Accepted Manuscript

Bio-controlling capability of probiotic strain Lactobacillus rhamnosus against some

common foodborne pathogens in yoghurt

Rania M. Kamal, Mohamed E. Alnakip, Salah F. Abd El Aal, Mohamed A. Bayoumi

PII: S0958-6946(18)30112-2
DOI: 10.1016/j.idairyj.2018.04.007
Reference: INDA 4308

To appear in: International Dairy Journal

Received Date: 18 October 2017

Revised Date: 20 April 2018
Accepted Date: 22 April 2018

Please cite this article as: Kamal, R.M., Alnakip, M.E., Abd El Aal, S.F., Bayoumi, M.A., Bio-controlling
capability of probiotic strain Lactobacillus rhamnosus against some common foodborne pathogens in
yoghurt, International Dairy Journal (2018), doi: 10.1016/j.idairyj.2018.04.007.

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 ACCEPTED MANUSCRIPT
1 Bio-controlling capability of probiotic strain Lactobacillus rhamnosus against some

2 common foodborne pathogens in yoghurt

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8 Rania M. Kamal, Mohamed E. Alnakip, Salah F. Abd El Aal, Mohamed A. Bayoumi*

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14 Food Control Department, Faculty of Veterinary Medicine, Zagazig University, Egypt
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* Corresponding author. Tel.: +201000526062

21 E-mail address: [email protected] (M. A. Bayoumi)

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24 ABSTRACT

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26 Despite there being many food preservation techniques, foodborne illnesses are still a

27 growing problem. Developing alternative natural methods to control foodborne

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28 pathogens is still necessary. The antimicrobial activity of Lactobacillus rhamnosus was

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29 studied against four common foodborne pathogens, both in vitro and in a food model

30 (yoghurt). Using an agar well diffusion assay, acidified L. rhamnosus cell free

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31 supernatant was shown to significantly inhibit all pathogens tested: Escherichia coli

32 O157:H7, Staphylococcus aureus, Yersinia enterocolitica and Salmonella enterica

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serovar Typhimurium. Moreover, neutralised supernatant also had an antimicrobial
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34 effect against most pathogens, which proved the presence of inhibitory compounds
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35 rather than acids. With addition of L. rhamnosus to pathogen-spiked yoghurt, complete

36 elimination or at least reductions of pathogen counts was achieved. Pathogen reduction

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37 by L. rhamnosus was correlated with the initial count. From this pilot study, addition of
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38 L. rhamnosus to yoghurt seems to be a beneficial and applicable bio-control strategy.

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40 1. Introduction

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42 Acidification has long been used as a simple method for extending food shelf-

43 life. Since low pH does not favour the growth of many foodborne pathogenic bacteria,

44 yoghurt and other acidified/fermented foods are generally recognised as

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45 microbiologically stable foods (Cutrim et al., 2016). However, many observations have

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46 shown the role of yoghurt and other fermented dairy products in transmission of

47 foodborne pathogens (Ahmed & Shimamoto, 2014; De Buyser, Dufour, Maire, &

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48 Lafarge, 2001). Moreover, certain foodborne pathogens, like Escherichia coli O175:H7

49 (E. coli O157:H7), Yersinia enterocolitica (Yer. entercolitica) and Listeria

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monocytogenes were found to survive the acidic conditions of yoghurt for up to 21 days
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51 of cold storage (Bachrouri, Quinto, & Mora, 2006; Gulmez & Guven, 2003). Another
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52 study showed that those pathogens were able to be cultured positively from a 40-day

53 stored yoghurt sample (McIngvale, Chen, McKillip, & Drake, 2000). Additionally,
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54 hygienic conditions worsen if yoghurt or fermented milks are made from raw milk, as
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55 has traditionally been the case of many developing countries.

56 Accordingly, there is an urgent need for alternative, effective and applicable food
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57 pathogen control methods. Recently, probiotic addition to foods is a fast growing trend

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(Liu et al., 2017a; Tripathi & Giri, 2014). Besides the numerous probiotic associated

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health benefits (Iglesias et al., 2017), their antimicrobial traits promote their

60 incorporation into different dairy and non-dairy based foods (Sireswar, Dey,

61 Sreesoundarya, & Sarkar, 2017). The main antimicrobial properties are mainly attributed

62 to production of bacteriocins (Bayoumi & Griffiths, 2010), organic acids (Beristain-

63 Bauza, Mani-López, Palou, & López-Malo, 2016; Chaveerach, Lipman, & Van Knapen,

64 2004,) and/or hydrogen peroxide (Lin, Hwang, Chen, & Tsen, 2006). However, before a

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65 probiotic strain can be incorporated into foods and commercialised (consumer

66 available), thorough functional, quality and safety studies must be performed.

67 Lactobacillus rhamnosus (L. rhamnosus) is a well-documented probiotic strains.

68 Many proven therapeutic functions have been attributed to L. rhamnosus, like efficient

69 intestinal colonisation (Reid & Burton, 2002), host immune stimulation

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70 (Patel, Shukla, & Goyal, 2015), anti-diarrhoeagenic effect (Gill, Rutherfurd, Prasad, &

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71 Gopal, 2000; Xie, Li, Wang, Li, & Chen, 2015) besides its bile and gastric acid

72 resistance (Gardiner et al., 2002). Despite well documented antipathogenic features of

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73 different probiotic strains, to our knowledge, scarce information is available regarding

74 the antipathogenic effect of L. rhamnosus against major foodborne pathogens. Thus, this

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study was aimed to characterise this property of L. rhamnosus.

76 Specifically, this study was designed to determine the capability of using L.

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77 rhamnosus as a food bio-controlling agent against certain foodborne pathogens [E. coli

78 O157:H7, Staphylococcus aureus (S. aureus), Salmonella enterica serovar

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79 Typhimurium (Sal. Typhimurium) and Yer. enterocolitica], in addition, the survivability

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80 of this probiotic strain in yoghurt as a food model was determined.

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82 2. Materials and methods

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84 2.1. Test strains and growth conditions

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86 Probiotic strain L. rhamnosus (LMG 23522, supplied from the Belgian Co-

87 ordinated Collections of Micro-organisms, BCCM), was used in this study. Following

88 supplier information, L. rhamnosus was grown anaerobically on De Man, Rogosa, &

89 Sharpe medium (MRS; Oxoid, Hampshire, UK). Four foodborne pathogenic strains

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90 were used in this study: E. coli O157:H7, S. aureus, Sal. Typhimurium and Yer.

91 enterocolitica (previously isolated and molecularly characterised; Bayoumi, Kamal, El

92 Aal, & Awad, 2012; Kamal, Bayoumi, & El-Aal, 2013). The selection of pathogens in

93 this study was based on their previous widespread prevalence in yoghurt and other

94 fermented dairy products. Pathogenic strains were aerobically grown at 37 °C on

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95 Tryptone Soya Agar (TSA, Oxoid) and/or broth (TSB, Oxoid). For yoghurt production,

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96 commercially available yoghurt starter cultures of Streptococcus thermophilus and

97 Lactobacillus delbrueckii spp. bulgaricus were purchased and used (Yoflex® YC-X11,

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98 batch No. 3337495, Chr. Hansen, Hørsholm, Denmark).

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2.2. Preparation of cell free supernatant

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102 L. rhamnosus was anaerobically grown in MRS broth at 37 °C for 48 h. A cell

103 free supernatant (CFS) was obtained through cold centrifugation (~4 °C) of grown broth
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104 at ~7800 × g for 10 min, followed by filtration through a 0.2 µm pore size filter
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105 (Whatman® Puradisc, Ann Arbor, MI, USA). Obtained CFS was divided into two parts,

106 one was adjusted to pH 7 using sterile 1 N NaOH solution (neutralised) and the other
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107 was left as was (acidified). Neutralised CFS was used to exclude the antimicrobial effect
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108 of any organic acids produced.

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110 2.3. In vitro antimicrobial activity of L. rhamnosus cell free supernatant

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112 Following a previously described method (Kim & Rajagopal, 2001) with slight

113 modifications, fresh pathogenic strain cultures were prepared by picking three to five

114 colonies of each strain from agar plated and suspending them in TSB. Broth tubes were

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115 incubated for 2–6 h and frequently spectrophotometrically measured at 600 nm

116 wavelength until the desired optical density was achieved (which corresponded ~ 8 log

117 cfu mL-1). Dipped sterile cotton swabs were used to spread test strains on the surface of

118 a Mueller Hinton agar plate (Thermo Scientific™, Schwerte, Germany). Following

119 spreading, wells of 5 mm diameter were cut into agar plates and 100 µL of both

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120 neutralised and acidified CFS were separately added to each well. Sterile MRS broth

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121 was used as a negative control. Plates were incubated at 37 °C and examined after 24 h

122 for presence of inhibition zones around wells (5 mm or more were considered positive).

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124 2.4. Antimicrobial activity of L. rhamnosus against foodborne pathogens during

125 controlled yoghurt fermentation

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127 Yoghurt starter culture (Yoflex® YC-X11) was inoculated into sterile UHT

128 whole cows’ milk at the manufacturer’s recommended level and incubated at 40 ± 1 °C
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129 until a final pH of 4.6 was reached. For each tested pathogen, 2 inoculation levels were
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130 used, 2 and 6 log cfu mL-1. Thus, 4 different batches of yoghurt were produced for each

131 pathogen (16 batches in total), two batches with different inoculation values and the
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132 other two with the addition of ~9 log cfu mL-1 of L. rhamnosus to the different pathogen
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133 inoculation levels (Fig. 1). Additionally, a batch was prepared with only addition of L.
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134 rhamnosus to serve as a control. After manufacturing, spiked and control yoghurt

135 batches were stored at 4 °C. Completely randomised samples were taken for pH

136 measurement and enumeration of probiotic and pathogenic strains at 0, 2, 4, 8, 24, 48

137 and 72 h after inoculation. A total of 105 samples were taken (5 samples at each

138 sampling time and repeated thrice). Samples were prepared for examination according to

139 standard methods: 11 g of each yoghurt sample was blended with 99 mL of 0.1%

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140 peptone water (Oxoid), followed by preparation of ten-fold dilutions. Then, 100 µL of

141 the appropriate dilution was used to in duplicate to determine microbial counts.

142 Pathogenic strains were counted on appropriate media at appropriate conditions [Baird-

143 Parker agar (Oxoid), for S. aureus; Sorbitol MacConkey agar for E. coli O157: H7; XLD

144 medium (Oxoid) for Sal. Typhimurium and Yersinia selective agar (Oxoid) for Yer.

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145 enterocolitica]. Incubation for all pathogens was done aerobically at 37 °C except for

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146 Yer. enterocolitica that was incubated at 32 °C.

147

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148 2.5. L. rhamnosus count and pH measurements

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Following a previously described method (Tharmaraj & Shah, 2003), L.
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151 rhamnosus was counted on MRS agar with addition of 10 mg mL-1 vancomycin (Sigma-
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152 Aldrich, Taufkirchen, Germany). The pH levels were measured using a combined glass

153 electrode pH meter (WTW, Weilheim, Germany). The pH levels were determined at
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154 previously indicated intervals.

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156 2.6. Statistical analysis
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All experiments (including inoculum preparation, product processing and

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storage) were performed in triplicate. Data were represented as mean ± standard

160 deviation (absolute). The mean values of pH of all samples as well as mean log counts of

161 the bacterial pathogens and L. rhamnosus were compared using one-way analysis of

162 variance (ANOVA). Significant statistical differences were considered at p < 0.05 and

163 under a liner model: Yij= µ+Ti+Pj+eijk (where Yij is reduction in different pathogens at

164 different pH values; µ is the overall means; Ti is the effect of pathogen where i=1-4; Pj is

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165 the effect of pH where j=1-2; and eijk is the random error). The acceptable C.V.% for the

166 current experiments was within the recommended ± 10%. Statistical analysis was done

167 using SPSS 13.0 for Windows (Apache Software Foundation, Forest Hill, MD, USA).

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169 3. Results and discussion

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171 3.1. Antimicrobial activity of L. rhamnosus CFS

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173 As shown in Table 1, both acidified and neutralised L. rhamnosus CFS were able

174 to significantly inhibit in vitro growth of all tested pathogens except Sal. Typhimurium.

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The inhibitory activity of L. rhamnosus CFS seems to be a group-specific. Whereas the
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176 Gram-positive pathogen (S. aureus) was inhibited by both CFS with no significant
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177 difference, Gram-negative pathogens showed different inhibition patterns. Growth

178 inhibition due to acidified CFS was significantly greater than neutralised CFS in the case
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179 of E. coli O157:H7 and Yer. enterocolitica. While in case of Sal. Typhimurium,
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180 neutralised CFS was not able to inhibit its growth.

181 Understanding of the antimicrobial behaviour of L. rhamnosus may explain these

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182 varied inhibition patterns. Different hypotheses have been proposed for the antimicrobial

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effect of L. rhamnosus. Antimicrobial activity of lactic acid bacteria (LAB) is primarily

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due to pH reduction by the secretion of lactic acid as a major end product (Axelsson,

185 2004). Also, it is well known that L. rhamnosus can produce high amounts of lactic acid

186 during fermentation (Beristain-Bauza et al., 2016). In addition to lactic acid, bacteriocin

187 production plays a role. Kankainen et al. (2009) described the presence of several

188 bacteriocin related genes in the genome of L. rhamnosus. While the inhibitory activities

189 of bacteriocins are mainly against Gram-positive bacteria (Abee, Krockel, & Hill, 1995),

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190 organic acids are known to have a general inhibitory activity against both Gram-positive

191 and -negative pathogens (Alakomi et al., 2000; De Keersmaecker et al., 2006; Fayol-

192 Messaoudi, Berger, Coconnier-Polter, Lie’vin-Le Moal, & Servin, 2005). However, Lu

193 et al. (2009) have isolated and characterised 7 peptides produced by L. rhamnosus, with

194 one of them (peptide NPSRQERR) having antibacterial properties, both on Gram-

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195 negative and Gram-positive bacteria. This may explain inhibition patterns of S. aureus,

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196 E. coli O157:H7 and Yer. enterocolitica. While regarding Sal. Typhimurium, the

197 influence of acidified CFS (pH≃5.1) was significantly less in comparison to other tested

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198 pathogens. This may be attributed to phenotypic acid tolerance response of Salmonella

199 spp. (Alvarez-Ordóňez, Broussolle, Colin, Nguyen-The, & Prieto, 2015). Burin, Silva Jr,

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and Nero (2014) cultured Sal. Typhimurium at three different pH levels (6, 5 and 4), and

201 found that it was able to tolerate pH≃5 and only pH≃4 was able to completely inhibit
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202 Sal. Typhimurium after 6 h of incubation. This documented phenotypic trait of Sal.

203 Typhimurium (Lianou, Nychas, & Koutsoumanis, 2017; Perez, Martins, Cara, Nicoli, &
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204 Tondo, 2012) could certainly account for our insignificant reduction of Sal.
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205 Typhimurium since acidified CFS had a pH of 5.1. Nevertheless, acidified CFS still had

206 an inhibitory activity comparable with that of the neutralised CFS.

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208 3.2. Pathogens elimination in yoghurt by addition of L. rhamnosus

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210 Fig. 2 represents L. rhamnosus ability to eliminate co-inoculated pathogens at

211 low and high inoculation levels in probiotic yoghurt (controlled fermentation) compared

212 with regular yoghurt. Generally, L. rhamnosus addition to yoghurt was significantly able

213 to both eliminate pathogens earlier and reduce their counts. Inhibition patterns seemed to

214 be dependent on pathogens’ initial numbers. The lower the pathogen’s count, the more

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215 the inhibitory effect. Complete elimination of 2 log cfu mL-1 of E. coli O157:H7, S.

216 aureus, Yer. enterocolitica and Sal. Typhimurium was obtained after 8, 4, 4 and 8 h in

217 probiotic yoghurt compared with 48, 24, 24 and 48 h, in control yoghurt, respectively

218 [Fig. 2A(i), B(i) and C(i)]. Furthermore, significant reductions (about 1 log cfu mL-1) in

219 E. coli O157:H7 and Yer. enterocolitica counts were observed at 2 h of fermentation in

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220 the presence of L. rhamnosus compared with control yoghurt. This difference widened

to about 2 log cfu mL-1 or more at 4 and 8 h of incubation, thus representing a highly

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222 significant reduction. Although inhibition patterns were less severe in the case of lower

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223 inoculation levels of both S. aureus and Sal. Typhimurium, they were still significant.

224 At higher initial pathogen counts [≃6 log cfu mL-1, Fig. 2 A(ii)–D(ii)], nearly

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similar inhibition patterns were observed except in case of Sal. Typhimurium. E. coli
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226 O157:H7, S. aureus and Yer. enterocolitica were successfully eliminated from L.
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227 rhamnosus fermented yoghurt after just 24 h of cold storage compared with regular

228 yoghurt where they were still detectable until the end of the experiment (except for S.
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229 aureus which was eliminated 24 h later). At the higher inoculation level of Sal.
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230 Typhimurium, addition of L. rhamnosus to yoghurt failed to completely eliminate Sal.

231 Typhimurium till 72 h of cold storage. In spite of this, L. rhamnosus addition still led to
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232 a significant reduction in Sal. Typhimurium count that was noticed from 24 h of storage.

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Several investigations have demonstrated that Salmonella spp. can survive in acidic
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234 foods at low pH values for long periods of time (Mugochi, Parawira, Mpofu, Simango,

235 & Zvauya, 1999). Overall, controlled fermentation of yoghurt with addition of L.

236 rhamnosus achieved its proposed purpose, even with incomplete inhibition of higher

237 Sal. Typhimurium counts, as it is unlikely extremely contaminated raw milk would be

238 used in production.

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239 Probiotic properties including antimicrobial activity of L. rhamnosus have been

240 extensively researched (Kaewiad, Kaewnopparat, & Kaewnopparat, 2015; Verdenelli et

241 al., 2009) and numerous associated health benefits have been reported (Rajoka et al.,

242 2017; Smith, Rigassio-Radler, Denmark, Haley, & Touger-Decker, 2013). Incorporation

243 of L. rhamnosus in different foods has been well studied and successfully applied

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244 (Dommels et al., 2009; Hekmat, Soltani, & Reid, 2009; Liu et al., 2017a). However,

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245 research into utilising the antimicrobial traits of L. rhamnosus in food biopreservation

246 are very scarce.

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247 Recently, Beristain-Bauza et al. (2016) developed an antimicrobial wrapping

248 material mounted with CFS of L. rhamnosus NRRL B-442 (probiotic strain) and tested it

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in vitro for its efficacy. They noticed significant inhibition against E. coli, S. aureus, Sal.
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250 Typhimurium and Lis. monocytogenes when a high concentration of L. rhamnosus CFS
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251 was used. In a recent attempt, the hygienic quality of Mutandabota, a traditional

252 southern African dairy product (with fruit pulp), was improved by addition of L.
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253 rhamnosus yoba (Mpofu et al., 2016). Compared with the traditional product, a
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254 significant log reduction of inoculated pathogens (Bacillus cereus, Campylobacter

255 jejuni, Lis. monocytogenes, Salmonella spp. and E. coli O157:H7) and complete
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256 elimination was achieved after nearly 24 h of fermentation using L. rhamnosus yoba,

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which resembles our findings. Regarding Salmonella, comparable findings were also

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obtained, whereas inoculated Salmonella (at similar log count) initially increased, it then

259 decreased till the end of potential consuming time where it was not detected. Mpofu et

260 al. (2016) attributed the final elimination Salmonella to the addition of acidic fruit pulp,

261 which acts as an extra hurdle.

262 The concept of using probiotics in food as biopreservative agents has acquired

263 food-processors and scientists’ attention (Ortiz-Rivera et al., 2017). The aim of

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264 biopreservation is to naturally extend the shelf life of food and ensure safety (Ananou,

265 Maqueda, Martínez-Bueno, & Valdivia, 2007). Among these natural biopreservative

266 methods is the addition of LAB, probiotics and/or their active metabolites (Deegan,

267 Cotter, Hill, & Ross, 2006). In addition to the biopreseravtive action of L. rhamnosus

268 demonstrated in this study, many probiotic species have been incorporated into different

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269 foods for their antimicrobial activities. Lactobacillus plantarum, a probiotic strain

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270 isolated from cows’ milk (Liu et al., 2017b), was found to have a wide spectrum

271 antimicrobial effect (Gram-positive and Gram-negative bacteria and some fungal

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272 species) and proved to be a useful bio-preservative agent for fresh chicken meat (Sabo,

273 Pérez-Rodríguez, Domínguez, & Oliveira, 2017) and fermented foods (Liu et al., 2017b;

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Zago et al., 2011). Reuterin, a bacteriocin produced in fermented milk inoculated by
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275 Lactobacillus reuteri, was demonstrated to have a good bio-preservative action to
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276 fermented milk (Ortiz-Rivera et al., 2017). Similarly, Lactobacillus acidophilus,

277 Lactobacillus fermentum, Lactobacillus paracasei, Enterococcus faecium and other

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278 probiotics have been used for food biopreservation (Aljewicz & Cichosz, 2015; Aspri et
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279 al., 2017; Tulumoglu et al., 2014).

280 From industrial/economical point of view, addition of probiotics to foods may

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281 represent the drawback of extra cost. However, this drawback can be offset by the

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advantageous features of addition of probiotics. Additionally, consumer preferences

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nowadays are shifting to functional and probiotics fortified foods (Zoumpopoulou, Pot,

284 Tsakalidou, & Papadimitriou, 2017). Lastly, sensorial acceptability of L. rhamnosus

285 probiotic yoghurt was comparable with that of a regular yoghurt (Hekmat & Reid,

286 2006).

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288 3.3. pH changes and L. rhamnosus count

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290 The pH drop in both probiotic and control yoghurt was non-significantly

291 different for the first 3 h of fermentation. Subsequently, a significant change was found

292 at 4 h that remained constant until the end of the experiment (Fig. 3). This lowering is

293 mainly attributed to the fermentation behaviour of L. rhamnosus, which produces a high

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294 amount of lactic acid (Beristain-Bauza et al., 2016). L. rhamnosus growth is largely

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295 affected by pH level. While a slightly acidic pH level (~5) does not favour L. rhamnosus

296 growth, a pH level of about 4.2 strongly promotes its growth and metabolic activities

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297 (Mpofu et al., 2014). Those findings might explain the significant variation in pH

298 between probiotic and control yoghurts at 4 h where starter microorganisms lowered the

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pH level to the appropriate one. The later constant pH level was mainly due to cold
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300 storage, which nearly halts all microbial metabolic activities.
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301 With respect to L. rhamnosus levels, it appears to be reversely correlated with

302 pH; lowering the pH resulted in an increased count (Fig. 4). This is supported by the
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303 previous explanation of L. rhamnosus activity at different pH levels (Mpofu et al.,

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304 2014). This is augmented with previous findings of Mpofu et al. (2016), who reported a

305 similar phenomenon. In this study, yoghurt as a food model for delivering probiotics
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306 was found to support the growth of L. rhamnosus. Unlike many probiotic strains (L.

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acidophilus, L. casei, Bifidobacterium bifidum, Bifidobacterium longum) that are

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suppressed by yoghurt acidic nature, growth of L. rhamnosus in yoghurt is not affected

309 (Shah, 2000). This is in accordance with other previous reports (Capela, Hay, & Shah,

310 2006; Hekmat et al., 2009).

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312 4. Conclusion

313

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314 To the best of our knowledge, this is among the first reports that proved the

315 applicability of using a probiotic strain of L. rhamnosus as a food bio-controlling agent.

316 This study indicated that addition of L. rhamnosus to yoghurt during fermentation is

317 capable of completely eliminating of many important foodborne pathogens (E. coli

318 O157:H7, S. aureus and Yer. enterocolitica) or at least significantly reducing higher

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319 counts of Sal. Typhimurium. These findings support the exploiting of L. rhamnosus’

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320 antimicrobial abilities in biopreservation of different fermented foods, especially those

321 produced in developing countries. Further studies should follow to reveal the actual

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322 mechanism of this phenomenon.

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324 Acknowledgement
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326 This study is financially supported by the Science and Technology Development

327 Fund, Egypt (project ID, 25454, short term fellowship/Capacity Building Grant), to
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328 whom the corresponding author and grant recipient would like to express his thanks.
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1 Figure legends

2
3 Fig. 1. Schematic flowchart of the proposed protocol of pathogens’ elimination during

4 controlled yoghurt fermentation.

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6 Fig. 2. Antagonistic reduction effect of co-inoculation of L. rhamnosus with yoghurt

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7 starter against tested pathogens: (A) E. coli O157:H7, (B) S. aureus, (C) Yer.

8 enterocolitica and (D) Sal. Typhimurium at two inoculation levels: (i) 2.0 log cfu mL-1

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9 and (ii) 6.0 log cfu mL-1. Black triangles with dotted line represent pathogens grown in

10 regular yoghurt, while open circles with dotted line represent pathogens grown in

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probiotic yoghurt. Reported values are the mean ± SEM of three independent trials.
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12 Counts were expressed as log cfu mL-1. The S and HS represent significant or highly
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13 significant difference at each sampling time, respectively.

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15 Fig. 3. Change in pH of regular ( ) and probiotic () yoghurt over experiment storage
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16 period. The S indicates a significant difference at each sampling time.

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18 Fig. 4. Change in L. rhamnosus count during experiment storage period. Reported

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values are the mean ± SEM of three independent trials. Counts were expressed as log cfu

20 mL-1.
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1 Table 1

2 In-vitro antimicrobial activity of L. rhamnosus acidified (pH≈5.1) and neutralised

3 (pH≈7.0) cell free supernatant (CFS). a

Pathogen Inhibition zone (mm)

Acidified CFS Neutralised CFS P value

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E. coli O157:H7 10.8±0.3 *aA 8.9±0.2 *bA 0.03

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S. aureus 12.4±0.3 *aB 11.3±0.4 *aB 0.08

Yer. enterocolitica 11.7±0.3 *aA 10.1±0.2 *bA 0.03

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Sal. Typhimurium 8.2±0.2 *aC 4.3±0.1 bC 0.01

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5 a

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6 against all tested pathogens. Results are the mean of three independent replicates;
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7 different superscript lowercase letters in the same species and different uppercase letters

8 in the same column represent significant differences (P < 0.05). An asterisk shows
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9 significant results (P < 0.05) compared with all results obtained.

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