Journal Pre-proofs

Antifungal activity of probiotic Lactobacillus strains isolated from natural fer‐

mented green olives and their application as food bio-preservative

Houssam Abouloifa, Sara Gaamouche, Yahya Rokni, Ismail Hasnaoui, Reda

Bellaouchi, Nabil Ghabbour, Salwa Karboune, Milena Brasca, Guy
D'Hallewin, Riadh Ben Salah, Ennouamane Saalaoui, Abdeslam Asehraou

PII: S1049-9644(20)30677-0
DOI: https://doi.org/10.1016/j.biocontrol.2020.104450
Reference: YBCON 104450

To appear in: Biological Control

Received Date: 2 July 2020

Revised Date: 17 September 2020
Accepted Date: 21 September 2020

Please cite this article as: Abouloifa, H., Gaamouche, S., Rokni, Y., Hasnaoui, I., Bellaouchi, R., Ghabbour, N.,
Karboune, S., Brasca, M., D'Hallewin, G., Ben Salah, R., Saalaoui, E., Asehraou, A., Antifungal activity of
probiotic Lactobacillus strains isolated from natural fermented green olives and their application as food bio-
preservative, Biological Control (2020), doi: https://doi.org/10.1016/j.biocontrol.2020.104450

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1 Antifungal activity of probiotic Lactobacillus strains isolated from natural fermented

2 green olives and their application as food bio-preservative

4 Houssam Abouloifaa, Sara Gaamouchea, Yahya Roknia, Ismail Hasnaouia, Reda

5 Bellaouchia, Nabil Ghabboura, Salwa Karbouneb, Milena Brascac, Guy D’Hallewind,

6 Riadh Ben Salahe, Ennouamane Saalaouia, Abdeslam Asehraoua*

8 a: Laboratory of Bioresources, Biotechnology, Ethnopharmacology and Health, Faculty of

9 Sciences, Mohammed Premier University, Oujda 60 000, Morocco.

10 b: Department of Food Science and Agricultural Chemistry, Macdonald Campus, McGill

11 University 21,111 Lakeshore, Ste Anne de Bellevue, Quebec H9X 3V9, Canada

12 c: Institute of Sciences of Food Production, National Research Council of Italy, Via Celoria 2,

13 20133, Milano, Italy.

14 d: CNR-ISPA UOS Sassari, Traversa la Crucca, 3 Loc. Baldinca 07040 Sassari, Italy.

15 e: Laboratory of Microorganisms and Biomolecules, Centre of Biotechnology of Sfax, BP:

16 1177, 3018, Tunisia.

17

18 ⁎Corresponding author: Abdeslam Asehraou, Laboratory of Bioresources, Biotechnology,

19 Ethnopharmacology and Health, Faculty of Sciences, Mohammed Premier University, Oujda

20 60 000, Morocco. Email: a.asehraou@ump.ac.ma

21
23 Abstract

24 The aim of this work was to characterize the antifungal activity of 14 probiotics Lactobacillus

25 strains of (L. plantarum, L. pentosus and L. brevis), isolated from traditional fermented green

26 olives. All the Lactobacillus strains and their cell-free-supernatants (CFS) showed high

27 antifungal activity against molds (Aspergillus niger, Penicillium sp, Fusarium oxysporum,

28 Rhizopus sp.) including biomass and mycelium growth inhibition, and against yeasts

29 (Candida pelliculosa and Rhodotorula sp.). Some (9/14) of these Lactobacillus strains

30 showed resistance and maintained their antifungal activity, against C. pelliculosa and P.

31 digitatum, after 5 hours of exposure to gastro-intestinal conditions (combination of 0.3% bile

32 salts and pH 2.5). The antifungal compounds produced by the Lactobacillus strains were heat

33 resistant (pasteurization and autoclaving), active in large pH values (3.8 to 7), with a

34 proteinaceous, lipid or other nature. Among the Lactobacillus strains, L. plantarum S61 and

35 its CFS showed important effectiveness as bio-preservative agent, and delayed the growth of

36 P. digitatum in tomato puree and pear fruits for 15 and 10 days, respectively; while in apple

37 juice, it decreased the C. pelliculosa population from 5 to 2.5 log CFU/mL, after 48 hours of

38 inoculation.

39

40

41 Keywords: Antifungal, Bio-preservation, Lactobacillus, Olive, Fermentation.

43 1. Introduction

44 Fungi involved in food spoilages have a global concern, because they lead to high economic

45 losses, due to food waste and food poisonings (Gerez et al., 2009). FAO estimated annual

46 food loss and waste to 30% in the worlds, where fruits and vegetables are of 21.6% (FAO,

47 2019). Fruits and vegetables are perishables, and they are subject to fungal attacks along the

48 food supply chain. The fruits and vegetables losses due to postharvest diseases are estimated

49 between 35% and 55% of the total production (Sanzani et al., 2016), and they are mostly

50 caused by fungi (Mahunu et al., 2015). Fungal development leads to food sensory defects

51 varying from visual deterioration to noticeable odor, flavor or texture changes, and it also can

52 have negative impact on health via mycotoxin production (Leyva Salas et al., 2017).

53 The control of food spoilages, caused by yeasts and molds, is commonly achieved by

54 chemical preservatives (Thind, 2017). However, chemical fungicides are known for their

55 drawbacks on human health and environment (Compant et al., 2005). In addition, increase of

56 fungal resistance to chemical fungicides constitutes a great challenge for human health and

57 food security (Fisher et al., 2018; Schnürer and Magnusson, 2005). Therefore, the

58 development of efficient and safe fungicides as alternative becomes necessary. Bio-fungicides

59 of microbial (bacteria, yeasts and molds) and plants origins are promising alternatives in

60 control of fungal spoilage of foods (Leneveu-Jenvrin et al., 2019; Seshadri et al., 2020).

61 During the last years, there has been a growing interest in food bio-preservation by lactic acid

62 bacteria (LAB), because of their GRAS (Generally Recognized as Safe) status, and their

63 potential antifungal activity against various fungal species (Ahmad Rather et al., 2013). LAB

64 are known for their ability to produce various antimicrobial compounds, composed mainly of

65 lactic acid, acetic acid, phenyllactic acid, fatty acids, proteinaceous compounds, cyclic

66 dipeptides, bacteriocins, hydrogen peroxide and other compounds (Sadiq et al., 2019; Siedler

67 et al., 2019). These bioactive metabolites can be used to control pathogenic and spoilage
68 microorganisms (Abbaszadeh et al., 2015; Muhialdin et al., 2018) and multi-resistant bacteria

69 and influenza A virus (Kwak et al., 2018). However, the antifungal activity of LAB is

70 influenced mainly by strain genotype and origin (Sadiq et al., 2019), and also by

71 environmental factors (Batish et al., 1997). LAB strains naturally occurring in extreme

72 environments, such as traditional fermented olive, may have important antifungal activity,

73 which can be effective as biopreservative.

74 Traditional fermented green olives are basically processed by natural lactic fermentation of

75 non-alkali-treated green olives. This fermentation process is assured by LAB, naturally

76 associated to olive fruits and containers, in olive brine rich of sodium chloride (10-12%, w/v),

77 phenolic compounds and various competitors’ microorganisms (Rokni et al., 2015). LAB

78 strains dominating in this extreme environment, should have important physiological

79 properties (i.e. antifungal activity) to overcome the abiotic and biotic stress factors of this

80 biotope. In previous works some Lactobacillus strains, we isolated from traditional fermented

81 green olive brine, demonstrated important probiotic and technological properties, including

82 antifungal activity (Abouloifa et al., 2019; Abouloifa et al., 2020). The main objective of this

83 work is to characterize the antifungal activity of these probiotics Lactobacillus strains, and

84 evaluate their bio-protective effect against fungal spoilage of some food products.

85 2. Material and methods

86 2.1. Microorganisms and growth conditions

87 Fourteen probiotic Lactobacillus strains, isolated from natural fermented green olive brine,

88 and selected for their antifungal activity against Candida pelliculosa and Penicillium sp, were

89 studied in this work, and they included Lactobacillus brevis (5 strains), Lactobacillus

90 pentosus (2 strains) and Lactobacillus plantarum (7 strains). The biochemical and molecular

91 (16S rRNA gene) characterization of these Lactobacillus strains was reported in previous

92 works (Abouloifa et al., 2019; Abouloifa et al., 2020). These strains were maintained in 20%
93 glycerol (v/v) at -80ºC, they were routinely reactivated in de Man Rogosa and Sharpe (MRS)

94 broth (BIOKAR, FRANCE) at 30ºC for 18 hours before use.

95 The target strains used in this work were yeasts (C. pelliculosa, Rhodotorula sp) and molds

96 (Aspergillus niger, Penicillium digitatum, Fusarium oxysporum and Rhizopus sp). These

97 strains were cultured in peptone dextrose agar (PDA, Biokar, France) at 30°C during 2 days

98 for yeasts and at 25°C during 5 days for molds. The spores of molds were harvested, from 5-

99 day fungus cultures obtained on PDA, in sterile distilled water containing 0.05% Tween 80,

100 and the spores suspension was adjusted by a hematimeter to 106 spores/mL.

101 2.2. Antifungal activity determination

102 2.2.1. Plate confrontation method

103 The antifungal activity of Lactobacillus strains was determined by the plate confrontation

104 method described by (Ndagano et al., 2011), with some modifications. Briefly, 5 μl of each 18

105 h Lactobacillus strain culture, obtained in MRS broth, were spot-inoculated on MRS agar

106 plates. After 48 h of incubation at 30°C, the colonies of Lactobacillus were overlaid with 10

107 ml of PDA medium (0.75% agar) previously inoculated to 105 spores/mL of each target mold

108 (A. niger, P. digitatum, F. oxysporum and Rhizopus sp.) and to 105 CFU/mL of each target

109 yeast (C. pelliculosa, Rhodotorula sp.). The inhibition diameters of the targets were measured

110 after incubation at 30°C for 2 days for yeasts and 25°C for 5 days for molds. All inhibition

111 tests were realized in triplicate.

112 2.2.2. In-vitro stability of antifungal activity in gastro-intestinal tract conditions

113 The in-vitro stability of antifungal activity of Lactobacillus strains in gastro-intestinal tract

114 condition was studied by the method of (Abouloifa et al., 2019). Briefly, Lactobacillus strains

115 cultures obtained in MRS broth, containing bile-salts (0.3%, w/v) and adjusted to pH (2, 2.5

116 or 3) and incubated at 37°C for 5 h, were pour plated (0.1 mL) on MRS agar. After 24 hours

117 of incubation at 37°C, the Lactobacillus colonies were coated with 10 mL of soft PDA agar
118 (0.75% agar) previously inoculated to 105 spores/mL of the target mold (P. digitatum), and to

119 105 CFU/mL of the target yeast (C. pelliculosa). After incubation at 30°C during 2 days for

120 yeast and 25°C during 5 days for mold, the antifungal activity was evaluated visually, by

121 observing the inhibition zones of targets around colonies of Lactobacillus strains. All the

122 experiments were performed in triplicate.

123 2.3. Antifungal activity of cell-free supernatant (CFS)

124 2.3.1. Preparation of CFS

125 An aliquot (200 μL) of overnight cultures of Lactobacillus strains, obtained in MRS broth

126 after 18 h of incubation at 30°C, was inoculated in sterile MRS broth (20 mL) and allowed to

127 grow at 30°C for 48 h. Then, the cells were removed by centrifugation (8000×g, 10 min) at

128 4°C, and the CFS obtained was filter-sterilized using 0.20 μm pore-size filter (Minisart

129 syringe filter, Sartorius, Germany).

130 2.3.2. Antifungal activity of CFS

131 The antifungal activity of CFS of Lactobacillus strains was measured by the agar diffusion

132 method. Suspensions of 105 spores/mL of molds (A. niger, P. digitatum, F. oxysporum and

133 Rhizopus sp.) and 105 CFU/mL of yeasts (C. pelliculosa and Rhodotorula sp.) were pour-

134 plated separately as targets on PDA. Then, wells of 6 mm diameter were performed in PDA

135 and charged aseptically with 100 μl of sterile CFS of Lactobacillus strains. The cultures were

136 then incubated at 30°C during 2 days for yeasts and at 25°C during 5 days for molds. The

137 inhibition diameters of targets obtained around wells were measured. All the assays were

138 conducted in triplicate.

139 2.3.3. Fungal biomass growth inhibition

140 The inhibition of fungal biomass growth by CFS of Lactobacillus strains was determined in

141 MRS broth, based on the method of (Muhialdin and Hassan, 2011), with some modifications.

142 A volume of 5 mL of sterile CFS of each Lactobacillus strain was introduced in a flask
143 containing 40 mL of MRS broth (BIOKAR, France), then inoculated (5 ml) by 106 spores/mL

144 of a target mold (A. niger, P. digitatum, F. oxysporum and Rhizopus sp.). A culture of each

145 fungus conducted in MRS broth without CFS was used as control. After 5 days of incubation

146 at 25°C, the fungal biomass was collected on Whatman Grade 1 filter paper and dried in an

147 oven at 100°C for 18 h. All the tests were made in triplicate. The percentage of biomass

148 growth inhibition (BI, %) was calculated using the formula: (BI, %) = [(TC-TT)/TC] x100,

149 where (BI, %) is the percentage of fungal biomass growth inhibition, TC is the total fungal

150 biomass (g) obtained without CFS (control), and TT is the total fungal biomass (g) obtained

151 with CFS.

152 2.3.4. Mycelium growth inhibition

153 The inhibition of mycelium growth by the CFS of Lactobacillus strains was determined on

154 Potato Dextrose Agar (PDA) (BIOKAR, France). Plates containing PDA medium, added

155 (10%, v/v) with sterile CFS of each Lactobacillus strain, were inoculated in the center with

156 disc (5 mm) of colony of target mold (A. niger, P. digitatum, F. oxysporum or Rhizopus sp.).

157 After incubation at 25°C for 5 days, the diameters of fungal colonies were measured. Plates

158 containing PDA mixed with sterile distilled water (10%, v/v), inoculated and incubated in the

159 same conditions above, were used as controls. All the assays were realized in triplicate. The

160 percentage of mycelium growth inhibition (MI, %) was calculated using the formula: (MI, %)

161 = [(TC-TT)/TC] x100, where (MI, %) is the percentage of mycelial growth inhibition, TC is the

162 total fungal colony diameter (mm) obtained in control, and TT is the total fungal colony

163 diameter (mm) obtained with CFS.

164 2.4. Nature of antifungal compounds

165 The influence of enzymes on the antifungal compounds of CFS was investigated on filter

166 sterilized CFS separately treated with one of the following enzymes: catalase (Sigma-Aldrich,

167 CAS Number: 9001-05-2), proteinase K (Sigma-Aldrich, CAS Number: 39450-01-6) and
168 lipase (Fluka, CAS Number: 9001-62-1). The CFS of each Lactobacillus strain was adjusted

169 to pH 7 using 1 M NaOH, then added with 1mg/mL of the respective enzyme, and incubated

170 at 37°C for 3 h. After incubation, the samples were placed in water bath at 65°C for 5 min for

171 denaturation of enzymes. The antifungal activity of treated CFS was evaluated by agar well-

172 diffusion method, as described above. All the assays were conducted in triplicate.

173 2.5. Physicochemical stability of antifungal compounds

174 The physico-chemical stability of antifungal compounds was evaluated by exposure of CFS to

175 elevated temperatures and different pH values, and the antifungal activity was measured by

176 well diffusion method. The CFS of each Lactobacillus strain was subject to different

177 treatments and then filter sterilized; it was heated in water bath at different temperatures

178 (80°C and 100°C for 10 min), autoclaved at 121°C for 15 min, and adjusted with 1 M NaOH

179 to different pH values (4, 5, 6 and 7). The antifungal activity of treated CFS was determined

180 by agar well diffusion method as described above, and the results are expressed according to

181 the method of (Adedokun et al., 2016). All the assays were conducted in triplicate.

182 2.6. Application of antifungal Lactobacillus strains in food bio-preservation

183 2.6.1. Bio-preservation of tomato puree

184 The bio-preservation potential of the strain L. plantarum S61, selected for its important

185 antifungal activity, was evaluated on tomato puree purchased in local supermarket in Oujda-

186 city, Morocco, according to the method of (Adedokun et al., 2016), with some modifications.

187 Briefly, twenty grams of tomato puree, having an initial pH of 3.8, were introduced in sterile

188 plastic Petri dishes. A volume of 1 mL of Lactobacillus culture (105 CFU/mL) or CFS or

189 concentrated CFS was spread on the surface of tomato puree, and then drop inoculated, in the

190 center of the Petri dishes, with 100 μL of spore suspension (105 spore/mL) of P. digitatum.

191 The control sample was added with 1 mL of sterile distilled water and inoculated in the same

192 conditions of the other assays. The concentrated CFS of L. plantarum S61 was obtained by
193 10-fold concentration of the CFS (10x-CFS) at 60°C using a concentrator plus (eppendorf

194 speed vac), and sterilized by 0.20 µm pore size filter (Minisart syringe filter, Sartorius,

195 Germany). The inoculated tomato puree of the assays, made in triplicate, were incubated at

196 25°C for 15 days, and the diameter of fungal colonies was measured. The percentage of

197 mycelium growth inhibition (I, %) was calculated using the following formula: I (%) = [(TC-

198 TT)/TC] x100, where TC is the total fungal colony diameter (mm) obtained in control (distilled

199 water), and TT is the total fungal colony diameter (mm) obtained with L. plantarum S61

200 (culture, CFS or 10x-CFS).

201 2.6.2. Bio-preservation of pear fruits

202 The CFS of L. plantarum S61 was tested for its protective effect of pear fruit against P.

203 digitatum, using the wound inoculation method. The pear fruits purchased from a local

204 supermarket in Oujda-city, Morocco, were disinfected by ethanol (90%) and exposed to UV

205 for 20 min in Safety Cabinet Class-II. After that, small wounds (3 mm wide x 5 mm deep)

206 were aseptically made by pinching sterile paper pins onto the pear fruit. A volume of 50 μL of

207 L. plantarum S61 culture or its CFS, obtained in MRS broth after 48 h of incubation at 30°C,

208 was introduced into the wounds, and after 15 min they were inoculated with 50 μL of a spore

209 suspension (105 spores /mL) of P. digitatum. The control was maintained by inoculating the

210 spore suspension in wounds added with 50 μL of sterile distilled water. The pear fruits were

211 incubated at room temperature for 10 days and the fungal growth was examined visually. All

212 treatments were realized in triplicate.

213 2.6.3. Bio-preservation of apple juice

214 Apple fruits, purchased from a local fruit supermarket (Oujda, Morocco), were washed with

215 distilled water and dried, then the apple juice was prepared by mixing apple fruits with

216 distilled water using a juicer, and then filtered with Whatman Grade 4 filter paper and stored

217 at -20°C. Apple juice in flasks, with the initial pH 4.2, was autoclaved at 121°C/15 min and
218 then cooled to room temperature. Sterilized CFS of L. plantarum S61 was added (10%, v/v) to

219 juice and inoculated (1%, v/v) to a concentration of 5 Log10 CFU/mL of C. pelliculosa. Apple

220 juice, without CFS was used as control. The assays, made in triplicate, were incubated at

221 30°C for 48 h and the concentration of C. pelliculosa was determined on PDA medium and

222 recorded as colony forming units /mL (CFU/mL).

223 2.7. Statistical analysis

224 The results obtained were presented as means ± standard deviation. The one-way ANOVA

225 analysis was used to compare the means with a significant difference of p<0.05. To identify

226 the groups of means, the Student-Newman-Keuls (S-N-K) comparison post hoc test was used.

227 The analyses were carried out using GraphPad Prism 8 software (San Diego, California

228 USA).

229 3. Results

230 3.1. Antifungal activity of Lactobacillus strains against molds and yeasts

231 The inhibition diameter values of yeasts and molds obtained with the Lactobacillus strains,

232 using the overlay method, are reported in Table 1, and the inhibitory effect of some

233 Lactobacillus strains against P. digitatum is indicated in Fig. 1. All Lactobacillus strains

234 showed important antifungal activity, with inhibition diameter ranges of 18.15-20.5 mm,

235 20.3-21.8 mm, 20.15-22.8 mm and 16.75-21.1 mm against A. niger, P. digitatum, F.

236 oxysporum and Rhizopus sp., respectively; while the inhibition diameter ranges obtained

237 against yeasts were 21.5-23.8 mm and 21.3-22.9 mm against Rhodotorula sp. and C.

238 pelliculosa, respectively. The inhibition diameters obtained, with most of the Lactobacillus

239 strains, against yeasts were slightly higher than those obtained against molds. L. plantarum

240 S61 and L. brevis S27 showed, in most cases, the highest (p<0.05) inhibition diameters

241 against 5 of 6 targets studied. C. pelliculosa was the most sensitive strain, and demonstrated
242 the highest (p<0.05) inhibition diameters towards most (11/14 strains) of the Lactobacillus

243 strains.

244 3.2. Antifungal activity of Lactobacillus strains in gastro-intestinal tract conditions

245 The stability of antifungal activity of Lactobacillus strains after their exposure during 5 hours

246 to gastro intestinal tract conditions was evaluated, and the results are indicated in Table 2. The

247 inhibition of P. digitatum by L. plantarum S61, after 5 hours of exposure to bile salts (0.3%)

248 and pH3, is indicated as example in Fig. 2. All Lactobacillus strains were able to survive and

249 still exerted antifungal activity, against both target strains, after 5 hours of exposure to the

250 combination of bile salts (0.3%) and pH 3. Among the 14 Lactobacillus strains, the same 9

251 Lactobacillus strains demonstrating their survival, exhibited antifungal activity against both

252 target strains (C. pelliculosa and P. digitatum) after 5 h of exposure to the combination bile-

253 salts (0.3%) and pH 2.5. No Lactobacillus strain was able to growth after 5 h of exposure to

254 the combination of bile salts (0.3%) and pH 2.

255 3.2. Antifungal activity of cell-free supernatant (CFS) of Lactobacillus strains

256 The inhibition diameter values of yeasts and molds, obtained for the CFS of Lactobacillus

257 strains, are reported in Table 3. All the CFS of Lactobacillus strains exhibited antifungal

258 activity against the targets studied, and the inhibition diameter ranges obtained were 12.95-

259 14.4 mm, 16.1-17.3 mm, 14.05-15.95 mm, 12.15-13.3 mm, against A. niger, P. digitatum, F.

260 oxysporum, and Rhizopus sp., respectively; while against yeasts, the inhibition diameter

261 ranges were 20.5-22.5 mm and 20.2-21.75 mm, against Rhodotorula sp. and C. pelliculosa,

262 respectively. The inhibition diameters obtained, for all Lactobacillus strains, against yeasts

263 were higher than those obtained against molds. Among the Lactobacillus strains, L.

264 plantarum S61, demonstrated the highest (p<0.05) inhibition diameter values against most of

265 target strains (yeasts and molds). P. digitatum and C. pelliculosa were the most sensitive
266 targets, based their highest (p<0.05) inhibition diameter values, towards 8 and 7 Lactobacillus

267 strains, respectively.

268 3.4. Biomass and mycelium growth inhibition

269 The biomass and mycelium growth inhibition values of mold strains obtained with the CFS of

270 Lactobacillus strains are reported in Table 4. The biomass inhibition values ranges obtained

271 were 76.13-83.32 mm, 75.46-88.64 mm, 67.07-80.10 mm and 42.05-70.59 mm, against A.

272 niger, P. digitatum, F. oxysporum and Rhizopus sp., respectively. The mycelium growth

273 inhibition ranges obtained were 22.73-33.27 mm, 27.04-39.45 mm, 25.54-35.33 mm and

274 21.29-32.07 mm, against A. niger, P. digitatum, F. oxysporum and Rhizopus sp., respectively.

275 Among the Lactobacillus strains, L. plantarum S61 showed the highest (p<0.05) biomass and

276 mycelium growth inhibition values against all the mold strains used as target. P. digitatum

277 exhibited high sensitivity towards L. plantarum S61, with biomass and mycelium growth

278 inhibitions values of 88.64% and 39.45%, respectively.

279 3.5. Nature and physico-chemical stability of antifungal compounds

280 The effect of different pH, high temperatures and enzymes on the antifungal activity of the

281 CFS of the Lactobacillus strains against C. pelliculosa was evaluated, and the results are

282 reported in Table 5. The CFS of the Lactobacillus strains demonstrated the highest and

283 similar inhibitory effect against C. pelliculosa at pH 3.8, 4 and 5, with inhibition diameters

284 higher than 17mm (Table 5). However, the inhibition diameters of neutralized CFS to pH 6

285 and 7, of all the Lactobacillus strains, decreased to the range of 11-17mm.

286 The CFS of the Lactobacillus strains was treated with 80°C/10 min, 100°C/10 min and

287 autoclaved at 121°C/15 min, and its antifungal activity against C. pelliculosa was determined.

288 The results obtained showed inhibition diameters higher than 17 mm for all the Lactobacillus

289 strains and after all the heat treatments tested (Table 5).
290 The effect of enzymes treatment (catalase, proteinase-K and lipase) on the antifungal activity

291 of the neutralized (pH7) CFS, of Lactobacillus strains, was evaluated against C. pelliculosa,

292 and the results are reported in Table 5. These results indicated that the treatment of the CFS

293 with different enzymes led to different antifungal activities between Lactobacillus strains. The

294 catalase enzyme didn’t affect the antifungal activity of the CFS of all Lactobacillus strains,

295 with inhibition diameter ranges of 11-17 mm, which are similar to those obtained with

296 neutralized CFS. However, among the 14 Lactobacillus strains, the CFSs of L. brevis (S14,

297 S18, S27 and S82), L. pentosus (S42 and S75) and L. plantarum (S49, S61 and S62) lost their

298 antifungal activity after treatment with proteinase-K. Excepting the strains S23 and S45 of L.

299 plantarum, the treatment of the CFS of the other Lactobacillus strains with lipase led to

300 opposite result to that obtained with proteinase-K. Thus, no antifungal activity was detected in

301 lipase-treated CFSs of L. brevis S63 and L. plantarum S71 and S72, while the other strains of

302 L. brevis (S14, S18, S27 and S82), L. pentosus (S42, S75) and L. plantarum (S49, S61 and

303 S62), which were sensitive to proteinase-K, demonstrated inhibition diameter ranges of 11-17

304 mm. The CFSs of L. plantarum S23 and S46 demonstrated their resistance to proteinase-K

305 and lipase treatments, and their inhibition diameters ranges were of 11-17 mm.

306 3.6. Application of Lactobacillus and CFS in food bio-preservation

307 Among the Lactobacillus strains, L. plantarum S61 was selected, for its high antifungal

308 activity against a wide spectrum of yeasts and molds, to be tested for its bio-preservative

309 effect of tomato puree, pear fruits and apple juice. The microbial culture and its CFS were

310 tested for this purpose.

311 3.6.1. Tomato puree

312 The results of the inhibition values (%) of P. digitatum, inoculated in tomato puree, are

313 reported in Table 6. Pictures of bio-preservative effect of L. plantarum S61 on tomato puree

314 obtained after 15 days of incubation at 25°C are reported in Fig. 3. Total inhibition (100%) of
315 P. digitatum was observed in tomato puree samples containing L. plantarum S61 and the 10-

316 fold concentrated CFS (10x-CFS). However, the CFS led to significantly (p<0.05) lower

317 inhibition values of P. digitatum, which were 88% and 57.25%, respectively after 7 and 15

318 days of incubation at 25°C. Tomato puree samples containing 10x-CFS were darker than

319 those containing L. plantarum S61 and CFS (Fig. 3).

320 3.6.2. Pear fruits

321 The protective effect of L. plantarum S61 and its CFS was further evaluated on pear fruits

322 contaminated with P. digitatum and maintained at ambient temperature, and the results are

323 reported in Fig. 4. In absence of L. plantarum S61 and its CFS (control), almost 50% of the

324 fruits surface was covered by P. digitatum after 5 days of incubation, and after 10 days the

325 fruits were totally spoiled by the fungi. However, in presence of L. plantarum S61 and its

326 CFS, the growth of P. digitatum was limited during 10 days of incubation, and the decay

327 covered small surface of the fruits, compared to the control. The culture of L. plantarum S61

328 appeared to be slightly more efficient than its CFS.

329 3.6.3. Apple juice

330 The control of Candida pelliculosa growth in apple juice, using the CFS of L. plantarum S61

331 was evaluated, and the results are reported in Fig. 5. The results showed a rapid increase of C.

332 pelliculosa in apple juice from 5 log CFU/mL to achieve 10.47 log CFU/mL during 48 h of

333 incubation at 25°C. However, in presence of CFS, C. pelliculosa population decreased from 5

334 log CFU/mL to 2.5 log CFU/mL after 48 hours of incubation.

335 4. Discussion

336 The antifungal activity of 14 Lactobacillus strains was evaluated and preliminary

337 characterized. These Lactobacillus strains were isolated from traditional fermented olive

338 brine, considered as extreme environment, and demonstrated important antimicrobial and

339 other probiotic properties (Abouloifa et al., 2019). In this study, all the Lactobacillus strains
340 and their CFS showed important antifungal activity against target strains of yeasts (Candida

341 pelliculosa and Rhodotorula sp.) and molds (Aspergillus niger, Fusarium oxysporum,

342 Penicillium digitatum and Rhizopus sp.). Their activity against molds concerned biomass and

343 mycelium growth inhibition of all target molds tested. The inhibitory effect of the

344 Lactobacillus strains against yeasts was mostly higher than that obtained against molds. The

345 anti-yeasts and anti-molds activity was previously reported in Lactobacillus strains (Falguni

346 et al., 2010; Magnusson and Schnurer, 2001; Nayyeri et al., 2017), and mycelium and

347 biomass inhibition (Deepthi et al., 2016; Muhialdin and Hassan, 2011). The Lactobacillus

348 strains studied in this work, showed their capacity of production of extracellular antifungal

349 compounds, demonstrating high inhibition diameters against a wide spectrum of yeasts and

350 molds. This antifungal activity may explain in part their natural dominance in spontaneous

351 lactic fermentation of non-alkali treated green olive. Therefore, olive brine and other

352 traditional fermented foods, realized in extreme conditions of osmotic pressure and low

353 nutrient contents, can be good sources of Lactobacillus strains with important antifungal

354 activity.

355 Among the Lactobacillus strains studied, 9 strains showed tolerance and antifungal activity,

356 against C. pelliculosa and P. digitatum, after 5 h of exposure to simulated gastro-intestinal

357 conditions (0.3% bile salts and pH 2.5) demonstrating their tolerance to gastro-intestinal tract

358 conditions. These findings indicate the possible use of these probiotic Lactobacillus strains as

359 antifungal agents against gastrointestinal diseases of fungal origin.

360 The CFS of all Lactobacillus strains, with pH of 3.8, 4 and 5, demonstrated the highest

361 antifungal activity against C. pelliculosa and P. digitatum. Their neutralization, to pH 6-7, led

362 to the reduction of their inhibitory effect, which corresponded to inhibition diameter of 11-17

363 mm. This result is similar to those reported in literature (Magnusson and Schnurer, 2001;

364 Muhialdin et al., 2018). The reduction of antifungal activity of neutralized CFS may be due to
365 the neutralization of carboxylic acids and other antifungal pH-dependent compounds (Axel et

366 al., 2016), such as lactic acid, acetic acid and phenyllactic acid (Barman et al., 2017; Gerez et

367 al., 2010). The CFS of these probiotic Lactobacillus strains may be used as antifungal agent in

368 acidic foods, such as fruit juices. In addition, the antifungal compounds of all Lactobacillus

369 strains were stable and resisted to different heat treatments (80°C and 100°C for 10 min and

370 autoclaving 121/15 min). This result is in agreement with the results of (Gerez et al., 2013;

371 Magnusson and Schnurer, 2001; Zhongjun et al., 2017), and indicates the possible use of the

372 CFS of these Lactobacillus strains as antifungal agents in pasteurized or sterilized food

373 products.

374 The antifungal activity of neutralized CFS of all Lactobacillus strains was not affected by

375 treatment with catalase, indicating that hydrogen peroxide is not responsible for the antifungal

376 activity observed. The treatment with proteinase K led to the disappearance of antifungal

377 activity in CFS of L. brevis (S14, S18, S27 and S82), L. pentosus (S41 and S75) and L.

378 plantarum (S49, S61 and S62), indicating the proteinaceous nature (proteins, peptide or

379 bacteriocin) of their antifungal compounds. The production of antifungals of proteinaceous

380 nature was demonstrated in Lactobacillus strains (Ahmad Rather et al., 2013; Bazukyan et al.,

381 2018; Gerez et al., 2013; Magnusson and Schnurer, 2001; Muhialdin et al., 2018). The

382 resistance of the antifungal compounds of these CFS to various heat treatments, demonstrated

383 in this work, indicates their possible peptide or bacteriocin nature, which are known for their

384 resistance to various heat treatments, including autoclaving at 121°C/15 min (Ahmadova et

385 al., 2013; Falguni et al., 2010).

386 The bioactive compounds of L. brevis S63 and L. plantarum S71 and S72 lost their antifungal

387 activity after lipase treatment, indicating their lipid nature or the presence of lipid moiety in

388 their antifungal compounds. These Lactobacillus strains were previously demonstrated for

389 their capacity of reduction of surface tension, due to biosurfactants (Abouloifa et al., 2019).
390 The biosurfactans are composed of amphiphilic compounds, such as glycolipids, lipopeptides,

391 glycolipopeptides and glycoprotein (Satpute et al., 2016), and they are advantageous as

392 antibacterial and antifungal agents (Sharma et al., 2016). These Lactobacillus and/or their

393 CFS can be used as antifungal preservatives in food emulsions, such as dairy products,

394 mayonnaise and other heat sensitive food products.

395 The antifungal activity of neutralized CFS of L. plantarum S23 and S46 was not affected by

396 proteinase K nor lipase enzymes, indicating that organic acids, proteinaceous and lipid

397 compounds were not involved in the antifungal activity observed in these strains. The

398 production of antifungal compounds other than carboxylic acids, proteinaceous and lipids are

399 reported in LAB, such as polycyclic lactates in Lactobacillus harbinensis K.V9.3.1Np

400 (Mosbah et al., 2018), Benzeneacetic acid, 2-propenyl ester in Lactobacillus plantarum

401 IMAU10014 (Wang et al., 2012), and a phenolic compound in Pediococcus acidilactici

402 LAB 5 (Mandala et al., 2007).

403 Among the Lactobacillus strains studied, L. plantarum S61 was selected to be tested as bio-

404 preservative in some food products, because of its important antifungal activity against a wide

405 spectrum of target yeasts and molds, the proteinaceous property of its antifungal compounds,

406 and for its capacity to survive and maintain its antifungal activity in gastrointestinal

407 conditions (0.3% bile salt combined with pH2.5). These important properties indicate the

408 possible use of this probiotic L. plantarum S61 strain as bio-preservative in foods, and to

409 prevent gastro-intestinal diseases caused by yeasts and molds, at least those used as targets in

410 this work.

411 The application of L. plantarum S61 and its CFS as bio-preservative agent in tomato puree,

412 pear fruits and apple juice, against P. digitatum and C. pelliculosa contamination, was

413 evaluated. In tomato puree, the culture of L. plantarum S61 and its 10-fold concentrated CFS

414 showed total inhibition of P. digitatum, and extend the shelf life of tomato puree at least for
415 15 days at 30°C; while lower preservative effect was obtained with the CFS. Previous works

416 demonstrated the antifungal activity of Lactobacillus strains and their CFS against P.

417 digitatum (Ma et al., 2019), and delayed the growth of Aspergillus oryzae in tomato puree

418 (Muhialdin et al., 2011). In this work the L. plantarum S61 culture and its 10-fold

419 concentrated CFS showed more effectiveness against P. digitatum than the CFS. This may be

420 due to the high concentration of antifungal compounds in 10-fold concentrated CFS and their

421 continuous production by L. plantarum S61 culture in tomato puree. However, the 10-fold

422 concentrated CFS affected the color of the tomato puree, by increasing dark color of the

423 product.

424 In pear fruits, L. plantarum S61 and its CFS showed important bio-protective effect against P.

425 digitatum attack, during 10 days of incubation at room temperature. The protective effect of

426 Lactobacillus strains was demonstrated in pear fruits against P. expansum spoilage (Crowley

427 et al., 2013), and in citrus fruits against P. digitatum (Ma et al., 2019). The L. plantarum S61

428 culture appeared more effective than its CFS against P. digitatum spoilage, which may be due

429 to their continuous production of antifungal compounds on pear fruits.

430 In apple juice, the CFS of L. plantarum S61 led to high reduction of almost 50% of initial

431 population of C. pelliculosa, indicating its effectiveness in decontamination of apple juice

432 against Candida, considered as one of the main genera involved in fruit juices spoilages

433 (Pandey and Negi, 2018). Apple juice, added with this CFS, may require low heat treatment

434 for its preservation against fungal contamination. High thermal processing is known for its

435 negative effect on organoleptic and nutritional values of apple and other fruits juices (Petruzzi

436 et al., 2017).

437 The L. plantarum S61 culture and its CFS demonstrated important in-vitro antifungal activity

438 and in in-vivo experiments on tomato puree, pear fruit and apple juice. The Lactobacillus

439 culture and 10-fold concentrated CFS were more efficient than CFS, due to lower
440 concentration of antifungal compounds. Therefore, the optimization of antifungal compounds

441 production by L. plantarum S61, allowing the increase of their contents, should improve their

442 effectiveness in food bio-preservation against fungal spoilages.

443 5. Conclusion

444 The 14 Lactobacillus strains, isolated from traditional fermented green olives, and their CFS

445 showed important antifungal activity against a wide spectrum of yeasts (Candida pelliculosa

446 and Rhodotorula sp.) and molds (Aspergillus niger, Fusarium oxysporum, Penicillium

447 digitatum and Rhizopus sp.). Some of these Lactobacillus strains were able to resist and to

448 maintain their antifungal activity in gastro-intestinal conditions. The antifungal compounds

449 produced by these Lactobacillus strains were heat resistant, active in large pH value, with a

450 variable nature (proteinaceous, lipid or other nature). Among the Lactobacillus strains, L.

451 plantarum S61 and its CFS showed important effectiveness as bio-preservative agent, and

452 delayed the growth of spoilage microorganisms (C. pelliculosa and P. digitatum) in different

453 foods (tomato puree, pear fruit and apple juice). This bio-preservative effect, of this probiotic

454 L. plantarum S61, can be improved by optimizing the production of antifungal compounds.

455 Conflict of interest

456 The authors declare no conflict of interest

457 Acknowledgments

458 The authors are grateful to the CNRST (PPR/19/2015), McGill University (Quebec), CNRST-

459 CNR (Morocco-Italy) and Morocco-Tunisia cooperation (17TM06) for their supports.

460
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619

620

621

622
623
624 Author Contributions
625 Abdeslam Asehraou selected the scope of the work and supervised the Lab work.

626 Houssam Abouloifa, Sara Gaamouche, Yahya Rokni, Ismail Hasnaoui, Reda Bellaouchi, and

627 Nabil Ghabbour conducted the experiments.

628 Abdeslam Asehraou, Salwa Karboune, Milena Brasca, Guy D’Hallewin, Riadh Ben Salah,

629 Ennouamane Saalaoui and Houssam Abouloifa wrote the manuscript.

630 All authors read and approved the manuscript.

631
632

633 Fig. 1. Antifungal activity of probiotic Lactobacillus plantarum (strains S61, S62 and S72)

634 against P. digitatum by overlay method

635

636

637

638 Fig. 2. Antifungal activity of probiotic L. plantarum S61 colonies against P. digitatum,

639 obtained by overly method, after 5 h of exposure to gastrointestinal tract conditions (0.3% bile

640 salts + pH 3)
641

Day 1 Day 5 Day 10

P. digitatum
(control)

642
P. digitatum P. digitatum P. digitatum + P. digitatum +
+ CFS 10x-CFS L. plantarum S61

643 Fig. 3. Bio-preservative effect of probiotic L. plantarum S61 against P. digitatum obtained on

644 tomato puree after 15 days of incubation at 25°C. (Legend: CFS, cell free supernatant; 10x-

645 CFS, 10-fold concentrated CFS)

646

647
648
 P. digitatum
+ CFS

P. digitatum
+ L. plantarum S61

649 Fig. 4. Bio-preservative effect of probiotic L. plantarum S61 and its CFS against Penicillium

650 digitatum attack of pear fruits maintained at room temperature. (Legend: CFS, cell free

651 supernatant)

652

653

654
655 Fig. 5. Growth of Candida pelliculosa in apple juice with CFS ( ) and without CFS ( )

656 of L. plantarum S61. (Legend: CFS, cell free supernatant)

657

658
659 Graphical Abstract
660

661 Antifungal activity against

662 yeasts and molds
Demonstrated

Highlightsstrains
663 Lactobacillus
from fermented olive Antifungal compounds:
664 (Probiotics) - Nature: proteinaceous, lipid or other
- Heat resistants to pasteurization and autoclaving
665  Lactobacillus strains from fermented olives showed
- active important
in large antifungal
pH values: 4-7 activity.

666 Bio-preservative
 Their antifungal activity is maintained effect
in gastro-intestinal on :
conditions.

667  Their antifungal compounds have variable nature (proteinaceous, lipid or other).

669
668  L. plantarum S61 Apple
and Juice
its CFS Pear fruits
showed Tomato puree

670 effectiveness in food bio-

671 preservation.

672
673 Table 1 Antifungal activity of probiotic Lactobacillus strains against molds and yeasts
674
Inhibition diameter (mm)
Lactobacillus
Molds
strains
A. niger P. digitatum F. oxysporum Rhizopus sp. Rhodotorula sp
L. brevis S14 18.5c±0.42 ab
21.5 ±0.71 bcd
21.15 ±0.21 18.4de±0.57 22.4bc±0.57
L. brevis S18 18.5c±0.71 20.3e±0.42 21.25bc±0.35 19.5bc±0.71 22.2bcd±0.28
L. brevis S27 19.5b±0.71 21.65ab±0.49 22.05a±0.07 20.8a±0.28 23.8a±0.00
L. brevis S63 18.4c±0.57 21.4abc±0.57 21.25bc±0.35 19.8b±0.28 22.25bcd±0.35
L. brevis S82 18.2c±0.28 20.3e±0.42 20.15f±0.21 18.45de±0.64 21.5d±0.71
L. pentosus S42 18.35c±0.49 21.1bc±0.14 20.4ef±0.57 20.5a±0.71 22.45bc±0.64
L. pentosus S75 18.5c±0.71 20.85cde±0.07 20.65def±0.49 17.6e±0.85 21.95bcd±0.07
L. plantarum S23 18.4c±0.14 21.65ab±0.49 22.8a±0.28 18.3de±0.42 23.3a±0.42
L. plantarum S46 18.15c±0.21 21.3abc±0.42 22.5a±0.71 18.5de±0.71 21.75cd±0.35
L. plantarum S49 18.25c±0.75 20.45de±0.64 21.1bcd±0.14 16.75f±0.35 22.65b±0.49
 L. plantarum S61 20.5a±0.71 21.15bc±0.21 22.5a±0.71 21.1a±0.14 23.8a±0.28
L. plantarum S62 18.3c±0.42 21.8a±0.28 22.65a±0.49 18.75cd±0.21 22.1bcd±0.14
L. plantarum S71 18.5c±0.71 20.4de±0.28 21.5b±0.71 19.5bc±0.71 21.75cd±0.35
L. plantarum S72 18.25c±0.35 20.9cd±0.14 20.5def±0.71 18.8cd±0.28 21.5d±0.71
675 Values are mean ± standard error of triplicates.
676 a-fMeans in same column at each parameter with different lowercase letters differed
677 significantly (p < 0.05).
678

679
680 Table 2 Antifungal activity of probiotic Lactobacillus strains after exposure to simulated
681 gastrointestinal tract conditions (bile salts and pH)
682
Candida pelliculosa Penicillium digitatum
Lactobacillus
0.3% bile salts 0.3% bile salts
strains
pH= 2 pH= 2.5 pH =3 pH= 2 pH= 2.5 pH= 3
L. brevis S14 - - + - - +
L. brevis S18 - - + - - +
L. brevis S27 - - + - - +
L. brevis S63 - + + - + +
L. brevis S82 - + + - + +
L. pentosus S42 - + + - + +
L. pentosus S75 - + + - + +
L. plantarum S23 - + + - + +
L. plantarum S46 - - + - - +
L. plantarum S49 - - + - - +
L. plantarum S61 - + + - + +
L. plantarum S62 - + + - + +
L. plantarum S71 - + + - + +
L. plantarum S72 - + + - + +
683 Legend: (+) presence of growth and antifungal activity; (-) Absence of growth
684

685
686 Table 3 Antifungal activity of Cell-Free-Supernatant of probiotic Lactobacillus strains
687 against molds and yeasts
688
Inhibition diameter (mm)
Lactobacillus
Molds
strains
A. niger P. digitatum F. oxysporum Rhizopus sp. Rhodotoru
c
16.1 ±0.14
L. brevis S14 13.4c±0.57 14.05f±0.07 12.15c±0.21 21.2bcd±0
L. brevis S18 12.95d±0.21 17.05a±0.07 14.15f±0.21 12.4c±0.57 21.2bcd±0
L. brevis S27 13.35c±0.49 17.25a±0.35 14.9de±0.14 13.05ab±0.07 22.3a±0
L. brevis S63 14.4a±0.57 17.15a±0.21 15.65abc±0.49 12.45c±0.64 21.4bcd±0
L. brevis S82 13.3c±0.42 16.8ab±0.28 14.85de±0. 21 13.3a±0.42 20.5d±0
L. pentosus S42 14.15b±0.21 16.1c±0.14 14.45ef±0.64 12.4c±0.57 21.5bc±0
 L. pentosus S75 14.2ab±0.28 16.35bc±0.49 15.2cd±0.28 13.1ab±0.14 20.95cd±0
L. plantarum S23 13.45c±0.64 17.3a±0.42 14.8de±0.28 12.35c±0.49 22.5a±0
L. plantarum S46 13.25c±0.35 16.2c±0.28 14.25f±0.35 13.1ab±0.14 20.5d±0
L. plantarum S49 14.3ab±0.42 16.7abc±0.42 15.3bcd±0.42 12.65bc±0.49 21.6bc±0
L. plantarum S61 14.25ab±0.35 17.3a±0.42 15.9a±0.14 12.75abc±0.35 22.05ab±0
L. plantarum S62 14.35ab±0.49 16.9ab±0.14 15.95a±0.07 12.3c±0.42 21.45bc±0
L. plantarum S71 13.4c±0.57 16.15c±0.21 15.75ab±0.35 12.25c±0.35 20.95cd±0
L. plantarum S72 13.2c±0.28 16.45bc±0.64 15.1d±0.14 12.35c±0.49 20.75cd±0
689 Values are mean ± standard error of triplicates.
690 a-fMeans in same column at each parameter with different lowercase letters differed

691 significantly (p < 0.05).

692

693
694 Table 4 Biomass and mycelium growth inhibition (%) of molds by CFS from probiotic
695 Lactobacillus strains
696
Inhibition (%)
Lactobacillus
Biomass M
strains
A. niger P. digitatum F. oxysporum Rhizopus sp. A. niger P. digitat
L. brevis S14 76.36 ±0.58 75.46 ±0.99 75.26c±0.08
e j 55.57c±0.30 25.77f±0.99 35.85b±0
L. brevis S18 79.17cd±0.41 77.53h±0.91 67.07h±0.78 46.82h±0.56 26.00ef±0.67 27.04e±0
L. brevis S27 79.90c±0.93 79.09fg±0.54 78.69b±0.72 58.66b±0.99 28.50c±0.54 38.95a±0
L. brevis S63 78.57d±0.37 86.74b±0.62 71.97e±0.49 49.14g±0.11 28.29cd±0.84 38.95a±0
L. brevis S82 76.98e±0.64 76.42i±0.42 75.04c±0.73 44.07i±0.12 27.14de±0.78 35.85b±0
L. pentosus S42 81.77b±0.93 81.06d±0.45 75.56c±0.56 53.19e±0.95 25.77f±0.99 35.85b±0
L. pentosus S75 79.18cd±0.83 79.94def±0.08 74.82c±0.72 42.05k±0.86 26.03ef±0.63 33.35c±0
L. plantarum S23 81.29b±0.98 82.90c±0.81 79.82a±0.90 54.86d±0.62 31.29b±0.75 38.95a±0
L. plantarum S46 80.05c±0.91 80.71de±0.34 69.25g±0.39 49.26g±0.37 22.73g±0.49 30.85d±0
L. plantarum S49 80.33c±0.31 80.40de±0.95 75.79c±0.13 43.07j±0.29 25.67f±0.85 37.40ab±0
L. plantarum S61 83.32a±0.80 88.64a±0.76 80.10a±0.88 70.59a±0.42 33.27a±0.78 39.45a±0
L. plantarum S62 76.13e±0.56 80.40de±0.50 74.82c±0.71 50.44f±0.61 28.78c±0.89 35.29b±0
L. plantarum S71 77.06e±0.49 79.69ef±0.82 73.29d±0.74 53.50e±0.69 28.29cd±0.84 38.95a±0
L. plantarum S72 78.39d±0.62 78.69g±0.82 71.05f±0.24 53.91e±0.43 28.29cd±0.84 38.95a±0
697 Values are mean ± standard error of triplicates.
698 a-kMeans in same column at each parameter with different lowercase letters differed

699 significantly (p < 0.05).

700

701
 702 Table 5 Effect of pH, temperature and enzymes treatment on antifungal activity of CFS from
703 probiotic Lactobacillus strains against Candida pelliculosa
704
tment Inhibition of C. pelliculosa
L. brevis L. pentosus L. plantarum
S14 S18 S27 S63 S82 S42 S75 S23 S46 S49 S61 S62 S7
upernatant
+++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++
=3.8)
of pH
=4 +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++
=5 +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++
=6 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
=7 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
mperature
10min +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++
/10min +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++
21°C/15 min +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++
enzymes
=7 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
Catalase ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
oteinase K - - - ++ - - - ++ ++ - - - ++
Lipase ++ ++ ++ - ++ ++ ++ ++ ++ ++ ++ ++ -
705 Note: Inhibition was measured by the agar well diffusion method against Candida
706 pelliculosa. (-): no inhibition diameter, (+): inhibition diameter between 5 and 11 mm, (++):
707 inhibition diameter between 11 and 17 mm, and (+++): inhibition diameter > 17 mm.
708

709
710 Table 6 Inhibition (%) of P. digitatum in tomato puree by probiotic L. plantarum S61 and its
711 CFS.
712
Inhibition (%)
Day 3 Day 7 Day 15
CFS 100a±00 88b±1.14 57.25b±0.35
10x-CFS 100a ±00 100a ±00 100a ±00
L. plantarum S61 100a±00 100a±00 100a±00
713 (Legend: CFS: Cell Free Supernatant, 10x-CFS: 10-fold concentrated CFS)
714 Values are mean ± standard error of triplicates.
715 a-bMeans in same column of each parameter with different lowercase letters differed significantly (p <

716 0.05)
717

718