Subsequently, the media were incubated at 37 °C for 24 h in anaerobic jars

201 (Oxoid, Basingstoke, UK). All the strains were grown in triplicate.

202 The optical density of the media was measured (densitometer DEN-1, Dynex,

203 Czech Republic) after inoculation (time zero), and at the end of cultivation (24 h). The

204 bacterial growth was evaluated as the change in the absorbance (at the wavelength of

205 540 nm) of the media during 24 h of fermentation. For statistical evaluation of the data,

206 Statgraphics® Centurion XV Software (StatPoint, Inc., Warrenton, USA) and the

207 multiple range comparison LSD test were used. A difference was considered to be

208 statistically significant at the level of p < 0.05.

209

210 3. Results and discussion

211 3.1 Fructan extraction

212 The greatest extraction efficiency was observed during the first 4 hours. The

213 precipitate fraction identified as inulin exhibited a yield of 4.5% and the soluble fraction

214 (FOS) presented a yield of 11%. Additional hours of extraction (8 and 12 h) did not

215 significantly increase the extraction yield of the polysaccharides. The carbohydrate

216 content in the factions was 35% and 60% for precipitate and supernatant fraction,

217 respectively.

218 Hot-water extraction is a method that ensures a good yield, as well as increasing

219 the solubility of the polysaccharides and the diffusion coefficient (Miao, Lin, Cao, He,

220 Qiao & Cao, 2011). Other studies consider an ideal extraction time of 4 hours, as
 10

221 beyond this time the polysaccharide yield does not significantly increase (Miao et al.,

222 2011). A similar data was observed in the present study.

223 The total fructans content was about 15%, similar to the value (15-20%)

224 observed in species currently used for commercial purposes, such as Jerusalem

225 artichoke (Helianthus tuberosus) and chicory (Cichorium intybus) (Chi et al., 2011).

226

227 3.2 Fractionation of the fructans crude extract

228 The fractionation of the fructan crude extract from S. rebaudiana stems allowed

229 to obtain two compounds with industrial interest, inulin and fructo-oligosaccharides.

230 The difference in the degree of polymerization (DP) of these molecules plays a key role

231 in the functionalities of fructans, resulting in different technological and nutritional

232 properties (Karimi, Azizi, Ghasemlou & Vaziri, 2015).

233 Inulin molecules are used as fat substitutes and texture agents (Karimi et al.,

234 2015), while FOS molecules are used as a low-calorie sugar replacement (1-2 kcal g-1)

235 (Villegas, Tárrega, Carbonell & Costell, 2010). The use of blends of short- and long-

236 chain fructans, which are selectively metabolized in the proximal and distal colon,

237 respectively, enhances the fermentative and prebiotic effects of these molecules (Karimi

238 et al., 2015; Tárrega, Rocafull & Costell, 2010).

239 The obtaining of two products (inulin and FOS) with different industrial

240 applications from the same source (S. rebaudiana stems) is a low cost option for

241 commercial extraction from industrial waste.

242

243 3.3 Inulin characterization

244 In the monosaccharide composition analysis by GC-MS, the peaks were

245 identified according to retention time in comparison with fructose (Fig. 1C) and glucose
 11

246 (Fig. 1D) standards, the fragmentation profile of the mass spectra of oxime-silylated

247 derivatives (Fig. S1) and literature data (Lopes et al., 2015).

248 The inulin chromatogram (Fig. 1A) showed peaks identified as fructose units

249 with retention times of 7.9, 8.0, 8.2, 9.6 and 9.9 min. Glucose units with retention times

250 of 10.2 and 12.6 min were observed. Retention times of 11.8, 15.3 and 15.9 min were

251 assigned to internal standard myo-inositol. Quantitative analysis by GC-MS showed a

252 higher content of fructose (32%) than glucose (2.6%). The DP calculated for inulin

253 obtained from S. rebaudiana stems by GC-MS was 12.

254 In the NMR analysis of inulin molecules, the H1’ signal of terminal glucose was

255 observed at low intensity in the anomeric region of the 1 H NMR spectrum (Fig. 2A) at δ

256 5.45 (1H, d, J = 3.8 Hz). Characteristic signals of fructose residues (→2-β-Fru) H3 and

257 H4 were observed at δ 4.27 (11H, d, J = 8.0 Hz) and δ 4.11 (14H, t, J = 8.4 Hz),

258 respectively.

259 The other fructosyl hydrogen signals (H1, H5 and H6) were assigned between

260 the δ 3.70-3.95 region (Table 1). All chemical shifts are consistent with the spectrum of

261 the inulin standard (Orafti® GR, Fig. S2) and literature data (Caleffi et al., 2015; Lopes

262 et al., 2015; Oliveira et al., 2011). The DP of inulin estimated through 1 H NMR analysis

263 was 12, a similar value was observed in the GC-MS analysis.
13
264 The C NMR spectrum (Fig. S3) showed six signals assigned to fructose

265 carbons. There was a peak of anomeric carbon at δ 103,20 (C2-Fru), methylene group

266 (CH2) peaks at δ 60.74 (C1-Fru) and 62.26 (C6-Fru), and peaks assigned to the C-H

267 group at δ 74.39 (C4-Fru), 77.00 (C3-Fru) and 81.02 (C5-Fru). These data were

268 confirmed using the DEPT 135 experiment (Caleffi et al., 2015; Lopes et al., 2015).

269 The correlation map HMBC (Fig. 3) showed the occurrence of a cross-peak

270 between H-1 and C-2, and the concomitant absence of correlations between C-2 and H-
 12

271 6 suggests a β-(2→1) linkage between fructosyl residues. The other correlations

272 observed, H3-Fru/C1-Fru, H4-Fru/C3-Fru, H3-Fru/C4-Fru, H4-Fru/C5-Fru, H6-Fru/

273 C5-Fru and H4-Fru/C6-Fru, contribute to the proposed assignments for the isolated

274 inulin molecule (Caleffi et al., 2015; Lopes et al., 2015).

275 From the degree of polymerization calculated by 1H NMR and GC-MS was

276 possible to estimated the molecular weight (Mw) of inulin molecule using the following

277 formula described by Suzuki et al., (2013): [C6nH10n+2O5n+1], where “n” is the average

278 value of DP of the inulin. Considering the DP = 12 and the monoisotopic mass of

279 atoms, the molecular weight of the inulin molecules was 1947 g/mol. This value is in

280 accordance with the literature which shows that inulin molecules with DP ranging from

281 2-13 have molecules with Mw in the range of 1400-2500 g/mol (Evans, Gallagher,

282 Ratcliffe and Williams, 2015).

283

284 3.4 FOS characterization

285 In monosaccharide composition analysis, the FOS chromatogram (Fig. 1B)

286 showed peaks with retention times of 7.7, 7.9, 8.1, 8.5, 9.5 and 9.7 min that were

287 assigned to fructose units. Signals relating to glucose units were observed at retention

288 times of 9.9, 10.1, 10.6, and 12.5 min. Similar retention times were observed for

289 fructose (Fig. 1C) and glucose (Fig. 1D) standards. The mass spectra of the majority

290 peaks of oxime-silylated derivatives are shown in Fig. S1. The data obtained for the

291 FOS molecules is consistent with literature (Lopes et al., 2016). The quantitative data

292 showed 45% fructose and 11% glucose concentration. The degree of polymerization by

293 GC-MS showed molecules with a DP = 4.

294 In the 1H NMR spectrum of FOS (Fig. 2B), characteristic signals (→2-β-Fru)

295 were observed at δ 4.27 (3H, d, J = 8.0 Hz) and δ 4.11 (6 H, t, J = 8.4 Hz), which
 13

296 corresponded to H3 and H4, respectively. The correlation 1H-13C of these signals were

297 observed in the HSQC Correlation Map (Fig. 4) at δ 4.27/76.93 (H3-Fru/C3-Fru) and

298 4.12/74.93 ppm (H4-Fru/C4-Fru). The H1’ of the terminal glucose was assigned in the

299 anomeric region, with a low intensity signal at δ 5.45 (1H, d, J = 3.8 Hz) (Lopes et al.,

300 2016).

301 In addition to the fructo-oligosaccharides, signals of mono- and disaccharides

302 were also observed in the extracts (Fig. 2B). These signals were confirmed using 2D

303 HSQC Correlation Map (Fig. 4). The anomeric signals due to free glucose were

304 observed at δ 5.24/91.75 ppm for α-glucose and at δ 4.65/95.81 ppm for β-glucose (its

305 H2 is present at δ 3.29 ppm), these correlations agree with data described by Matulová

306 et al., (2011).

307 The sucrose chemical shifts are shown in Table 1 and the main correlations for

308 →2)-β-Fruf residues were observed at δ 4.21/76.50 (H3-Fru/C3-Fru), 4.04/71.78 ppm

309 (H4-Fru/C4-Fru) in the HQSC Correlation Map (Fig. 4). These molecules are

310 commonly observed in aqueous plant extracts, mainly in soluble fractions, as was

311 observed in the present study.

312 The other fructosyl (H1, H5 and H6) and glucosyl hydrogens (H3’, H5’ and H6’)

313 of the fructo-oligosaccharide and sucrose molecules were found in the region between δ

314 3.40-3.95 (Table 1). The chemical shifts are consistent with FOS standard (Orafti® P95,

315 Fig. S2) and literature data (Lopes et al., 2016; Matulová et al., 2011). The FOS

316 molecules analyzed by 1 H NMR showed an average DP = 4.5.

317 The off-line ESI-MS spectrum (Fig. S4) showed fructo-oligosaccharide peaks

318 with potassium adducts [M + K]+. The peaks at m/z 544 and 706 corresponded to FOS

319 molecules with DP = 3 and DP = 4, that corresponding to 1-Kestose and 1-Nystose

320 compounds, respectively. These are the two first products of the biosynthesis of fructo-
 14

321 oligosaccharides molecules. The mass difference of 162 Da between the peaks

322 corresponds to hexose units (Evans et al., 2015; Lopes et al., 2016).

323 Some steviol glycosides were observed in the ESI-MS analysis of the extract

324 from the stems of S. rebaudiana, most notably the stevioside and rebaudioside A in the

325 adduct potassium form (Fig. S4). Jackson et al., 2009, also report the presence of these

326 glycosides in small amounts in other plant parts besides the leaves of S. rebaudiana.

327

328 3.5 Prebiotic activity testing

329 Inulin and fructo-oligosaccharides are prebiotic compounds that are widely used

330 in functional food formulations. They are defined as non-digestible food ingredients

331 which selectively stimulate the growth of lactobacilli and bifidobacteria, which improve

332 human health (Karimi et al., 2015; Roberfroid et al., 2010).

333 The present study evaluated the prebiotic activity in vitro of inulin and FOS

334 previously isolated and characterized from S. rebaudiana stems. The results were

335 expressed as the differences in the optical density of the growth media at inoculation

336 (time zero) and after 24 h of incubation. The resulting data is shown in Table 2.

337 The FOS isolated from S. rebaudiana stems were fermented by all the strains

338 tested, with a growth statistically (p < 0.05) higher than bacterial growth in the basal

339 medium (negative control). The average increases in the growth of the bifidobacteria

340 and lactobacilli strains were 2.28-fold and 1.7-fold, respectively.

341 Among the tested bifidobacteria, the strain B. bifidum CCDM 559 showed better

342 capacity to fermente FOS from S. rebaudiana and in this case was detected even better

343 growth in the medium containing S. rebaudiana FOS than in the medium with Orafti®

344 P95, a commercial prebiotic product containing FOS from chicory.

 15

345 From lactobacilli group, the strain L. gasseri PHM-7E1 was evaluated to utilize

346 FOS from S. rebaudiana stems most efficiently, where the highest increase in optical

347 density after 24 h incubation was measured. The strain L. fermentum RL25

348 demonstrated a similar ability to ferment S. rebaudiana FOS and chicory FOS (Orafti®

349 P95) as carbon sources. For the other tested strains the bacterial growth in the medium

350 containing S. rebaudiana FOS was significant (p < 0.05) lower than the medium

351 containing prebiotic control.

352 The inulin molecules isolated from S. rebaudiana stems was evaluated to be not

353 a suitable energy source neither for bifidobacteria nor for lactobacilli, because no

354 statistically significant increase (p < 0.05) in optical density of the medium containing

355 this sugar source compared to the basal medium (without sugar) was detected (Table 2).

356 The prebiotic control (Orafti® P95) was the most suitable for comparison with

357 FOS extracts of S. rebaudiana stems, because both are polysaccharides with similar

358 chemical structure and degree of polymerization that provide more similar fermentation

359 parameters, unlike the Wilkins Chalgren broth (WCB) where glucose molecules are

360 used as carbon source, this medium it is easily fermented and it is not a selective

361 medium. The WBC was used as a control of bacterial growth and viability.

362 Strain specificity in fermentation capacity was observed and each strain utilized

363 FOS from S. rebaudiana with varying intensity. This agrees with the general knowledge

364 about strain and substrate specificity (Gibson, Probert, Loo, Rastall & Roberfroid, 2004;

365 Moreno-Vilet et al., 2015).

366 The human gastrointestinal tract represents a competitive environment for

367 bacterias. Lactobacilli and bifidobacteria, belong to a group of saccharolytic bacteria

368 able to utilize a wide range of mono-, oligo- and polysaccharides as energy source

369 (Vernazza, Gibson & Rastall 2005). The capability of microorganisms to ferment sugars
 16

370 depends on their enzymatic equipment, on the presence of hydrolases and transportes.

371 As example, the ability to ferment fructan molecules by certain bacterial strains is

372 related to the presence of the β-fructofuranosidase, an inulinase responsible for the

373 hydrolysis of fructan chain in terminal position β-(2 → 1) (Karimi et al, 2015; Moreno -

374 Vilet et al, 2015).

375 In this study, FOS molecules with DP < 6 were better used as substrate by

376 probiotic strains than inulin molecules (DP = 12), both isolated from the same source. It

377 is also known, that carbohydrates utilization as substrate by bacteria is influenced to a

378 significant extent by their chemical structure, such as molecule branching, glycosidic

379 bonding and chain length, i.e. degree of polymerization (Caleffi et al., 2015). The latter

380 parameter has great influence on the rate of FOS and inulin fermentability by bacteria

381 where their growth increases with decreasing of the degree of polymerization

382 (Mclaughlin et al, 2015; Moreno-Vilet et al, 2014).

383 The fermentation process in the colon is the result of complex microbial

384 metabolic activity and in the ecosystem of the human colon, many different species and

385 genera of microorganisms live together. It is known that comensal relationships

386 between certain microorganisms exist. As for example, where strains able to cleave

387 oligofructose (B. longum) can provide monosaccharides for other strains (Anaerostipes

388 caccae) this condition provide mutual growth of bacterial strains (Pompei et al., 2008;

389 Ward et al., 2007). These kinds of interactions are obviously not reflected in in vitro

390 studies.

391 The FOS molecules isolated from S. rebaudiana stems, showed a chemical

392 profile and degree of polymerization similar to commercial FOS (Orafti® P95).

393 However, the carbohydrate content was significantly different between the samples. In

394 the total sugar determination, a higher concentration of sugars was found for the
 17

395 commercial FOS (100%) in comparison with FOS extract from S. rebaudiana (60%).

396 Therefore, the best prebiotic effect observed for the commercial FOS can be justified by

397 the industrial process of this product that results in a higher purity and high

398 carbohydrate content.

399 The Lactobacilli and Bifidobacteria populations play a key role in bowel

400 function in health and well-being. Therefore, the adequate knowledge of the metabolism

401 of certain types of polysaccharides as substrate by cetain bacteria strains, may provide

402 important information for the rational development of selective food ingredients for

403 bacterial strains as well as in the production of synbiotic products (Mclaughlin et al,

404 2015; Rossi et al., 2005).

405

406 4. Conclusions

407 The results of the present study suggested that S. rebaudiana stems can be a new

408 and promising source for obtaining inulin and fructo-oligosaccharides. The in vitro

409 prebiotic assay of these molecules indicated the strain specific ability to use fructans as

410 carbon sources. FOS molecules with low DP are preferably fermented in vitro by

411 beneficial microbiota than inulin molecules which higher DP.

412 S. rebaudiana stems can be further explored as an alternative source of the

413 extraction of bioactive compound with commercial application from waste of the

414 sweetener industry.

415

416 Acknowledgment

417 The authors thank the Brazilian agencies: Conselho Nacional de

418 Desenvolvimento Científico e Tecnológico (CNPq, process no. 481915/2013-3) as well

419 as the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the
 18

420 Complexo de Centrais de Apoio à Pesquisa (COMCAP) of the State University of

421 Maringá. Authors also thank the Ministry of Education, Youth and Sports of the Czech

422 Republic (COST LD 14123).

423

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545

546

547 Figure Caption

548

549 Fig. 1. GC-MS Chromatogram (TIC) of oxime-silylated derivatives: (A) inulin and

550 from S. rebaudiana stems, (B) fruto-oligosaccharides from S. rebaudiana stems, (C)

551 fructose standard, (D) glucose standard.

552 Fig. 2. 1H NMR spectra (500.0 MHz, D2O at 298 K) of the (A) inulin and (B) fruto-

553 oligosaccharides from S. rebaudiana stems.

554 Fig. 3. Heteronuclear Correlation Map Multiple-Bond (HMBC) of the inulin from S.

555 rebaudiana stems.

556 Fig. 4. Heteronuclear Single Quantum Correlation (HSQC) of the fruto-

557 oligosaccharides from S. rebaudiana stems.

558

559

560
 22

561

562 Fig.
563 1

564
565 Fig. 2.
566

567
A
 23

568
569

570

571

572

573

574

575

576

577 Fig. 3
578
 24

H-5
H-4
H-3
H-6
H-1 H-1

H-1'

C-4
C-4 - HMBC OH
H 6' H-3/C-1 55
Sheila - DFA
5' O H-4/C-6
C-1 HO 4' {4.28,60.80} {4.11,62.08}
H 60
C-6 H
HO H
3' 2' 1' 65
OH
H
H-3/C-4 70
C-4
O {4.27,74.26} H-4/C-3
HO H {4.11,76.73}
6 O HO 75
C-3
H 3 {4.11,81.01} {3.77,80.99}
80
C-5 5 H 2 H-4/C-5 H-6/C-5
H H 85
4
H C H
HO 1 90

n
HO 95
O H-1/C-2
6 H O HO H-1/C-2
{3.95,103.12} {3.71,103.27} 100
C- 2 H 3
5 H 2
105
H
H H C H
4 1 110
OH
OH 115

120
5.9 5.7 5.5 5.3 5.1 4.9 4.7 4.5 4.3 4.1 3.9 3.7 3.5 3.3 3.1
579 f2 (ppm)

580 Fig. 4
581
 25

582
583
 26

584 Table 1. 1H chemical shifts (δ) of inulin standard (Orafti® GR), FOS (Orafti® P95) standard, inulin and fructo-oligosaccharides (FOS) from S.
585 rebaudiana stems
1
Sample Sugar Unit H Chemical Shifts/δ
1 2 3 4 5 6
Orafti® GR →2)-β-Fruf 3.93-3.72 - 4.27 4.11 3.87 3.79-3.76
Orafti® P95 →2)-β-Fruf 3.92-3.71 - 4.25 4.11 3.87 3.78-3.76
Stevia Inulin →2)-β-Fruf 3.93-3.72 - 4.27 4.11 3.86 3.79-3.76
Stevia FOS →2)-β-Fruf 3.91-3.70 - 4.26 4.11 3.88 3.78
α-Glcp-(1→ 5.42 3.58 3.75 3.47 3.87 3.83
Sucrose*
→2)-β-Fruf 3.68 - 4.22 4.04 3.87 3.80
*
586 Chemical shifts (δ) of sucrose observed in the FOS from S. rebaudiana stems
587
 27

588
589 Table 2. Fermentability of inulin and fructo-oligosaccharides (FOS) from S. rebaudiana stems by bifidobacteria and lactobacilli strains.
Bacterial density
(change in A540)
Bacterial strains Inulin FOS Orafti® P95 WCH BM
B. bifidum CCDM 559 0,64 ± 0,21a 3.08 ± 0.28d 2.47 ± 0.16 b
7.19 ± 0.57c 1.82 ± 0.53c
B. animalis subsp. lactis Bb12 0,69 ± 0,15a 1.33 ± 0.10ab 4.95 ± 0.18d 3.98 ± 0.13a 0.30 ± 0.08a
B. breve CCDM 562 0,73 ± 0,23a 1.57 ± 0.07b 2.42 ± 0.23 b
6.83 ± 0.13c 0.50 ± 0.03a
Bifidobacterium average 0.68 ± 0.29¥ 1.99 ± 0.33¶ 3.28 ± 0.33 £
6.00 ± 0.33Ɛ 0.87 ± 0.33¥
L. fermentum RL25 0,95 ± 0,15ab 3.72 ± 0.29e 3.72 ± 0.11c 7.04 ± 0.12c 2.29 ± 0.20d
L. animalis CCDM 382 1,24 ± 0,23bc 2.52 ± 0.25c 5.47 ± 0.20 e
5.79 ± 0.17b 1.00 ± 0.05b
L. casei subsp. paracasei PE1TB-P 0,94 ± 0,21ab 1.11 ± 0.08a 1.38 ± 0.13 a
5.84 ± 0.12b 1.02 ± 0.14b
L. gasseri PHM-7E1 1,49 ± 0,30c 4.03 ± 0.20e 6.93 ± 0.21 f
6.84 ± 0.43c 2.34 ± 0.08d
Lactobacillus average 1.15 ± 0.29¥ 2.85 ± 0.33¶ 4.38 ± 0.33£ 6.38 ± 0.33Ɛ 1.67 ± 0.33¥
590 Data are expressed as increase in turbidity of the bacterial suspension estimated from the increase in absorbance (A540) during 24 h of incubation; values are means from
591 triplicate determination ± standard deviation (SD).
592 Orafti® P95 – commercial prebiotic (FOS positive control), WCH - Wilkins-Chalgren broth (positive control), BM – basal medium without sugar (negative control).
abcdef
593 – data in columns with different superscripts differ (p < 0.05)
Ɛ¥¶£
594 - data in lines with different superscripts differ (p < 0.05)
595

596

597
 28

598 Highlights
599

600 - Industrial waste as source for obtainment of bioactive compounds.

601 - We obtained inulin and FOS molecules with technological and nutritional

602 applications.

603 - Chemical characterization of inulin and FOS by GC-MS, NMR and off-line ESI-MS.

604 - FOS molecules with low DP are preferably fermented by beneficial microbiota.

605

606