Accepted Manuscript

Symbiotic microencapsulation to enhance Lactobacillus acidophilus survival

Karen Cristina Guedes Silva, Eliza Cristina Cezarino, Mariano Michelon, Ana Carla
Kawazoe Sato

PII: S0023-6438(17)30845-9
DOI: 10.1016/j.lwt.2017.11.026
Reference: YFSTL 6655

To appear in: LWT - Food Science and Technology

Received Date: 19 July 2017

Revised Date: 9 October 2017
Accepted Date: 11 November 2017

Please cite this article as: Silva, K.C.G., Cezarino, E.C., Michelon, M., Sato, A.C.K., Symbiotic
microencapsulation to enhance Lactobacillus acidophilus survival, LWT - Food Science and Technology
(2017), doi: 10.1016/j.lwt.2017.11.026.

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 Symbiotic microencapsulation to enhance Lactobacillus acidophilus survival

2 Karen Cristina Guedes Silva1, Eliza Cristina Cezarino1, Mariano Michelon2, Ana Carla

3 Kawazoe Sato1
1
4 Department of Food Engineering, Faculty of Food Engineering, University of
5 Campinas, 13083-862 Campinas, SP, Brazil.

PT
2
6 Department of Mechanical Engineering, Pontifical Catholic University of Rio de
7 Janeiro, 22451-900 Rio de Janeiro, RJ, Brazil.

RI
8
9 Corresponding author (Ana Carla Kawazoe Sato): E-mail: acksato@unicamp.br; Tel.:

SC
10 +55 19 35214088
11 Karen Cristina Guedes Silva: karen_cgs@hotmail.com
12 Eliza Cristina Cezarino: elizacezarino10@hotmail.com

U
13 Mariano Michelon: michelonmariano@gmail.com
AN
14

15 Abstract
M

16 Lactobacillus acidophilus was microencapsulated in alginate-gelatin (AG) and alginate-

gelatin-fructooligosaccharides (AGF) microbeads by external gelation, with the purpose

D

17

18 of increasing the viability of the probiotic culture when exposed to gastrointestinal tract
TE

19 (GIT) and during storage when added to yogurt. The microencapsulation provided
EP

20 greater protection of cells when exposed to simulated GIT. Moreover the addition of

21 fructooligosaccharide (FOS) to the matrix promoted the formation of a more

C

22 interconnected network, which contributed to better protection of cells and controlled

AC

23 delivery. Microencapsulation process proved to not affect cell viability. Microbeads AG

24 and AGF improved probiotic survival during the storage in yogurt compared to free L.

25 acidophilus (FLA). This study showed that symbiotic microencapsulation provided

26 greater viability of L. acidophilus during the storage in yogurt and in GIT, as well as

27 providing functional characteristics of yogurt with added of AGF microbeads.

28
 ACCEPTED MANUSCRIPT
29

30 Keywords

31 Microbeads, controlled delivery, digestibility, viability, probiotics

32

33 1. Introduction

PT
34 Nowadays functional products have been playing a major role in the segment of

RI
35 the food industry due to their beneficial health properties (Tripathi & Giri, 2014). In

36 order to meet the demand for functional products, the food industry and health

SC
37 professionals have focused on studying how such functional products can be controlled

38 more effectively to promote health, nutrition and well-being (Saarela, Lähteenmäki,

39
U
Crittenden, Salminen, & Mattila-Sandholm, 2002).
AN
40 Probiotics are defined as live microorganisms that confer health benefits to the
M

41 host, if administered in adequate amounts (FAO/WHO, 2001). When consumed,

42 probiotics colonize the intestinal tract conferring several benefits, such as stimulation of
D

43 the immune system, maintenance of mucosal integrity, production of important

TE

44 digestive enzymes and others (Holzapfel & Schillinger, 2002).

45 Nevertheless, the viability of probiotics may be suppressed by environmental

EP

46 stress, such as presence of oxygen, mechanical damage, high temperatures of storage

and processing, interaction with foods in which are added, as poor compatibility with
C

47
AC

48 traditional starter of yogurts during fermentation and milk products or when added in

49 products with high acidity. Besides the passage through the digestive system, due to

50 high acidity of the stomach, presence of salts and enzymes (Kailasapathy, 2002;

51 Krasaekoopt & Watcharapoka, 2014; García-Ceja et al., 2015). Thus, in order to

52 achieve benefits of probiotics, they must contain at least 106 CFU/g or be eaten in

53 sufficient amounts to yield a daily intake of 108 CFU (Chávarri et al., 2010). Besides
 ACCEPTED MANUSCRIPT
54 this, probiotics need to be metabolically stable and active in both, the product and the

55 host (Krasaekoopt & Watcharapoka, 2014).

56 Since the probiotic bacteria must be protected from adverse environment for

57 preserving its viability, microencapsulation has proven to be a promising method for

58 providing a suitable coating for these microorganisms so they can reach the site of

PT
59 action in adequate amounts (Douglas & Sanders, 2008; Ribeiro et al., 2014).

RI
60 Likewise, the supplementation of microcapsules with prebiotics may stimulate

61 probiotic growth. Prebiotics contribute to supply a fermentable carbohydrate for

SC
62 probiotic bacteria in the colon, stimulating the growth and activity (Gibson, Probert,

63 Loo, Rastall, & Roberfroid, 2004). The synergistic combination of probiotics and

64
U
prebiotics give rise to the concept of symbiotic (Rodrigues et al., 2011). Several reports
AN
65 proved that symbiotic microcapsules were more effective when exposed to simulated
M

66 GIT (Chávarri et al., 2010; Cook, Tzortzis, Charalampopoulos, & Khutoryanskiy,

67 2014). However, few reports have focused on the simulation of simbyotic

D

68 microcapsules along the GIT conditions and its application in food products, to evaluate
TE

69 their interaction with the medium and the viability of probiotics during storage.

70 Many polymers have been used for the production of encapsulation systems for
EP

71 application in the food industry (Kuhn, Picone, & Cunha, 2012). Alginate is a natural

polysaccharide that exhibits excellent biocompatibility, low cost, non toxicity and good
C

72
AC

73 stability when subjected to acid conditions. All these properties make the alginate one

74 of the most attractive biopolymers for use in microcapsules, applied for protection and

75 controlled delivery in the intestinal tract (Cook, Tzortzis, Charalampopoulos, &

76 Khutoryanskiy, 2011).

77 However, some drawbacks are attributed to alginate, as the large pores of the

78 biopolymer network wich can reduce barrier properties (Gouin, 2004). To solve this
 ACCEPTED MANUSCRIPT
79 problem, the blend of other compounds to the alginate matrix can promote greater

80 stability, strengthening and protection of the active, by reducing the pore size

81 (Etchepare et al., 2016). To reduce the alginate pores size, gelatin has also been

82 explored, since it can form structures via hydrogen bonding and ionic interaction with

83 the alginate chains. This blend can provide protection of sensitive compounds and

PT
84 contribute to a more resistant network (Dong, Wang, & Du, 2006; Li, Jia, Cheng, Pan,

RI
85 & Jiang, 2011).

86 Thus, this study aimed to produce probiotic and symbiotic alginate-gelatin

SC
87 microbeads by external gelation, with or without FOS addition as coating materials.

88 These systems were evaluated and characterized during refrigerated storage in yogurt

89
U
containing L. acidophilus in free and microencapsulated forms. The survival of the free
AN
90 and encapsulated microorganisms during the simulation of the passage through the
M

91 gastrointestinal tract was also assessed.

92
D

93 2. Material and methods

TE

94 2.1. Materials

95 The following ingredients were used for microbeads production: alginate

EP

96 (Danisco, France); gelatin A type - bloom 280 (Gelco gelatinas do Brasil, Brazil); CaCl2

dihydrate (Dinâmica - Química Contemporânea Ltda, Brazil); fructooligosaccharide -

C

97
AC

98 FOS (Corn Products, Brazil) and culture of Lactobacillus acidophilus LA-5 (Christian

99 Hansen, Brazil). For activating and enumerating the L. acidophilus cells, de Man,

100 Rogosa and Sharpe (MRS) culture medium (Merck Millipore, Germany) and agar-agar

101 (Merck Millipore, Germany) were used. Homogenized pasteurized milk A type (Rico -

102 Fazenda Colorado, Brazil) and semi-skimmed yogurt (Serramar - Fazenda Bela Vista,

103 Brazil) were acquired for yogurt production. For the preparation of simulated
 ACCEPTED MANUSCRIPT
104 gastrointestinal fluids, pepsin porcin (P6887), pancreatin (P7545) and bile salt (B8631)

105 (Sigma-Aldrich Co., United States) were used. All other reagents were analytical-grade.

106

107 2.2. Methods

108 2.2.1. Activation of culture of L. acidophilus

PT
109 Freeze-dried culture of L. acidophillus was inoculated into 10 mL MRS broth at

RI
110 37 °C for 18 h. The resulting cultures were transferred to 90 mL MRS broth and

111 incubated once more, under the same conditions. Cultures were harvested by

SC
112 centrifugation at 2400 g at 20 °C for 9 min and washed twice with 30 mL of sodium

113 citrate solution (2 g/100 mL). Supernatants were discarded and culture of L. acidophilus

114
U
was re-suspended in 30 mL of distilled water and submitted to centrifugation at the
AN
115 same conditions. Later, the collected bacterial cells were transferred to the biopolymer-
M

116 based solutions.

117
D

118 2.2.2. Biopolymer-based solutions

TE

119 Gelatin solution (1.5 g/100 mL) was prepared under magnetic stirring (500 rpm)

120 for 15 min, at 45 °C. Then, the solution was cooled to 25 °C before the addition of 1
EP

121 g/100 mL alginate to produce the AG formulation, and of 1 g/100 mL alginate and 3

g/100 mL FOS to produce the AGF formulation. The concentration of 3 g/100 mL FOS
C

122
AC

123 was determined based on preliminary studies (Silva & Sato, 2017). Before the addition

124 of L. acidophilus, the biopolymer-based solutions were maintained under constant

125 stirring until complete dissolution (2 h) and then stored for 24 h at 25 °C. The blend

126 (probiotic and biopolymer solutions) was diluted in water (1 g/10 mL) for enumerating

127 the colony forming units per gram (CFU/g) of free cells added initially to the
 ACCEPTED MANUSCRIPT
128 biopolymer-based matrix ( ) and for correlating with encapsulation yield (Section

129 2.3.2).

130

131 2.2.3. Biopolymer-based macrogels

132 AG and AGF macrogels were prepared by external gelation, where the wall

PT
133 materials solution was put into dialysis membranes, 3500 molecular weight cut-off

RI
134 (MWCO) (SnakeSkin Dialysis Tubing, United States) and dialyzed against 150 mM

135 CaCl2 solution, for 9 days, at room temperature. Alginate or gelatin solutions, with or

SC
136 without FOS addition were also prepared as a control. The gels without alginate, were

137 prepared by adding the gelatin in water, as described in the section 2.2.2. and stored

138
U
under refrigeration. The microstructure was characterized by scanning electron
AN
139 microscopy (SEM).
M

140

141 2.2.4. Biopolymer-based microbeads

D

142 AG and AGF microbeads were prepared by mixing cell suspension and 75 mL
TE

143 wall materials. The mixture was transported to a double fluid atomizer nozzle with 0.7

144 mm diameter (Labmaq do Brazil Ltda, Brazil) using a peristaltic pump (model 7518-00,
EP

145 Masterflex, United States), set at 90 rpm. The solution was atomized at room

temperature, into 150 mM calcium chloride solution at flow rate of 0.19 mL/s and 0.12
C

146
AC

147 mL/s for AG and AGF respectively. The distance between the atomizer nozzle and the

148 CaCl2 collecting solution was set at 30 cm, the compressor pressure was fixed at 1 bar

149 and the compressed air flow rate at 1.2 m3/ h (Perrechil, Sato, & Cunha, 2011). The

150 microbeads were maintained in CaCl2 solution for 30 min for gelation, sieved using a

151 0.053 mm mesh diameter and rinsed with distilled water, before being aseptically

152 transferred to sterile flasks for storage at 4 °C. To enumerate viable cells during storage
 ACCEPTED MANUSCRIPT
153 and characterize the encapsulation yield ( ), 1 g of microbeads was broken in 10 mL of

154 sodium citrate solution (0.06 mol/L), pH 8.18±0.02, under stirring for 45 min at 37 °C,

155 to release the microorganisms.

156

157 2.2.5. Yogurt production and viability of L. acidophilus

PT
158 For the yogurt preparation, 170 g of started semi-skimmed yogurt was

RI
159 inoculated into 1 L of homogenized pasteurized milk preheated to 42 °C and

160 subsequently incubated at 37 °C, for fermentative bacteria convert lactose into lactic

SC
161 acid. For this, the pH was monitored and the incubation was stopped when the pH

162 decreased to 4.6 (Lee & Lucey, 2010), which was achieved in 48 h. The yogurt was

163
U
distributed in 12 mL portions in sterile flasks containing 3 g of microbeads. Then, flasks
AN
164 were stored in a BOD (Biochemical Oxygen Demand) (Model: MA 415 UR, Marconi,
M

165 Brazil) at 4 °C for 28 days. Free cells were added to yogurt, for control, according to the

166 methodology proposed by García-Ceja et al. (2015). The activated cells were suspended
D

167 into 75 mL of sterile water (instead of AG and AGF biopolymer-based solutions) and
TE

168 then divided into sterile flasks as mentioned above.

169 For the storage viability studies, pH and viability of L. acidophilus were
EP

170 evaluated every 7 days of yogurt samples containing microbeads and FLA. pH was

mesured in triplicate by electrode immersion with a pH-meter (Model: Metrohm 827,

C

171
AC

172 Metrohm, Switzerland). To enumerate the viable cells in microbeads added to the

173 yogurt, samples were sieved using a 0.053 mm mesh diameter and washed with distilled

174 water. 1 g of AG and AGF microbeads were broken in sodium citrate as described in

175 section 2.2.4. Counts for FLA in yogurt were achieved by subtraction of colony forming

176 units observed from pure yogurt (control experiment).

177
 ACCEPTED MANUSCRIPT
178 2.2.6. In vitro digestibility

179 The in vitro resistance of FLA, AG and AGF to simulated gastric (SGF) and

180 intestinal fluids (SIF) was evaluated in an orbital shaker (Model: TE-420, Tecnal,

181 Brazil) at 100 rpm, maintained at 37±1 °C (Mun, Park, Kim, & McClements, 2016).

182 The protocol of Minekus et al. (2014) was used with slight modifications, considering

PT
183 that, according to the authors, the mouth step can be eliminated for liquid samples.

RI
184 Initially, the microbeads and the solution containing free probiotics were diluted in

185 water at a ratio of 1:4, to reproduce dilution of food on oral step, as described by

SC
186 Hoebler et al. (2002). 25 mL of each sample was incubated for 2 h with 25 mL of SGF,

187 the pH was adjusted to 3 with hydrochloric acid and monitored. SGF stock solution was

188
U
prepared by the addition of 0.3 mol/L sodium chloride and pepsin 2000 U/mL in
AN
189 distilled water. Then 50 mL of gastric phase (sample+SGF) were mixed with 50 mL of
M

190 SIF, the pH was adjusted to 7 with sodium hydroxide and monitored. The samples were

191 incubated at 37 °C for 2 h under continuous stirring. SIF was prepared with SIF stock
D

192 solution with addition of 0.3 mol/L sodium chloride, 800 U/mL pancreatin and 10 mM
TE

193 bile salt, suspended in distilled water. Counts were performed at 30 min intervals over

194 the 4 h of shaker incubation. The enumeration of the released cells along GIT was
EP

195 determined using the drop plate technique and the morphology of the microbeads were

evaluated by optical microscopy.

C

196
AC

197

198 2.3. Characterization

199 2.3.1. Viable L. acidophilus cell counts

200 The enumeration of viable cells of L. acidophilus was performed in triplicate

201 using the drop plate technique in a medium containing 1.5 g/100 mL of agar in MRS

202 broth. Firstly, dilutions of the culture were made by successive addition of 1 mL of the
 ACCEPTED MANUSCRIPT
203 samples to a tube containing 9 mL of peptone water 0.1 g/100 mL, followed by its

204 homogenization and the removal of another 1 mL for the next dilutions. Then, 20 µL of

205 each dilution was plated into numbered sectors. After the drop absorption, the plates

206 were inverted and incubated at 37 °C for 48 h in anaerobic jars. The results were

207 expressed in CFU/g. To enumerate viable cells of L. acidophilus of gelled systems, a

PT
208 fraction of the microbeads was broken with sodium citrate in a magnetic stirrer for 45

RI
209 min and plated as previously described.

210

SC
211 2.3.2. Encapsulation yield (EY)

212 Entrapped bacteria in AG and AGF microbeads were released by homogenizing

213
U
1g of filtered microbeads in 10 mL of sodium citrate as previously described. The
AN
214 encapsulation yield was calculated as described by Chávarri et al. (2010), which is a
M

215 combination of the efficacy of entrapment and survival of viable cells during the

216 microencapsulation procedure (Equation 1):

D

217
TE

% = ⁄ .100 (1)

218
EP

219 Where is the number of viable entrapped cells released from the microbeads, and
C

220 is the number of free cells added to the biopolymer-based matrix, during the production
AC

221 of the microbeads.

222

223 2.3.3. Microestructure and surface morphology

224 The morphology of microbeads and microstructure of macrogels, were

225 determinated by SEM according to the methodology described by (Pires Vilela,

226 Cavallieri et al. 2011), with some modifications. The samples were dried at critical
 ACCEPTED MANUSCRIPT
227 point (Balzers Critical Point Dryer CPD03, Liechtenstein) and coated with gold

228 (Balzers Sputter Coater SCD 050, Liechtenstein) prior to obtaintion of images using a

229 SEM (JEOL JSM 5800 LV (Tokyo, Japan) operated at 10 kV. The shape of the

230 microbeads, was also assessed using optical microscopy (Carl Zeiss Axio Scope.A1,

231 Zeiss, Germany).

PT
232 2.4. Statistical analysis

RI
233 Statistical analysis was performed by ANOVA and Tukey's mean comparison

234 tests (p < 0.05), using SISVAR: a computer statistical analysis system (Ferreira, 2011),

SC
235 to identify significant differences regarding to L. acidophilus viability, pH and size of

236 beads.

237
U
AN
238 3. Results and discussion
M

239 3.1 Entrapment process and particles characterization

240 To evaluate the effect of the encapsulation process on probiotic viability, as well
D

241 as the characteristics of the particles formed entrapment efficiency, macrogels

TE

242 microstructure and microbeads morphology were studied. The results of encapsulation

243 yield did not present a significant diference (p > 0.05), showing that microorganisms
EP

244 were resistant to the atomization process (Tab. 1), presenting a very high cell loading

98.32±5.81 and 99.28±6.47 for AG and AGF microbeads respectively. Besides that,
C

245
AC

246 FOS addition did not influence this stage of the process, once the atomization process

247 occurs very quickly, so that in this step the prebiotic does not have enough time to act as

248 substrate source, improving the growth and probiotic viability. In the same way,

249 Krasaekoopt and Watcharapoka (2014) showed that entrapment efficiency of the

250 microencapsulation process is not dependent on the type, concentration or presence of

251 prebiotics in the matrix.

 ACCEPTED MANUSCRIPT
252 The SEM images (Fig. 1) show the network morphology of gels produced by

253 alginate or gelatin, as well as the combination of alginate and gelatin, with and without

254 FOS addition. It is possible to observe that the alginate network (Fig. 1a) has relatively

255 large pores and continuous structure, while the gelatin network is uniform and regular,

256 with small pores (Fig. 1b). The association of alginate-gelatin promoted the formation

PT
257 of a more cohesive structure, with a slight reduction of pores (Fig. 1c) compared to

RI
258 alginate, indicating better characteristics for protection of compounds of smaller sizes

259 and molecular weight. However, micro phase separation have occurred, which was

SC
260 associated to the fact that gelatin and alginate solutions were produced in a pH

261 condition (around 6.0), in which they both have negative charge (-10 and -40 mV,

262
U
respectively). This micro phase separation, have led to the formation of two distinct
AN
263 structures, one spongious and other denser and more continuous, which were associated
M

264 to pure gelatin and alginate, respectively. The inhomogeneous morphology of alginate-

265 gelatin hydrogels can also be attributed to the crosslinking reaction between the
D

266 compounds, which can destroy the regular structural pattern of alginate (Sarker, 2014).
TE

267 Other studies also indicate a possible thermodynamic incompatibility during gel

268 formation. In this case, gelatin is reorganized by cross-link association, forming a

EP

269 fractal-type gel network during the gelation process, excluding its interaction with other

polymers (Firoozmand, Murray, and Dickinson (2007).

C

270
AC

271 The FOS addition to alginate promoted reduction in pore size (Fig. 1d), whereas

272 its association with gelatin resulted in a thicker network, with slight larger pores (Fig.

273 1e). Finally, it is possible to observe that FOS acts on the alginate-gelatin matrix (Fig.

274 1f) reducing the pore size and forming a more interconnected network, indicating that,

275 possibly, the FOS acts filling the interstitial spaces between the alginate-gelatin gel
 ACCEPTED MANUSCRIPT
276 network, reducing the effects of phase separation. This interconnected network may

277 have been formed through hydrogen bonds between alginate-FOS (Ivanovska, 2012).

278 The optical microscopy (Fig. 2.1) and SEM observations (Fig. 2.2) clearly

279 demonstrated that the produced microbeads were spherical. Large amount of L.

280 acidophilus were homogeneously distributed inside the microsphere (Fig. 2.1) providing

PT
281 a good biocompatible growth environment for L. acidophilus, due to prebiotic source,

RI
282 also avoiding the probiotics from coming into contact with gastric fluids, reducing their

283 viability (Mei et al., 2014).

SC
284

285 3.2 Stability in yogurt

286
U
In order to verify the survival of probiotics added in yogurt during 28 days of
AN
287 controlled temperature storage, the pH and the viable cell count were evaluated at
M

288 intervals of 7 days. Over the 28-day storage, no significant pH differences (p > 0.05)

289 were observed in any yogurt samples, that remained around 4.25. These results can be
D

290 explained once the addition of the microbeads and FLA occurred after the conversion of
TE

291 the sugars present in milk into lactic acid by the natural yogurt bacteria (Bosnea,

292 Moschakis, & Biliaderis, 2017). Vivek (2013) also reported that Dahi yogurt pH did not
EP

293 change during the storage at 4 °C for 20 days, when added of L. paraplantarum and B.

bifidum encapsulated in alginate with FOS.

C

294
AC

295 The stability results at the 28-day storage are shown in Fig. 3. Both microbeads

296 were effective in maintaining the viability of L. acidophilus, improving its survival

297 when compared to FLA, in yogurt. It was recognized that although the probiotic count

298 in the systems without FOS were higher than the therapeutic level, the AGF microbeads

299 increased the survival of the entrapped cells. Comparing both microbeads,

300 microencapsulated cells with FOS provided better results during the storage, with a
 ACCEPTED MANUSCRIPT
301 decrease of entrapped cells as low as 0.9 logarithmic cycles (12.85%), while a reduction

302 of 1.1 logarithmic cycles (18.64%), was observed for AG along the 28 days.

303 FLA presented a significant reduction (p < 0.05) of 1.84 logarithmic cycles in

304 their viability after 7 days of storage, while after 28 days a decrease of 28.80% of viable

305 cells (~2.5 logarithmic cycles) was observed. This may have occurred by factors

PT
306 affecting to the survival of probiotics in yogurt, such cells competition by yogurt

RI
307 cultures and probiotic cells, inhibitory substances produced by the yogurt culture or an

308 excess of dissolved oxygen (Grosso & Fávaro-Trindade, 2004; Cruz et al., 2013;

SC
309 Krasaekoopt & Watcharapoka, 2014). Besides the reduction in survival of probiotics,

310 poor sensory characteristics and undesirable changes has been reported for free L.

311
U
acidophilus added in yogurt (García-Ceja et al., 2015), which justifies the importance of
AN
312 encapsulation systems for protection and delivery of this probiotics.
M

313 The microbeads provided cells protection from yogurt products such as organic

314 acids, fermentative microorganisms and others, since microbeads withstanded the low
D

315 pH environment, presenting viable cells counts higher than the therapeutic level (106
TE

316 CFU/g). The highest survival for AGF microbeads could be attributed to the FOS

317 addition, due to the prebiotic source, that acted as a substrate, imparting a synergistic
EP

318 interaction with probiotic culture (Rodrigues et al., 2011). Moreover FOS also promoted

the formation of a more interconnected network, with smaller pores (Fig. 1f), as
C

319
AC

320 previously discussed, and reinforced network (Silva & Sato, 2017) which may have led

321 to the additional protection of the encapsulated L. acidophilus. In the same way, this

322 tendency was observed for probiotic microcapsules of alginate and modified starch,

323 which acted synergistically in gelling, resulting in additional protection to entrapped

324 bacterial, enhancing their survival as compared to pure alginate matrix (Sultana et al.,

325 2000).
 ACCEPTED MANUSCRIPT
326

327 3.3 In vitro evaluation of gastric and intestinal fluids

328 For simulating the behavior of the microbeads and evaluating their resistance

329 during their transit through the stomach, an in vitro gastro intestinal simulation was

330 performed. Such analysis indicates the ability of the particles to protect the probiotics

PT
331 and release them at intestine, in their viable form, as well to investigate the role of FOS

RI
332 in L. acidophilus viability.

333 Evaluating the L. acidophilus counting in the microbeads (AG and AGF), the

SC
334 number of viable cells was in agreement with the requirements for classifying them as

335 probiotics benefits, after the passage through the gastric tract (FAO/WHO, 2001). After

336
U
2 h under simulated gastric conditions, the reduction on the viability of probiotic in AG,
AN
337 AGF and FLA was of 26.0 %; 40.1 % and 49.7 %, that corresponds to logarithmic cycle
M

338 reduction of 0.62, 1.51 and 3.07 respectively (Fig. 4). Even though the probiotics were

339 encapsulated, the gels showed a porous network (Fig. 1), which may have allowed for
D

340 the diffusion of the SGF into the particle, reducing the viability of the microorganisms.
TE

341 However, the reduction was lower than the observed for the free microorganism,

342 indicating that the encapsulation process improved the viability of the probiotics. Some
EP

343 studies have presented reduction of approximately 2 logarithmic cycles for L.

acidophilus in alginate and chitosan beads (Urbanska, Bhathena, & Prakash, 2007) and
C

344
AC

345 3 logarithmic cycles for alginate particles exposed to SGF (Ortakci & Sert, 2012).

346 Fig. 5 presents microbeads microscopy during the in vitro digestibility. Along

347 the gastric digestion, both microbeads, with and without FOS, were able to maintain

348 their structure during the passage through the gastric step (SGF). After 2 h, entire

349 microbeads were still observed. After 2 h under simulated enteric conditions (Fig. 4), an

350 increase in probiotic viability was observed: 0.19 (2.6 %) and 0.51 (7.0 %) logarithmic
 ACCEPTED MANUSCRIPT
351 cycles for AG and AGF, respectively, possibly due to the gradual delivery promoted by

352 the encapsulating systems. For FLA, a reduction of 0.39 logarithmic cycles (5.1 %) was

353 recorded during the enteric step (Fig. 4).

354 Despite the viable count cell at the end of gastrointestinal simulation have

355 registered reduction for AG and AGF, it should be noted that the survival of the L.

PT
356 acidophilus in microbeads was significantly (p < 0.05) greater than FLA. Both AG and

RI
357 AGF beads promoted a protection to the L acidophilus, which maintained their viability

358 along the intestinal step (Fig. 4).

SC
359 Along the enteric step, the protective effect of the microbeads was reduced, once

360 swelling and collapse of the microbeads were observed (Fig. 5). However, even at the

361
U
end of SIF conditions, AGF microbeads were able to maintain part of their structure,
AN
362 indicating that the network is stronger than AG microbeads. As can be observed, even at
M

363 120 min (Fig. 5), particle borders are apparent and the particles are still able to retain the

364 L acidophilus. During the counting, the non-released L. acidophilus were still protected
D

365 by microbeads, and thus, not counted in the plating technique, resulting in a lower AGF
TE

366 viable cell measurements (Fig. 4).

367 Even though alginate microbeads swell when submitted to SGF, they remain
EP

368 stable in this stage, collapsing only during the intestinal stage, which is associated to the

instability of particles when exposed to the neutral pH. In this condition, the carboxyl
C

369
AC

370 groups from calcium-alginate network are exposed to bile salts, starting an ion exchange

371 process. As a result, repulsion between COO- alginate groups occurs, with consequent

372 relaxation of chains, leading to the swell and rupture of the gels (Li et al., 2011). The

373 same authors also reported that the combination of alginate with other compounds can

374 reduce the collapse of microbeads, which was also observed for FOS addition to

375 alginate-gelatin at intestinal phase (Fig. 5). Although the ion exchange process occurs in
 ACCEPTED MANUSCRIPT
376 the intestinal tract, the AGF network remained resistant during the simulation of

377 intestinal fluids, which may have occurred due to the filling of the interspaces

378 delineated by the alginate-gelatin networks in the microbeads with FOS. As a result, the

379 resistance to the diffusional process across the microgel is high, and the release of the

380 probiotic across the AGF microgel is smaller, providing a more sustained release. The

PT
381 sustained release is important and desirable for insertion of compounds that need to be

RI
382 released gradually and slowly, since it allows longer residence time and the

383 achievement of a greater extent along the intestinal mucosa, providing greater efficiency

SC
384 (Agüero et al., 2017; Flores & Kong, 2017).

U
385
AN
386 4. Conclusions

387 FOS addition to the alginate-gelatin matrix resulted in an improved network,

M

388 since the FOS filled the interstitial spaces of the alginate-gelatin matrix, resulting in

389 smaller pores and a more interconnected network. The results showed that both
D

390 microbeads were able to protect probiotics, improving their survival in the conditions of
TE

391 storage and digestibility evaluated, when compared to free probiotics. The addition of
EP

392 prebiotic (FOS) to microbeads improved the viability of L. acidophilus in yogurt as it

393 acted as a substrate source and promoted the formation of an encapsulation matrix more
C

394 resistant to disintegration when subjected to gastrointestinal conditions, indicating a

AC

395 promising application of microbeads for protecting compounds that require slow and

396 gradual release.

397

398 Acknowledgments

399 The authors thank CAPES (DEA/FEA/PROEX), CNPq (453986/2014-5) and

400 FAPESP (2007/58017-5; EMU2009/54137-1) for their financial support. Karen Cristina
 ACCEPTED MANUSCRIPT
401 Guedes Silva thanks CNPq (130498/2014-9) for the assistantship. The authors also

402 thank the Gelco (Pedreira, São Paulo, Brazil), Danisco (Landerneau, France), Christian

403 Hansen, Brazil) and Corn Products (Brazil) for donation of samples.

404

PT
RI
U SC
AN
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT
405 5. References

406 Agüero, L., Zaldivar-Silva, D., Peña, L., & Dias, M. L. (2017). Alginate microparticles
407 as oral colon drug delivery device: A review. Carbohydrate Polymers, 168, 32-43.
408 Bosnea, L. A., Moschakis, T., & Biliaderis, C. G. (2017). Microencapsulated cells of
409 Lactobacillus paracasei subsp. paracasei in biopolymer complex coacervates and their
410 function in a yogurt matrix. Food & Function, 8(2), 554-562.
Chávarri, M., Marañón, I., Ares, R., Ibáñez, F. C., Marzo, F., & Villarán, M. d. C.

PT
411
412 (2010). Microencapsulation of a probiotic and prebiotic in alginate-chitosan capsules
413 improves survival in simulated gastro-intestinal conditions. International Journal of

RI
414 Food Microbiology, 142(1), 185-189.
415 Cook, M. T., Tzortzis, G., Charalampopoulos, D., & Khutoryanskiy, V. V. (2011).
416 Production and evaluation of dry alginate-chitosan microcapsules as an enteric delivery

SC
417 vehicle for probiotic bacteria. Biomacromolecules, 12(7), 2834-2840.
418 Cook, M. T., Tzortzis, G., Charalampopoulos, D., & Khutoryanskiy, V. V. (2014).

U
419 Microencapsulation of a synbiotic into PLGA/alginate multiparticulate gels.
420 International Journal of Pharmaceutics, 466(1), 400-408.
AN
421 Cruz, A. G., Castro, W. F., Faria, J. A. F., Bolini, H. M. A., Celeghini, R. M. S., Raices,
422 R. S. L., Mársico, E. T. (2013). Stability of probiotic yogurt added with glucose oxidase
423 in plastic materials with different permeability oxygen rates during the refrigerated
M

424 storage. Food Research International, 51(2), 723-728.

425 Etchepare, M. A., Raddatz, G. C., de Moraes Flores, É. M., Zepka, L. Q., Jacob-Lopes,
D

426 E., Barin, J. S., de Menezes, C. R. (2016). Effect of resistant starch and chitosan on
427 survival of Lactobacillus acidophilus microencapsulated with sodium alginate. LWT -
TE

428 Food Science and Technology, 65, 511-517.

429 Flores, F. P., & Kong, F. (2017). In Vitro Release Kinetics of Microencapsulated
EP

430 Materials and the effect of the Food Matrix. Annual Review of Food Science and
431 Technology, 8(1), 237-259.
432 Dong, Z., Wang, Q., & Du, Y. (2006). Alginate/gelatin blend films and their properties
C

433 for drug controlled release. Journal of Membrane Science, 280(1–2), 37-44.
AC

434 Douglas, L. C., & Sanders, M. E. (2008). Probiotics and prebiotics in dietetics practice.
435 Journal of the American Dietetic Association, 108(3), 510-521.
436 FAO/WHO. (2001). Food and Agriculture Organization of the United Nations and
437 World Health Organization. Evaluation of health and nutritional properties of
438 probiotics in food including powder milk with live lactic acid bacteria. Córdoba.
439 Ferreira, D. F. (2011). Sisvar: a computer statistical analysis system (Version Sisvar 5.3
440 Build 77). Science and agrotecnology (UFLA).
441 Firoozmand, H., Murray, B. S., & Dickinson, E. (2007). Fractal-Type particle gel
442 formed from gelatin + starch solution. Langmuir, 23(8), 4646-4650.
 ACCEPTED MANUSCRIPT
443 García-Ceja, A., Mani-López, E., Palou, E., & López-Malo, A. (2015). Viability during
444 refrigerated storage in selected food products and during simulated gastrointestinal
445 conditions of individual and combined lactobacilli encapsulated in alginate or alginate-
446 chitosan. LWT - Food Science and Technology, 63(1), 482-489
447 Gibson, G. R., Probert, H. M., Loo, J. V., Rastall, R. A., & Roberfroid, M. B. (2004).
448 Dietary modulation of the human colonic microbiota: updating the concept of
449 prebiotics. Nutrition Research Reviews, 17(2), 259-275.

PT
450 Gouin, S. (2004). Microencapsulation. Trends in Food Science & Technology, 15(7),
451 330-347.
452 Grosso, C. R. F., & Fávaro-Trindade, C. S. (2004). Stability of free and immobilized

RI
453 Lactobacillus acidophilus and Bifidobacterium lactis in acidified milk and of
454 immobilized B. lactis in yoghurt. Brazilian Journal of Microbiology, 35, 151-156.

SC
455 Hoebler, C., Lecannu, G., Belleville, C., Devaux, M. F., Popineau, Y., & Barry, J. L.
456 (2002). Development of an in vitro system simulating bucco-gastric digestion to assess
457 the physical and chemical changes of food. International Journal of Food Sciences and

U
458 Nutrition, 53(5), 389-402.
AN
459 Holzapfel, W. H., & Schillinger, U. (2002). Introduction to pre- and probiotics. Food
460 Research International, 35(2–3), 109-116.
461 Ivanovska, T. P., Mladenovska, K., Kavrakovski, Z., Bogdanovska, L., Grozdanov, A.,
M

462 Popovski, E., & Petrushevska-Tozi, L. (2012). Effect of prebiotic content on functional
463 and physicochemical properties of Lactobacillus casei loaded chitosan-Ca-alginate
464 microparticles. Macedonian Pharmaceutical Bulletin, 58, 45-52.
D

465 Kailasapathy, K. (2002). Microencapsulation of probiotic bacteria: technology and

TE

466 potential applications. Current Issues in Intestinal Microbiology, 3(2), 39-48.

467 Krasaekoopt, W., & Watcharapoka, S. (2014). Effect of addition of inulin and
468 galactooligosaccharide on the survival of microencapsulated probiotics in alginate beads
EP

469 coated with chitosan in simulated digestive system, yogurt and fruit juice. LWT - Food
470 Science and Technology, 57(2), 761-766.
C

471 Kuhn, K. R., Picone, C. S. F., & Cunha, R. L. d. (2012). Biopolymer Engineering in
472 Food Processing. In T. F. group (Ed.), Biopolymer Engineering in Food Processing (pp.
AC

473 111-144): CRC Press.

474 Lee, W. J., & Lucey, J. A. (2010). Formation and Physical Properties of Yogurt. Asian-
475 Australas J Anim Sci, 23(9), 1127-1136.
476 Li, Y., Jia, H., Cheng, Q., Pan, F., & Jiang, Z. (2011). Sodium alginate–gelatin
477 polyelectrolyte complex membranes with both high water vapor permeance and high
478 permselectivity. Journal of Membrane Science, 375(1), 304-312
479 Minekus, M., Alminger, M., Alvito, P., Ballance, S., Bohn, T., Bourlieu, C., . . .
480 Brodkorb, A. (2014). A standardised static in vitro digestion method suitable for food -
481 an international consensus. Food & Function, 5(6), 1113-1124.
 ACCEPTED MANUSCRIPT
482 Mei, L., He, F., Zhou, R.-Q., Wu, C.-D., Liang, R., Xie, R., Chu, L.-Y. (2014). Novel
483 Intestinal-Targeted Ca-Alginate-Based Carrier for pH-Responsive Protection and
484 Release of Lactic Acid Bacteria. ACS Applied Materials & Interfaces, 6(8), 5962-5970.
485 Mun, S., Park, S., Kim, Y.-R., & McClements, D. J. (2016). Influence of
486 methylcellulose on attributes of β-carotene fortified starch-based filled hydrogels:
487 Optical, rheological, structural, digestibility, and bioaccessibility properties. Food
488 Research International, 87, 18-24.

PT
489 Ortakci, F., & Sert, S. (2012). Stability of free and encapsulated Lactobacillus
490 acidophilus ATCC 4356 in yogurt and in an artificial human gastric digestion system.
491 Journal of Dairy Science, 95(12), 6918-6925.

RI
492 Perrechil, F. A., Sato, A. C. K., & Cunha, R. L. (2011). κ-Carrageenan–sodium
493 caseinate microgel production by atomization: Critical analysis of the experimental

SC
494 procedure. Journal of Food Engineering, 104(1), 123-133.
495 Vilela, J. A. P., Cavallieri, Â. L. F. and Lopes da Cunha, R. (2011). "The influence of
496 gelation rate on the physical properties/structure of salt-induced gels of soy protein

U
497 isolate-gellan gum. Food Hydrocolloids, 25(7), 1710-1718.
AN
498 Ribeiro, M. C. E., Chaves, K. S., Gebara, C., Infante, F. N. S., Grosso, C. R. F., &
499 Gigante, M. L. (2014). Effect of microencapsulation of Lactobacillus acidophilus LA-5
500 on physicochemical, sensory and microbiological characteristics of stirred probiotic
M

501 yoghurt. Food Research International, 66, 424-431.

502 Rodrigues, D., Rocha-Santos, T. A. P., Pereira, C. I., Gomes, A. M., Malcata, F. X., &
503 Freitas, A. C. (2011). The potential effect of FOS and inulin upon probiotic bacterium
D

504 performance in curdled milk matrices. LWT - Food Science and Technology, 44(1), 100-
108.
TE

505
506 Saarela, M., Lähteenmäki, L., Crittenden, R., Salminen, S., & Mattila-Sandholm, T.
507 (2002). Gut bacteria and health foods—the European perspective. International Journal
EP

508 of Food Microbiology, 78(1–2), 99-117.

509 Sarker, B., D. G. Papageorgiou, R. Silva, T. Zehnder, F. Gul-E-Noor, M. Bertmer, J.
510 Kaschta, K. Chrissafis, R. Detsch and A. R. Boccaccini (2014). Fabrication of alginate
C

511 gelatin crosslinked hydrogel microcapsules and evaluation of the microstructure and
AC

512 physico-chemical properties. Journal of Materials Chemistry B 2(11), 1470-1482.

513 Silva, K. C. G., & Sato, A. C. K. (2017). Biopolymer gels containing
514 fructooligosaccharides. Food Reserach International, 101, 88-95.
515 Sultana, K., Godward, G., Reynolds, N., Arumugaswamy, R., Peiris, P., &
516 Kailasapathy, K. (2000). Encapsulation of probiotic bacteria with alginate–starch and
517 evaluation of survival in simulated gastrointestinal conditions and in yoghurt.
518 International Journal of Food Microbiology, 62(1), 47-55.
519 Tripathi, M. K., & Giri, S. K. (2014). Probiotic functional foods: Survival of probiotics
520 during processing and storage. Journal of Functional Foods, 9, 225-241.
 ACCEPTED MANUSCRIPT
521 Urbanska, A. M., Bhathena, J., & Prakash, S. (2007). Live encapsulated Lactobacillus
522 acidophilus cells in yogurt for therapeutic oral delivery: preparation and in vitro
523 analysis of alginate-chitosan microcapsules. Can J Physiol Pharmacol, 85(9), 884-893.
524 Vivek, K. B.; Reddy, K. K.; Reedy, P. V. M.; Kumar, M. S.; Narsaiah, K. (2013). Effect
525 of co-encapsulation of probiotics with prebiotics and their survivability in Dahi during
526 storage. International Journal of Agriculture and Food Science Technology, 4(1), 67-
527 75.

PT
RI
U SC
AN
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT
Table 1. Encapsulation yield of alginate-gelatin (AG), alginate-gelatin-
fructooligosaccharide (AGF) microbeads by atomizer.
Viable cells (log CFU/g)
Formulations EY (%)
Before After
AG 7.68±1.08a 7.53±0.95a 98.32±5.81A
AGF 6.75±0.46a 6.68±0.21a 99.28±6.47A

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

a b c
FOS (0 g/100 mL)

PT
d e f
FOS (3 g/100 mL)

RI
SC
Figure 1. Scanning electron micrographs of macrogels after 9 days of salt diffusion, (a)
alginate, (b) gelatin, (c) alginate-gelatin, (d) alginate-fructooligosaccharide, (e) gelatin-
fructooligosaccharide, (f) alginate-gelatin-fructooligosaccharide. Magnitude of 1k and
scale bar equal to 10 μm.
U
AN
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT

a1 b1

PT
a2 b2

RI
U SC
AN
Figure 2. Optical (1) and scanning electron (2) micrographs of microbeads, with
magnitude of 2k and scale bar equal to 10 μm; (a) alginate-gelatin , (b) alginate-gelatin-
fructooligosaccharide.
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT
120 aA
abA aA abAB bcA abA
aA abA
100 aB cA
bA
cAB
80 bB
bB
Survival (%) bB

60 AG
AGF

PT
40 FLA

20

RI
0
0 7 14 21 28

SC
Time (d)

Figure 3. Viability of alginate-gelatin (AG) and alginate-gelatin-fructooligosaccharide

U
(AGF) microbeads and free L. acidophilus (FLA) added in yogurt over 28 days under
refrigeration at 4 °C. Lowercase letters represent differences for the same formulation
AN
evaluated over the 28 days. Uppercase letters represent the differences between the
formulations compared at the same day (p< 0.05).
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT
100

90

80

70

60
Survival (%)

PT
50 FLA
AG
40

RI
AGF
30
Stomach Intestine
20

SC
10

U
0 0.5 1 1.5 2 2.5 3 3.5 4
AN
Time (h)

Figure 4. Viability of free L. acidophilus (FLA) and L. acidophilus in alginate-gelatin

M

(AG) and alginate-gelatin-fructooligosaccharide (AGF) microbeads under in vitro

digestibility for a period of 4 h.
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT

SGF SIF
0 (min) 60 (min) 120 (min) 0 (min) 60 (min) 120 (min)

PT
FLA

RI
U SC
AN
AG

M
D
TE
AGF

C EP

Figure 5. Optical micrographs of free L. acidophilus (FLA) and L. acidophilus in alginate-gelatin (AG) and alginate-gelatin-
AC

fructooligosaccharide (AGF) microbeads during simulated in vitro digestion. Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF).
 ACCEPTED MANUSCRIPT
Highlights

Ionic gelation for encapsulating symbiotic and probiotic cells.

Blends of alginate, gelatin and FOS are suitable wall materials.

Biopolymer-based beads protects the L. acidophilus during storage and simulated GIT.

The use of FOS allowed gradual and controlled delivery along the GIT.

PT
RI
U SC
AN
M
D
TE
C EP
AC