1 Characterization of lactic acid bacteria isolated from wheat bran sourdough

2 F. Maninia, M.C. Casiraghia, K. Poutanenb,c, M. Brascad, D. Erbaa, C. Plumed-Ferrerb

3 a
Dept. of Food, Environmental and Nutritional Science, University of Milan, Milan, Italy,
4 b
University of Eastern Finland, Institute of Public Health and Clinical Nutrition, PO Box 1627, FI-70211 Kuopio,
5 Finland.
6 c
VTT Technical Research Centre of Finland, PO Box 1000, FI-02044 VTT, Finland
7 d
Institute of Sciences of Food Production - Italian National Research Council, Milan, Italy
8
9 Corresponding Author: Maria Cristina Casiraghi- Dept. of Food, Environmental and Nutritional Science, University of
10 Milan, Via G. Celoria 2, 20133 Milan Italy. Phone: +390250316647
11 maria.casiraghi@unimi.it
12
13 Abstract

14 Spontaneously fermented foods, such as sourdough, represent a source of lactic acid bacteria with

15 potential interesting functional and technological properties as well as a potential source of

16 probiotics. The choice of the starter cultures has a critical impact on the palatability, processability

17 and nutritional attributes of fermented products. The aim of this study was to characterize the

18 predominant microbial species previously isolated from a sourdough-like spontaneous fermented

19 wheat bran. Lactic acid bacteria, such as Leuconostoc mesenteroides, Leuconostoc citreum,

20 Lactobacillus brevis, Lactobacillus curvatus, Lactobacillus sakei, Lactobacillus plantarum and

21 Pediococcus pentosaceus, were phenotypically characterized by their growth and acidification rate,

22 antifungal activity, carbohydrate metabolism, exopolysaccharide production, as well as safety. The

23 strains were also tested for xylan- and phytate-degrading activities. Moreover, probiotic properties,

24 such as acid and bile tolerance, anti-listeria activity and adhesion ability to Caco-2 cells were

25 examined. L. plantarum CE42, CE60 and P. pentosaceus CE65, CE23 showed interesting

26 technological application potential due to their antifungal activity and exopolysaccharide

27 production. Some strains also exhibited phytate degrading activity and could be exploited to

28 improve mineral bioavailability of fermented products. Moreover, P. pentosaceus CE65 seems to be

29 a candidate for use as probiotic.

30

31 Keywords: lactic acid bacteria; probiotic; wheat bran sourdough; starter cultures.

1
32 1. Introduction

33 The demand for faster, more efficient, controllable and large-scale fermentation has resulted in a

34 careful selection of starter microorganisms to guarantee the reproducibility of fermentation at

35 industrial scale and to obtain a product with specific properties (Carnevali et al., 2007). The choice

36 of starter culture has critical impact on the final quality of fermented foods. Fermentation with well-

37 characterized cultures, yeast or lactic acid bacteria (LAB), could be a potential tool to improve the

38 palatability, processability and the nutritional value of fermented cereal products or high-fiber

39 ingredients, such as sourdough bread, fermented wheat bran and whole-meal flours (Salmenkallio-

40 Marttila et al., 2001).

41 The main criteria used to select microbial starters are desirable technological, sensory and

42 nutritional aspects. Technological factors of interest for fermentation are growth and acidification

43 rate (Coda et al., 2010), synthesis of antimicrobial compounds (Messens & De Vuyst, 2002),

44 antifungal activity (Coda et al., 2013) and exopolysaccharide production (e.g. glucan and fructan)

45 (Galle & Arendt, 2014). Among nutritional properties, degradation of anti-nutritional factors (e.g.

46 phytic acid) and increased availability of functional compounds (e.g. soluble fiber, soluble

47 arabinoxylans, free phenolic acids, bioactive peptides) are desirable (Katina & Poutanen, 2013).

48 Technologically interesting potential starter strains are usually selected from the food matrix they

49 are going to be used for. Some recent studies have shown that the use of selected autochthonous

50 LAB to ferment sourdough is a suitable biotechnological approach to exploit the potential of cereals

51 and pseudo-cereals in bread making (Coda et al., 2010). Moreover, fermentation of bran with yeasts

52 and LAB or with enzymes has been shown to improve loaf volume, crumb structure and shelf life,

53 as well as nutritional properties of bread supplemented with fermented bran (Katina et al., 2012,

54 Salmenkallio-Marttila et al., 2001).

55 LAB could also be exploited because of their probiotic properties in order to contribute to the health

56 and wellbeing of the hosts by maintaining or improving their intestinal microbial balance (Asahara
2
57 et al., 2004). In the case of baked products, the probiotic cultures should be added to the final

58 product and/or their survival and viability should be guaranteed throughout the process steps

59 involved in the manufacture and during the storage conditions (Soukoulis et al., 2014). The aim of

60 the current study was to characterize the 13 LAB strains previously isolated from a spontaneous

61 fermented sourdough-like wheat bran (Manini et al., 2014) for their potential use as starter cultures

62 in food applications such as wheat bran fermentation and sourdough bread, and as potential

63 probiotics.

64

65 2. Materials and methods

66

67 2.1 Microorganisms
68 Thirteen LAB strains were previously isolated from spontaneously fermented wheat bran

69 sourdough-like and identified by phenotypic and molecular techniques (Manini et al., 2014). The

70 following species were found: Lactobacillus brevis (n = 2), Lactobacillus plantarum (n = 3),

71 Lactobacillus curvatus (n = 1), Lactobacillus sakei (n = 1), Leuconostoc mesenteroides (n = 2),

72 Leuconostoc citreum (n = 2), and Pediococcus pentosaceus (n = 2) (Table 1). As control,

73 Lactobacillus rhamnosus GG was used as well as two recently isolated potential probiotic strains

74 Lactobacillus plantarum Q823 and Lactobacillus casei Q11 (unpublished results).

75

76 2.2 Growth and acidification rate

77 The fermentation capacity of the strains was evaluated measuring microbial counts, pH and Total

78 Titratable Acidity (TTA) using wheat bran (raw, untreated) (mean particle size 475-633 µm -

79 Molino Quaglia, Vighizzolo D’Este, PD, Italy) as a substrate.

80 An overnight culture (1% v/v) of each test strain was individually inoculated into a sample (100 g)

81 of wheat bran dough (15% w/v of bran and 85% of water) and incubated for 8h at 30°C. The

3
 82 microbial counts were evaluated before and after fermentation. The LAB were determined on de

83 Man, Rogosa and Sharpe agar (MRS agar) (LAB M, Lancashire, UK) and the yeasts were evaluated

84 on Plate Count Agar (PCA) (LAB M, Lancashire, UK). Plates were incubated at 30 °C for 48-72 h.

85 pH and TTA developments were measured during fermentation. A sample (10 g) of bran was taken

86 every 2h and suspended in 100 mL of distilled water. For the determination of TTA, this suspension

87 was titrated with 0.1M NaOH to a final pH of 8.5, detected by a pHmeter (PHM 250, Radiometer,

88 Copenhagen); TTA was expressed as mL of 0.1 M NaOH needed to achieve the final pH of 8.5. All

89 samples were analyzed in duplicate.

90

91 2.3 Technological properties and antibiotic resistance

92 2.3.1 Carbohydrate metabolism

93 Carbohydrate fermentation profiles of the strains were determined by using API 50 CH system

94 (BioMèrieux, Marcy-l’Etoile, France). The test was performed according to the Manufacturer’s

95 instructions.

96

97 2.3.2 Phytase activity

98 The LAB strains were preliminary inoculated in MRS broth (LAB M, Lancashire, UK) and

99 incubated at 30 °C for 24 h. Strains were then grown at 30 °C for 24 to 48 h in modified Chalmers

100 broth without neutral red and with 1% of sodium phytate (Sigma-Aldrich, Milan, Italy). The

101 phytase activity was determined on modified Chalmers agar plates without CaCO3 and with 1% of

102 phytic acid calcium or sodium salt (Sigma-Aldrich) as described by Anastasio et al. (2010).

103

104 2.3.3 Xylanase activity

105 For the screening of xylanase producing microorganisms, an agar medium was prepared by adding

106 0.1% (w/v) of the dyed substrate (Remazolbrilliant Blue R treated Azo-Xylan - birchwood),

4
107 (Megazyme International Ireland Ltd, Co. Wicklow, Ireland), as the only carbon source, to a

108 sodium phosphate buffer, 100 mM, pH 6 and/or a sodium acetate buffer, 100 mM, pH 4.5 (to test

109 the activity at different pH).

110 LAB were preliminary inoculated in MRS broth and incubated at 30 °C for 24 h. The cell cultures

111 were inoculated in wells made in triplicate in the agar plates and examined, after 48h of incubation

112 at 30 °C, for clearing zones around the holes.

113

114 2.3.4 Antifungal activity

115 The antifungal activity of the strains was determined using the overlay method described by

116 Magnusson & Schnurer (2001), slightly modified.

117 The moulds Aspergillus oryzae ATCC 66222 and Aspergillus niger 25541 were used for this test. A

118 spore suspension was prepared by growing the moulds on PCA at 30°C for 3-4 days and then

119 collecting the conidia by vigorously shaking the slants with sterile peptone water.

120 LAB were inoculated as two 2-cm-long lines on MRS plates and incubated at 30°C for 48 h. The

121 plates were then overlaid with 10 mL of malt extract soft agar (3% malt extract, 1.5% bacto

122 peptone, 0.75% agar) inoculated with fungal spore suspension. After solidification, the plates were

123 incubated aerobically at 30°C for 48 h. The plates were examined for zones of inhibition around the

124 bacterial streaks.

125

126 2.3.5 Exopolysaccharides

127 LAB strains were plated on different MRS agar plate with glucose, sucrose, raffinose, maltose,

128 lactose and starch as the only carbon source. Plates were incubated for 2 days at 30°C. Duplicate

129 plates containing 25 to 250 colonies were scored for mucoid properties (scale of ++ = excess EPS to

130 – = no visible mucoid). Colonies were scored as ropy if strings of 5 mm or more were detected

5
131 when the colony was touched once with a wire inoculating loop (Ruas-Madiedo & De Los Reyes-

132 Gavilán, 2005).

133

134 2.3.6 Antibiotic resistance

135 The minimum inhibitory concentrations (MICs) of ten antibiotics (gentamicin, kanamycin,

136 streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin, neomycin,

137 vancomycin) were determined by the microdilution method as reported by the ISO 10932:2010

138 standard method (ISO 10932/IDF 223, 2010).

139 Briefly, strains were prepared by suspending single colonies (picked up from fresh cultures on LSM

140 agar plates incubated for 48 h at 37°C) in a tube with 3 mL of 0.85% saline suspension and the

141 density was adjusted spectrophotometrically to an OD625 of 0.16–0.20 and subsequently diluting

142 them 1:500 in the medium. This suspension density corresponds approximately to McFarland

143 standard 1 (McF 1), 3 x 108 CFU/ mL.

144 Inoculation of manually pre-made MIC microtiter test plates (containing the different antibiotic

145 concentrations in 50 µl of LSM broth per well), with the standardized strain suspensions, was

146 performed to obtain a final LAB inoculum of 102 bacteria mL-1. The microtiter plates were

147 subsequently incubated anaerobically either at 28°C or at 32°C (P. pentosaceus) for 24 h. The MICs

148 were evaluated as the lowest concentration of a given antibiotic at which no growth of the test

149 organism was observed.

150 Epidemiological cut-off values were defined according to the FEEDAP Panel (EFSA-FEEDAP,

151 2012).

152

153 2.4 Potential probiotic properties

154 2.4.1 Growth at 30 °C and 37 °C
155 The growth performances at 30° C and 37 °C for 24 h in MRS broth were monitored using a

156 Thermo Bioscreen C automatic turbidometer (Labsystems Oy, Helsinki, Finland). Growth was
6
157 measured using 100-well Honeycomb microplates (TermoLabsystems, Helsinki, Finland). Each

158 strain was tested in five replicates in a total volume of 300 µL per well.

159 2.4.2 Acid tolerance

160 The ability of the strains to survive at low pH was evaluated in triplicate as described by Lee et al.

161 (2011), in acidified MRS broth (final pH 2.5). The pH-adjusted MRS broth was inoculated with an

162 overnight culture of the different LAB strains (1% v/v) to a final cell concentration of

163 approximately 1.0 x 107 CFU/mL. pH tolerance was evaluated by measuring survival after 2h of

164 incubation at 37 °C to simulate intestinal conditions. Bacterial counts were determined in duplicate,

165 on MRS agar plates and incubated at 30 °C for 48 h.

166

167 2.4.3 Bile tolerance

168 Bile tolerance was measured in triplicate by means of the method described by Sabir et al. (2010) in

169 MRS broth containing 0.3% oxgall (Sigma–Aldrich, Steinheim, Germany), inoculated (1% v/v) and

170 incubated at 37 °C. Samples were taken at 24h and plated on duplicate MRS agar plates. The

171 survival rate was determined after a 48 h incubation at 30 °C.

172

173

174

175 2.4.4 Adhesion to human colon carcinoma cell-line Caco-2

176 The human colon carcinoma cell-line Caco-2 cells (ATTC HTB-37) were grown in 75 cm3 cell

177 culture bottles (Sarstedt, Inc., Newton, NC, USA) using DMEM supplemented with 10% (v/v) heat

178 inactivated fetal bovine serum, 2mM L-glutamine, 1% (v/v) non-essential amino acids and 100 IU

179 penicillin/mL and 100 g streptomycin/mL (EuroClone, Siziano, Italy). Caco-2 cells were

180 subsequently seeded to 24-well culture plates at a concentration of 2.5x105 cells per well. Cells

7
181 were differentiated for 2 weeks, changing medium every 2-3 days. Cells were always incubated at

182 37ºC in a 5% CO2 atmosphere.

183 Four bacterial strains were chosen for this test based on the results obtained in previous tests. L.

184 curvatus CE83, L. plantarum CE84, L. brevis CE85, P. pentosaceus CE65 were grown overnight at

185 37ºC in MRS broth. After incubation, bacterial cells were collected by centrifugation, washed twice

186 with PBS and suspended in PBS to an appropriate dilution (Abs625nm of 0.2, approx. 2x108

187 CFU/mL) then added to each well and incubated for 2 hours. After incubation, the cells were

188 washed four times and lysed with 0.1% Triton X-100 (Sigma-Aldrich). Cell lysates were serially

189 diluted, plated in duplicate on MRS agar and then incubated at 37ºC for 2 days. The adhesion

190 capacity of the strains was calculated as percentage of the bacteria counted from the cell lysates

191 divided by the total bacteria added to the well. Three biological replicates made in different days

192 and four replicates for each biological replicates were used for this test.

193

194 2.4.5 Anti-Listeria activity

195 The capacity of the strains to inhibit Listeria was determined using the agar spot test described by

196 Jacobsen et al. (1999), with some modifications. The assayed strains included Listeria innocua, L.

197 monocytogenes and L. welshimeri, isolated respectively from food, animal and human.

198 A 100-µl of an overnight culture of the pathogen strains was plated on PCA and dried. The LAB

199 strains were then spotted (10 µl) in triplicate on the surface of the PCA plate and incubated at 37°C

200 to develop the spots. After 48h the inhibition zones were evaluated. A clear zone of more than 1

201 mm around a spot was scored as positive. Each test was performed in duplicate.

202

203 3. Results and discussion

204 3.1 Growth and acidification rate

8
205 The growth of all the tested LAB increased substantially after 8 h of bran fermentation (average
-1
206 increase approximately 2.7 log CFU.g , Table 2). In particular, L. sakei CE47 and Ln.

207 mesenteroides CE48 showed the highest growth (3.6 and 3.7 log CFU.g-1 respectively), while the

208 lowest growth was observed with Ln. citreum CE54 (1.6 log CFU.g-1). L. plantarum CE84, CE42,

209 CE60, L. curvatus CE83, L. sakei CE47, and P. pentosaceus CE65, CE23 showed the highest

210 acidification rate, the smallest pH reached being pH 4. The lowest acidification rate was obtained

211 with obligate heterofermentative L. brevis CE94, CE85, in which pH reached values 5.5 and 5.3

212 respectively.

213

214 3.2 Technological properties and antibiotic resistance

215 3.2.1 Carbohydrate metabolism

216 The carbohydrate metabolism profiles of the LAB are shown in Table 3. All the tested strains were

217 able to ferment D-galactose, D-glucose, D-fructose, D-maltose and N-acetylglucosamine. Mixtures

218 of strains with different carbohydrate metabolism are frequently used because they may guarantee

219 optimal acidification and sensory properties (Gobbetti, 1998). Among the tested strains, Ln. citreum

220 CE88, Ln. mesenteroides CE48, L. curvatus CE83, L. brevis CE94, CE85 and P. pentosaceus CE65

221 were able to use both arabinose and xylose confirming similar findings reported before (Gobbetti et

222 al., 1999). Among L. plantarum strains, only CE60 was able to use L-arabinose and to a lesser

223 extent also D-arabinose.

224 Despite that none of the L. plantarum tested metabolized xylose, these strains showed the widest

225 carbohydrate utilization spectrum. This species is commonly found in sourdoughs ecosystems and

226 its prevalence in cereal fermentations has been mainly attributed to the versatile metabolism of

227 carbohydrates (Minervini et al., 2010).

228

229 3.2.2 Enzymatic activities

9
230 The indigenous microbiota of sourdough is a source of considerable genetic diversity representing

231 different enzymatic activities useful in biotechnological applications (Pepe et al., 2004). Enzymes,

232 such as xylanase and phytase are examples of the technological and nutritional potential of the

233 microbial biomass of sourdough.

234 None of the tested strains showed endo-xylanase activity in the plate assay (Table 4). Xylanolytic

235 enzymes are involved in the hydrolysis of xylan and arabinoxylan polymers, and consequently in

236 their solubilization (Gruppen et al., 1993). Moreover, in bread and bakery industry, xylanases are

237 used to increase the dough viscosity, bread volume and shelf life (Poutanen et al., 2009).

238 Considering the nutritional importance of this enzymatic activity, related to “solubilization” of bran

239 dietary fibre, further investigation has been planned to evaluate the presence and the expression of

240 genes codifying for this activity.

241 Regarding phytate activity, the ability to degrade sodium phytate was prevalent among all the LAB

242 tested, except for L. plantarum CE60. This is consistent with a study of De Angelis et al. (2003) in

243 which phytase activity was prevalent in all the 12 species representing LAB isolated from

244 sourdough. Moreover, 8 out of 13 LAB strains tested were able to hydrolyze both hexacalcium and

245 sodium phytate (phy+), the most abundant phytate forms in cereals and legume-based foods. In

246 particular, Ln. citreum CE54 exhibited potent activity both on calcium and sodium phytates.

247 Although some P. pentosaceus strains have been reported to be able to degrade both sodium and

248 calcium phytate (De Angelis et al., 2003), in the present study the two P. pentosaceus strains

249 degraded only one of the phytate types, reflecting a variability among strains of phytase activity.

250 The strains that have shown phytate degrading ability could be exploited as starter cultures in

251 fermented foods to improve mineral bioavailability and thus upgrading the nutritional quality of

252 phytate-rich foods.

253

254 3.2.3 Antifungal activity

10
255 The culture overlay assay carried out on the LAB strains showed different levels of inhibition

256 against the two fungal target strains Aspergillus oryzae and Aspergillus niger (Table 5), which are

257 capable of rapid growth on the surface of bakery products (Smith et al., 2004).

258 L. plantarum CE42, CE60, CE84, L. curvatus CE83 and P. pentosaceus CE65, CE23 showed the

259 highest activity against Aspergillus oryzae and a moderate activity against Aspergillus niger. The

260 latter was particularly inhibited by Ln. mesenteroides CE52 and L. brevis CE94. In previous studies,

261 some L. plantarum have been shown to possess antifungal activity due to the formation of lactic

262 acid, acetic acid, and hydrogen peroxide, to the competition for nutrients and the production of

263 bacteriocins (Gupta & Srivastava, 2014). During the last few years, there has been a growing

264 interest in the use of microorganisms and/or their metabolites to prevent spoilage and to extend the

265 shelf-life of bakery products (Garofalo et al., 2012).

266

267 3.2.4 Exopolysaccharide production

268 An interesting property of sourdough LAB is their ability to synthesize a large structural variety of

269 exopolysaccharides (EPS), such as glucan and/or fructans (Galle & Arendt, 2014). EPS have a

270 positive effect on the texture, mouthfeel, taste perception, and stability of fermented food and for

271 certain EPS, prebiotic effects have also been described (Dal Bello et al., 2001).

272 In accordance to Tieking & Gänzle (2005), who postulated that any sourdough microbiota contains

273 at a high probability at least one EPS producing strain, only 3 out of 13 LAB strains studied here

274 did not produce EPS (CE47; CE52; CE85), the major EPS producers being L. plantarum and P.

275 pentosaceus. As shown in Table 6, the two P. pentosaceus strains were able to produce EPS while

276 growing on glucose and raffinose (CE65), and maltose (CE23). The L. plantarum tested produced

277 EPS with different carbohydrates, indicating that in each case the most suitable carbohydrate is

278 largely strain-dependent. L. plantarum CE84 was able to produce EPS in presence of different

279 carbon sources, such as sucrose, raffinose, maltose and in particular lactose, while L. plantarum

11
280 CE42 and CE60 were able to produce EPS in presence of starch, usually the most abundant carbon

281 source in cereal products. Ln mesenteroides CE48 produced EPS using glucose and maltose as

282 carbon source and probably synthesized dextran as reported in other studies (Lacaze et al., 2007); in

283 Panettone, a traditional Italian sweet bread, dextran from Ln. mesenteroides is responsible for the

284 long storage stability (Decock & Cappelle, 2005). Differently, Ln. mesenteroides CE52 did not

285 show any EPS production.

286

287 3.2.5 Antibiotic resistance

288 Although LAB have been 'generally recognized as safe (GRAS), it has been shown that genes

289 coding for antibiotics resistance can be transferred among bacteria of different genera and thus to

290 human commensal flora and to pathogenic bacteria, temporarily residing in the hosts, which

291 consequently cannot be treated with previously successful antibiotics (Adimpong et al., 2012).

292 The results of antibiotic susceptibility testing are shown in Table 7. The bacteria were considered

293 resistant to a particular antibiotic when the MIC (µg/mL) values obtained were higher than the

294 recommended breakpoint value, defined at species level by the FEEDAP Panel (EFSA-FEEDAP,

295 2012). In the present study, L. plantarum CE84, L. curvatus CE83 and L. brevis CE94, CE85 were

296 resistant to Clindamycin. This antibiotic is used to treat infections caused by anaerobic bacteria but

297 also aerobic gram-positive cocci such as Staphylococcus or Streptococcus. However, cases of

298 clindamycin resistant and methicillin-resistant Staphylococcus aureus are being reported (Cadena et

299 al., 2012) and thus these strains may require further molecular investigation to find out the cause of

300 these resistance patterns and they should not be used as starter cultures in foods, unless the type of

301 resistance can be shown to be non-transmissible. The investigated strains had MIC values for

302 vancomycin of 64 or 128 μg mL-1, which can be expected in the case of Lactobacillus, Pediococcus

303 and Leuconostoc species (MIC values 128 μg mL-1) because this is due to the absence of D-Ala-D-

304 lactate in their cell wall (Ammor et al., 2007). Thus, the resistance mechanisms observed among

12
305 these strains are probably inherent or intrinsic to their species and could therefore not be attributed

306 to acquisition of resistance genes. No other strain showed to be resistant to the antibiotic tested and

307 thus these strains are safe for the use as starter cultures and/or probiotics.

308

309 3.3 Screening for probiotic properties

310 Spontaneously fermented foods, such as sourdough, may constitute a reservoir for new LAB strains

311 with potential probiotic characteristics. The LAB strains were screened for their ability to survive to

312 the passage through the gastro-intestinal tract (GIT), to adhere to epithelial surfaces and for their

313 antagonistic activity towards pathogens.

314

315 3.3.1 Growth at 30 °C and 37 °C

316 All the tested strains have grown both at 30 °C and 37 °C, except for Ln. mesenteroides CE52 that

317 did not grow at 37°C (Table 8).

318

319 3.3.2 pH and bile resistance

320 According to Fuller (1992), bile, even at low concentrations, can inhibit the growth of

321 microorganisms. Gilliland et al. (1984) reported that 0.3% is considered to be a critical

322 concentration for screening for resistant strains. All strains were able to survive in 0.3% (w/v) bile

323 and L. plantarum CE60, CE84, L. curvatus CE83, Ln. mesenteroides CE48 and L. brevis CE94,

324 CE85 were even able to replicate in presence of bile salt after 24h (Table 8). In accordance with

325 Delgado et al. (2007), the acidic condition (pH 2.5) seemed to be more damaging to the bacteria,

326 with only 6 out of 13 strains surviving 2h of exposure and none of them growing.

327 In conclusion, L. curvatus CE83, Ln. mesenteroides CE48, L. brevis CE94, CE85 and P.

328 pentosaceus CE65, CE23 were able to survive when exposed to the conditions resembling those of

329 the GIT, in terms of the low pH and the presence of bile salts.

13
330 Our study showed that the acidic tolerance is not necessarily related to the species of LAB, but may

331 also be strain-specific; in fact, differences were observed among strains belonging to the same

332 species, such as L. plantarum, Ln mesenteroides and P. pentosaceus, in terms of acid and bile

333 tolerance. Moreover, although strains of L. plantarum have previously been shown to survive

334 gastric transit (Mathara et al., 2008), our results indicate that L. plantarum may have strong bile

335 tolerance but lower ability to survive at low pH.

336 These tests are, however, rather qualitative, and the resistances of probiotic cultures to low pH and

337 bile in food matrices during passage through the GIT might be greater than those seen in the

338 physiological solutions used in the present study.

339

340 3.3.3 Adhesion to Caco-2 cells

341 The capacity of bacteria to adhere to the intestinal mucosa is a key factor in a strain’s ability to

342 function as desired in the intestine and thus have probiotic effects.

343 The adhesion ability to Caco-2 cells, which simulates the morphological and physiological

344 characteristics of human enterocytes (Blum et al., 1999), was evaluated for 4 LAB strains (Table 8),

345 belonging to different species, selected according to their ability to survive to the conditions

346 resembling those of the GIT: L. curvatus CE83, L. brevis CE85, P. pentosaceus CE65, and also L.

347 plantarum CE84 was tested despite its poor resistance to low pH. L. rhamnosus GG, L. plantarum

348 Q823 and L. plantarum CE42 were used as positive and negative control, respectively. All the

349 tested strains strongly adhered to the Caco-2 cells with adhesive properties even higher than those

350 assessed in the positive control. L. curvatus CE83, L. brevis CE85 and P. pentosaceus CE65, due to

351 their ability to survive to the conditions of the GIT and their capacity to adhere to the intestinal

352 mucosa, could be suitable candidates to be used as probiotics.

353

354 3.3.4 Anti-listeria activity

14
355 The inhibition of pathogenic bacteria, causing diarrhea or other diseases in the human intestine, is a

356 desirable property for probiotic bacteria (Delgado et al., 2007) in order to balance the intestinal

357 environment, and thereby improve host health. L. plantarum CE42, CE60, CE84 and P.

358 pentosaceus CE65 showed high inhibitory activity against the Listeria species tested (L. innocua, L.

359 monocytogenes, L. welshimeri) (Table 9). Ln. citreum CE88, CE54 were able to inhibit only L.

360 monocytogenes, and L. sakei CE74 showed activity against L. monocytogenes and L. welshimeri.

361 This inhibition could be due to the production of inhibitory substances, such as organic acids,

362 bacteriocins or peroxide hydroxide (Juven et al., 1992).

363

364 4. Conclusions

365 Wheat bran sourdough proved a good source of several interesting microbial strains with potential

366 for future applications. Excluding the strains resistant to clindamycin, L. plantarum CE42, CE60

367 and P. pentosaceus CE65, CE23 could have interesting technological applications due to their

368 antifungal activity and EPS production. These strains also shown phytate-degrading activity on

369 calcium and/or sodium phytate salts and could thus also be useful to improve mineral bioavailability

370 of fermented products. Moreover, P. pentosaceus CE65 seems to be a suitable candidate for use as

371 probiotic. Further studies will be needed in order to test the effectiveness of these strains in

372 improving the quality of fermented products and to better determine their suitability for specific

373 applications.

374

375 5. References

376 Adimpong, D. B., Nielsen, D. S., Sørensen, K. I., Derkx, P. M., & Jespersen, L. (2012). Genotypic

377 characterization and safety assessment of lactic acid bacteria from indigenous African fermented

378 food products. BMC Microbiology, 12, 75-86.

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379 Ammor, M.S., Belén Flórez, A., & Mayo, B. (2007). Antibiotic resistance in non-enterococcal

380 lactic acid bacteria and bifidobacteria. Food Microbiology, 24,559-570.

381 Anastasio, M., Pepe, O., Cirillo, T., Palomba, S., Blaiotta, G., & Villani, F. (2010). Selection and

382 Use of Phytate‐Degrading LAB to Improve Cereal‐Based Products by Mineral Solubilization

383 During Dough Fermentation. Journal of Food Science, 75, M28-M35.

384 Asahara, T., Shimizu, K., Nomoto, K., Hamabata, T., Ozawa, A., & Takeda, Y. (2004). Probiotic

385 bifidobacteria protect mice from lethal infection with Shiga toxin-producing Escherichia coli

386 O157: H7. Infection and Immunity, 72,2240-2247.

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