1 Inhibiting potential of selected lactic acid bacteria isolated from Costa 1 Rican agro-industrial waste against Salmonella sp. in yogurt 2 3 Valeria Piedra1, Jessie Usaga2, Mauricio Redondo-Solano3, Lidieth Uribe-Lorío4, Carol 4 Valenzuela-Martínez2,3, Natalia Barboza1,2 5 1Food Technology Department, University of Costa Rica (UCR), Costa Rica. 6 2National Center of Food Science and Technology (CITA), UCR, Costa Rica. 7 3Research Center for Tropical Diseases (CIET) and Food Microbiology Research and 8 Training Laboratory (LIMA), College of Microbiology, UCR, Costa Rica. 9 4Agronomic Research Center (CIA), Agronomy Department, UCR, Costa Rica. 10 11 Correspondence: Natalia Barboza, Food Technology Department, University of Costa 12 Rica, Costa Rica 13 Tel.: +(506) 2511-7222 14 E-mail: natalia.barboza@ucr.ac.cr 15 16 Key words: bioprotective culture, food pathogens, in vitro assays. 17 18 Contributions: approved the final version of the manuscript, and agreed to be 19 accountable for all aspects of the manuscript, and agreed to be accountable for all aspects 20 of the work. 21 22 Conflict of interest: the authors declare no conflict of interest. 23 24 Ethics approval and consent to participate: not applicable. 25 about:blank 2 26 Funding: This research was sponsored by the Vicerrectoría de Investigación of the 27 University of Costa Rica, Project number 735-B9-457. 28 29 Availability of data and materials: data used to support the findings of this study are 30 included within the article. 31 32 Acknowledgements: This work was supported by the technical assistance of Marcelo 33 Jiménez at the Food Microbiology Laboratory in CITA, UCR. 34 35 36 37 38 39 40 3 Abstract 41 This study aimed to characterize lactic acid bacteria (LAB) isolated from Costa Rican 42 agro-industrial waste and explore their bioprotective potential against Salmonella in 43 yogurt. A total of 43 LAB isolates were identified using the 16S rRNA region. In vitro 44 inhibition of Salmonella, Listeria monocytogenes, Staphylococcus aureus, and 45 Escherichia coli was determined. Fifteen of the 43 isolates showed a good to strong 46 antimicrobial effect against at least two pathogens. Fourteen selected isolates were 47 evaluated for antibiotic resistance, gelatinase, and hemolytic activity. The bioprotective 48 effect of the most promising strain, Lactiplantibacillus pentosus, was assessed against 49 Salmonella sp. during yogurt fermentation. All the isolates were resistant to vancomycin 50 and showed variable degrees of susceptibility to other antibiotics. All of the isolates were 51 negative for gelatinase, and five isolates had no hemolytic activity. A significant 52 inhibitory effect of L. pentosus_58(6)-2I (P < 0.05) against Salmonella during 53 fermentation was found, but pathogen reduction was limited to 0.611 log CFU/mL. 54 55 56 57 4 Introduction 58 In Costa Rica, more than 6.3 billion tons of organic waste are generated in the 59 primary economic sector alone (Miranda-Durán et al., 2020), underscoring the urgent 60 need for innovative waste management strategies. Agro-industrial waste is any substance 61 or object that the holder discards or intends or is required to discard, considering residue 62 as everything that is not the final (main) product of the process (Okino Delgado et al., 63 2015). 64 The isolation of lactic acid bacteria (LAB) from agro-industrial waste could be a 65 feasible option to obtain microorganisms adapted to local conditions. Recent research (de 66 Melo Pereira et al., 2018; Todorov et al., 2024) have been shown that bacteria isolated 67 from sources other than the gastrointestinal tract such as strains from local foods, 68 traditional drinks, fruits, fermented process, and agroindustry waste (Amenu et al., 2024; 69 Prihanto et al., 2024; Santos et al., 2016; Taboada et al., 2024, Wu et al., 2021), could be 70 an option to obtain isolates with probiotic potential. Bioprotective potential is a desired 71 feature which can offer an additional safety barrier against pathogenic microorganisms in 72 addition to the thermal treatments used by the food industry. 73 The use of LAB in fermented products can contribute to preventing food borne 74 diseases (Martin-Garcia et al., 2023). Some members of the genera Lactobacillus and 75 Bifidobacterium are characterized by the production of organic acids, specifically lactic 76 and acetic acids, and some strains have been studied to prevent the growth of pathogenic 77 bacteria such as Salmonella (Motahari et al., 2017). 78 Salmonella spp. is one of the most frequent bacterial etiological agents of 79 foodborne diseases in the European Union and the United States (EFSA and ECDC, 2021; 80 Williams et al., 2023). It is transmitted by the fecal-oral route, either directly or through 81 food. Milk and milk derivative products have been implicated in the transmission of 82 https://www.sciencedirect.com/science/article/pii/S0924224422004770#bib44 5 Salmonella, mostly due to the use of raw or inadequately pasteurized milk or 83 contaminated after pasteurization (Olsen et al., 2004; Singh et al., 2018). A survival of S. 84 Typhimurium for 23 days and S. Typhi during 16 days in a refrigerated (4°C) Egyptian 85 yogurt has been previously reported (El-Gazzar and Marth, 1992). 86 Yogurt presents unfavorable conditions for the growth of Salmonella, however, a 87 research study shows a maximum specific growth rate of Salmonella during yogurt 88 fermentation that ranged from 0.26 to 0.38 for S. Enteritidis and from 0.50 to 0.56 log 89 CFU/g/h for S. Typhimurium (Savran et al., 2018a). Moreover, it has been confirmed that 90 S. Enteriditis is able to survive longer during yogurt storage when temperatures are low 91 (e.g. 304 h at 4 °C, 60 h at 25 °C) (Savran et al., 2018b). The use of contaminated raw 92 milk or the incorrect application of hygiene practices could be a source of contamination 93 (Kumbhar et al., 2009). Despite only one outbreak of salmonellosis in yogurt has been 94 reported associated with cross contamination due to an open, blood-stained yogurt pots 95 stored beneath a rack of raw lamb (Evans et al., 1995), recent data show the presence of 96 Salmonella in raw milk, yogurt, and other dairy products (Asfaw et al., 2023). 97 This research aimed to characterize LAB isolated from Costa Rican agro-98 industrial residuals, and explore the bioprotective potential of a selected one against 99 Salmonella during yogurt fermentation. 100 101 Materials and Methods 102 Isolation of LAB from Agro-industrial waste 103 Agro-industrial wastes were collected from Costa Rican companies that produce 104 value-added products from coffee, pineapple, orange, coffee, cocoa, and carrot (Table 1, 105 Supplementary Table 1). The colonies of LAB for each agro-industrial waste were 106 obtained according to Wu et al. (2021). Selected colonies were identified by Gram 107 6 staining and morphology. The cultures were preserved as glycerol stocks (20% v/v) at -108 80 °C until examination. 109 110 DNA extraction and PCR amplification 111 Lactic acid bacteria were grown in MRS agar (Oxoid) for 22 ± 2 h at 35.0 ± 0.5 112 °C. Using a miniprep extraction protocol (Birnboim and Doly, 1979), total nucleic acids 113 were extracted from each isolate. The primer pair 27F/1492R was used to obtain a 1.5-kb 114 fragment of the 16S rRNA gene (Edwards et al., 1989). PCR was conducted according to 115 Wu et al. (2021). 116 117 Antimicrobial activity of LAB isolates against foodborne pathogens 118 A modified methodology of the overlay assay (Hütt et al., 2006; Soleimani et al., 119 2010) was used to evaluate the in vitro antagonistic capacity of the LAB isolates against 120 Salmonella, L. monocytogenes, S. aureus, and E. coli. Microorganisms used in the study 121 included five L. monocytogenes strains (ATCC 19116 and wild strains isolated from meat 122 products), five Salmonella isolates (Salmonella Typhimurium, Salmonella Typhi, and 123 three wild isolates of an undefined serotype); all of them isolated from clinical samples, 124 E. coli ATCC 25922 and S. aureus ATCC 25923. Pathogens were provided by the 125 bacteriology collection of the Food Microbiology Research and Training Laboratory from 126 the Faculty of Microbiology at the University of Costa Rica. In the case of Salmonella or 127 L. monocytogenes, a cocktail suspension was used to inoculate. Before the experiments, 128 the plates were incubated at 35.0 ± 0.5 °C for 24 ± 2 h in MRS (Oxoid) or Tryptic Soy 129 Broth (TSB) (Oxoid), respectively. After incubation, each LAB isolate was inoculated in 130 a straight line 7 cm long and 0.5 cm from the edge, using MRS agar. The plates were 131 incubated under capnophilic conditions at 35.0 ± 0.5 ◦C for 24 ± 2 h. Before, 5 mL of 132 7 Brain Heart Infusion agar (BHI) (Oxoid) was added. After solidification, a cocktail 133 suspension prepared with the overnight cultures of each pathogen was added. The Petri 134 dishes were incubated at 35.0 ± 0.5 ◦C for 24 ± 2 h under aerobic conditions and they 135 were examined for the presence of an antagonistic interaction between each LAB isolate 136 and the pathogens. Antagonistic effect was visualized as a clear inhibition zone around 137 the line of each LAB. Clear zones were measured (Pan et al., 2009; Wu et al., 2021; 138 Duche et al., 2023) and the isolates were classified according to the size of the inhibition 139 zone. The 14 isolates showing the strongest inhibition halo against the pathogens were 140 selected for safety testing. 141 142 Safety assays 143 Antibiotic resistance 144 Antibiotic resistance of the LAB isolates was evaluated. A total of nine antibiotics 145 of the main classes were used (Table 2). Each isolate was grown in MRS broth (Oxoid) 146 incubated at 35.0 ± 0.5 ºC for 24 ± 2 h and they were swabbed on Mueller-Hinton agar 147 (Oxoid) using a sterile cotton swab. Disks, impregnated with each antibiotic, were placed 148 on the agar plates that were incubated at 35.0 ± 0.5 ºC for 24 ± 2 h in capnophilic 149 conditions. Diameter of the inhibition zones was measured after incubation and 150 interpreted according to the standards established by the Clinical and Laboratory Standard 151 Institute (Sharma et al., 2016). Experiments were performed in duplicate. 152 153 Gelatinase and hemolytic activity 154 The LAB isolates were grown on MRS agar (Oxoid) at 35.0 ± 0.5 ºC for 48 ± 2 155 h. For the gelatinase test, one colony from each isolate was inoculated into nutritive 156 gelatin tubes and incubated for 7 days at 35.0 ± 0.5 ºC. Every 48 ± 2 h, tubes were placed 157 8 in an ice bath for 15 ± 2 min and observed for gelatin hydrolysis. Isolates were considered 158 gelatinase negative if the gel remained solid after 7 days of incubation, or positive if there 159 was hydrolysis (Klamm, 2019). The experiment was performed in duplicate. S. aureus 160 ATCC 25923 and E. coli ATCC 25922 were used as positive controls, and an 161 uninoculated tube as a negative control. 162 For the hemolysis test, 0.5 McFarland standard suspension was prepared for each 163 isolate in 0.1% sterile peptone water. The suspensions were streaked as pure cultures on 164 Columbia agar with 7% sheep blood and incubated at 35 °C for 48 h (Maasjost et al., 165 2019). Color changes in zones on the blood agar indicated hemolytic activity: green zone 166 (α-hemolysis), light zone (β-hemolysis), and no color change (γ- hemolysis). Two 167 replicates were performed. S. aureus ATCC 25923 was used as a positive control (Aziz 168 et al., 2021). 169 170 Bioprotective effect of L. pentosus against Salmonella sp. in yogurt 171 Pathogen inoculation 172 Five Salmonella strains (S. enterica serovar Typhimurium 93, S. enterica 750, S. 173 enterica 80, and Salmonella DA36) were grown individually in TSB at 35.0 ± 0.5 ºC for 174 24 ± 2 h. Stationary phase cultures were then mixed in equal proportions. From the initial 175 Salmonella sp. cocktail decimal dilutions were made to obtain a population of 7 log 176 CFU/mL. Finally, 1 mL was inoculated into 1 L of yogurt targeting an initial pathogen 177 population of 4 log CFU/mL. 178 179 Lactiplantibacillus pentosus inoculation 180 9 L. pentosus_58(6)-2I was grown for 24 h in MRS broth at 35 ± 0.5 ºC. The 181 inoculum was added to the yogurt (20 mL for 2 L of product) for an initial population of 182 6-7 log CFU/mL. 183 184 Yogurt manufacture and inoculation 185 A formulation of 95% skim milk and 5% skim milk powder was used. The mixture 186 was pasteurized at 90 ± 2 ºC for 10 min and then cooled to 43-44 ºC. The commercial 187 culture Yo-Flex (CHR HANSEN), (Streptococcus thermophilus and Lactobacillus 188 bulgaricus), was added in the amount recommended by the supplier. The yogurt was 189 divided into four portions and used in the following treatments: 1) uninoculated yogurt, 190 2) yogurt inoculated with the Salmonella sp. cocktail 3) yogurt supplemented with 6 log 191 CFU/mL of L. pentosus_58(6)-2I, and 4) yogurt supplemented with 6 log CFU/mL of L. 192 pentosus_58(6)-2I and Salmonella sp. All treatments were incubated at 41 ºC in 8 oz 193 containers until a pH of approximately 4.5 was reached. The assay was performed in 194 triplicate. 195 196 Microbiological analysis 197 Treatments were sampled hourly during 6 h of fermentation. Decimal dilutions of 198 each sample were made in 0.1% phosphate saline solution (PSA). LAB counts (including 199 L. pentosus_58(6)-2I) were performed with the 3M Petrifilm method whereas Salmonella 200 s p. was quantified on xylose-lysine/deoxycholate agar (XLD) (Oxoid) using the spread 201 plate technique. Plates were incubated at 35 ºC for 48 ± 3 h. The yogurt pH was monitored 202 in the four treatments to observe the effects of Salmonella sp. or L. pentosus_58(6)-2I on 203 the acidification curve during fermentation. The pH of the uninoculated yogurt, and of 204 the yogurt with added L. pentosus was measured every 30 min using a HI2002-01 edge 205 10 pH meter (Hanna Instruments, Woonsocket, RI) equipped with a HI10530 electrode 206 (Hanna Instruments). The pH of the treatments inoculated with Salmonella spp. was 207 measured every hour. The uninoculated yogurt’s moisture, ash, protein and sodium 208 contents were determined using standard AOAC International methods (AOAC 209 International, 2012). Fat content was determined in yogurt as previously described 210 (Carpenter et al., 1993), and carbohydrate content was determined by calculation. 211 212 Statistical analysis 213 An analysis of variance (ANOVA) was performed to determine differences 214 between the growth or death curves (log CFU/mL) at time 0 and 6 h of L. pentosus_58(6)-215 2I and Salmonella sp. in the yogurt treatments. ANOVA was also performed for the 216 acidification curves (pH values at time 0 and 6 h). A significance level of 5% was 217 established, with values of P<0.05 considered significant. When significant differences 218 were identified, an HSD-Tukey multiple comparison of means test was performed to 219 determine the difference between treatments. 220 221 Results 222 At least 10 different species of LAB were identified from the agro-industrial 223 wastes (Table 1, Supplementary Table 1). Out of 43 isolates obtained from culture, 17 224 showed some degree of antagonistic activity against at least one of the tested pathogens. 225 However, just 14 isolates were selected for further trials based on their antimicrobial 226 effect against at least two pathogens (inhibition diameter larger than 6 mm). The only 227 exception was L. argentoratensis_79(4)-2C (Table 1). 228 For antibiotic resistance, all the selected LABs were resistant to vancomycin. L. 229 paracasei subsp. tolerans strains were susceptible to tetracycline but they were resistant 230 11 to streptomycin, chloramphenicol, erythromycin and penicillin whereas the isolates L. 231 plantarum_17-(4D), L. plantarum subsp. plantarum_71-6(2F), L. argentoratensis_57(7)-232 1H, and L. argentoratensis_79(4)-2C were resistant to ciprofloxacin (Table 2). 233 In the case of hemolytic and gelatinase activity, L. paracasei subsp. tolerans 234 (2A2-B, IA2P, II-CI-C Y 11-C1-B) and L. casei ATCC 393 did not produce beta 235 hemolysis and were negative for gelatinase activity (Table 3). 236 237 Biopreservative effect of Lactobacillus pentosus during yogurt processing 238 Based on the previous results, L. pentosus_58(6)-2I was selected as a potential 239 biopreservative for yogurt. The nutritional profile of the yogurt used is summarized in 240 Table 4. Figure 1 shows the pH of the four yogurt treatments during fermentation. The 241 acidification curves were consistent with the profile provided by the reference starter 242 culture after 6 h of fermentation at 41 °C. There were no significant differences among 243 treatments (P= 0.338). 244 Total LAB counts differed significantly (P = 0.010) among three of the treatments 245 (yogurt inoculated with L. pentosus and Salmonella sp., yogurt inoculated with 246 Salmonella spp., and uninoculated yogurt) after 6 h of fermentation. Specifically, there 247 were differences in bacterial counts after 6 h of fermentation, between yogurt inoculated 248 with L. pentosus and Salmonella sp. and yogurt with Salmonella sp. (P = 0.008) (Figure 249 2). However, these two treatments did not differ from the control (uninoculated sample) 250 (P = 0.538 and P = 0.108, respectively). As expected, the initial LAB population was 251 higher in yogurt inoculated with L. pentosus and Salmonella sp. than in yogurt inoculated 252 with Salmonella. However, the LAB population stabilized after 2 h of fermentation and 253 remained constant until the end of the process. This was consistent with the acidification 254 curves since the pH values did not change with the addition of L. pentosus (Figure 1). 255 12 Salmonella sp. survival in yogurt inoculated with L. pentosus_58(6)-2I was 256 significantly lower (P = 0.019) compared to the control (Figure 3). However, the HSD-257 Tukey test did not show differences between pathogen populations at times 0 and 6 in 258 either of the treatments (P = 0.331 and P =1.00, respectively), and differences between 259 the two treatments at time 6 h were not significant (P < 0.05). There was a pathogen 260 reduction of 0.611 log CFU/g in the treatment with L. pentosus_58(6)-2I and 0.017 log 261 CFU/g in the negative control. The Salmonella population increased during the initial 262 stage of fermentation (after 2 h of fermentation in the positive control and after 3 h in the 263 negative control). However, after a longer period of fermentation, this population 264 decreased, especially in the presence of L. pentosus_58(6)-2I. 265 266 Discussion and Conclusions 267 L. paracasei frequently exhibit broad-spectrum antimicrobial activity with 268 simultaneous inhibitory effects against L. monocytogenes, E. coli, S. aureus, and 269 Salmonella (Akpinar and Yerkliyaka, 2021), that is related with the production of 270 antimicrobial compounds such as organic acids, bacteriocins and exopolysaccharides 271 (Amini et al., 2022). The antagonistic activity of L. argentoratensis is closely related to 272 L. plantarum and it was recently classified as a new species (McFrederick et al., 2018). 273 Literature about the antimicrobial capacity of this species is relatively scarce; however, 274 some studies have confirmed the antimicrobial capacity of some isolates against Gram 275 positive and Gram negative bacteria (Siangpro et al., 2023). Recent advances in whole 276 genome sequencing of L. argentoratensis are providing insights about the potential of 277 this species as a biocontrol agent (Syrokou et al., 2021). 278 Vancomycin resistance found in this research was similar to previous reports 279 (Guo, 2017). This resistance is intrinsic in nature and is given by the vanX gene which 280 13 codes for the dipeptide ligase enzyme (Ddi) (Guo, 2017; Zhang et al., 2018), and transfer 281 to foodborne pathogens is not expected (Álvarez and Poce, 2018). LAB normally have 282 more than 70% resistance to amynoglicosides (gentamicin and streptomycin) and 283 ciprofloxacin, and low resistance to penicillin, tetracycline and chloramphenicol. 284 Variability in antibiotic resistance among species may be related to intrinsic traits. For 285 example, more than 68% of Lactobacillus species are resistant to ciprofloxacin due to the 286 gyrA gene. The tet(M) and erm(B) genes of L. paracasei confer resistance to tetracycline 287 and erythromycin (Guo et al., 2017). 288 Bacteria that produce total hemolysis in agar may contribute to anemia, 289 inflammation, and edema, mostly due to decreased iron availability (Rastogi et al., 2021). 290 Therefore, non-hemolytic LAB strains are considered safer for food applications. Some 291 isolates from this study were classified as partial-hemolytic strains; however, this trait is 292 normal in Lactobacillus and it is attributed to the generation of hydrogen peroxide (Aziz 293 et al., 2021). Also, no gelatinase activity was found in Lactobacillus (Aziz et al., 2021) 294 due to its low capacity to hydrolyze tissue components. This feature supports that LAB 295 strains are safe for food applications (Hashem et al., 2020). 296 The pathogen reduction observed in this study in the presence of L. 297 pentosus_58(6)-2I was greater than the decrease in Salmonella sp. due to the effect of 298 low pH reported by Savran et al. (2018a). Other mechanisms not studied here that may 299 explain the pathogen reduction include the synthesis of biosurfactant compounds, 300 bacteriocins, and hydrogen peroxide, which have in vitro inhibitory effects against 301 Salmonella sp. (Liu et al., 2018). Moreover, the bioprotective effect of L. pentosus against 302 Salmonella sp. was demonstrated by Motahari et al. (2017) using another L. pentosus 303 strain. Further analyses are required to elucidate the causes behind the greater decrease 304 in Salmonella spp. in the presence of L. pentosus_58(6)-2I. 305 14 The effect of a higher load of L. pentosus_58(6)-2I on Salmonella sp. survival 306 during yogurt fermentation should be tested. If effective, this approach may be suitable if 307 the sensorial properties of yogurt and acidification curves are not affected, and consumer 308 acceptance is not compromised. L. pentosus_58(6)-2I should also be evaluated in other 309 foods such as dairy products and fermented meats. 310 Likewise, other properties should be assessed to identify bacteria with probiotic 311 potential. According to Todorov et al. (2023), evaluation under simulated gastrointestinal 312 tract conditions, antagonism against pathogens, resistance to enzymes, presence of 313 transferable antibiotic resistance genes, ability to reduce pathogen adhesion to surfaces, 314 removal of cholesterol from surfaces, as well as taking into account the evaluation of the 315 shelf life of foods and their sensory characteristics, are some characteristics that should 316 be considered when evaluating the properties of new isolates. 317 318 References 319 320 Akpinar A, Yerlikaya O, 2021. 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Inhibition halo of Salmonella enterica, Listeria monocytogenes, Staphylococcus aureus 29213 and Escherichia coli 25922 grown on 497 culture media pre-inoculated with selected LAB strains isolated from agro-industrial waste. 498 Halo LAB strain GenBank code Isolation source Salmonella L. monocytogenes S. aureus E. coli Lacticaseibacillus paracasei subsp. tolerans_2A2-B ON763280 MFC of coffee effluent +++ +++ +++ +++ Lacticaseibacillus paracasei subsp. tolerans_ IA2-P ON763283 MFC of coffee effluent +++ +++ +++ +++ Lacticaseibacillus paracasei subsp. tolerans_1-C1 ON763284 MFC of coffee effluent +++ +++ +++ +++ Lacticaseibacillus paracasei subsp. tolerans_11-CI-C ON763282 MFC of coffee effluent +++ +++ +++ +++ Lacticaseibacillus paracasei subsp. tolerans_11-C2-C ON763287 MFC of coffee effluent +++ +++ + ++ Lacticaseibacillus paracasei subsp. tolerans_11-C1-B ON763286 MFC of coffee effluent +++ +++ ++ +++ Lacticaseibacillus paracasei subsp. tolerans_I-C2 ON763285 MFC of coffee effluent +++ +++ +++ +++ Leuconostoc pseudomesenteroides_17-(2D) ON763309 Coffee brush ++ + +++ +++ Lactiplantibacillus plantarum_17-(4D) ON763301 Coffee brush +++ +++ ++ +++ Lactobacillus plantarum subsp. plantarum_71-6(2F) ON763308 Orange waste residuals +++ +++ +++ + Lactiplantibacillus argentoratensis_57(7)-1H ON763326 Trinitario cocoa +++ ++ +++ + Lactiplantibacillus pentosus_58(6)-2I ON763304 Trinitario cocoa +++ + + + Lactiplantibacillus pentosus_58(6)-1I ON763303 Trinitario cocoa +++ ++ ++ ++ Lactiplantibacillusargentoratensis_79(4)-2C ON763328 Trinitario cocoa ++ ++ ++ + 23 + Inhibition zone 0- 3 mm in diameter (weak), ++ inhibition zone 3- 6 mm in diameter (good), +++ inhibition zone larger than 6 mm in diameter 499 (strong). MFC=microbial fuel cells. 500 501 502 503 504 505 506 507 508 509 510 511 512 513 24 Table 2. Antibiotic resistance/susceptibility of selected isolates from agro-industrial waste (S, susceptible. R, resistant. I, intermediate). 514 Isolate Antibiotic (concentration) Amoxicilli n with clavulanic acid (30 μg) Streptomycin (15 μg) Chloramphenicol (30 μg) Gentamicin (10 μg) Erythromycin (15 μg) Tetracycline (30 μg) Ciprofloxacin (5 μg) Vancomycin (30 μg) Penicillin (10 IU) L. paracasei subsp. tolerans_2A2-B S R S S S S S R S L. paracasei subsp. tolerans_IA2-P S R R S R R S R R L. paracasei subsp. tolerans_1-C1 S S S S S S S R S L. paracasei subsp. tolerans_II-CI-C S R S S S S S R S L. paracasei subsp. tolerans_11-C2-C S I S S S S S R S L. paracasei subsp. tolerans_11-C1-B S R S S S S S R S L. paracasei subsp. tolerans_I-C2 S R S S S S S R S L. pseudomesenteroides_17-(2D) S S S S S S S R S L. plantarum_17-(4D) S S S S S S R R S L. plantarum subsp. plantarum_71- 6(2F) S S S S S S R R S L. argentoratensis_57(7)-1H S S S S S S R R S L. pentosus_58(6)-2I S S S S S S I R S L. pentosus_58(6)-1I S S S S S S S R S L. argentoratensis_79(4)-2C S S S S S S R R S L. casei_ATCC 393 S S R R R R S R S L. paracasei_6714 S R S S S S S R S 515 25 Table 3. Results of hemolytic activity and gelatinase activity to evaluate the probiotic 516 profile of selected lactic acid bacteria isolated from agroindustrial waste. 517 Isolate Hemolytic Gelatinase Lacticaseibacillus paracasei subsp. tolerans_2A2-B γ Neg Lacticaseibacillus paracasei subsp. tolerans_IA2-P γ Neg Lacticaseibacillus paracasei subsp. tolerans_1-C1 α Neg Lacticaseibacillus paracasei subsp. tolerans_11-CI-C γ Neg Lacticaseibacillus paracasei subsp. tolerans_11-C2-C α Neg Lacticaseibacillus paracasei subsp. tolerans_11-C1-B γ Neg Lacticaseibacillus paracasei subsp. tolerans_I-C2 α Neg Leuconostoc pseudomesenteroides_17-(2D) α Neg Lactobacillus plantarum_17-(4D) α Neg Lactobacillus plantarum subsp. plantarum_71-6(2F) α Neg Lactiplantibacillus argentoratensis_57(7)-1H α Neg Lactiplantibacillus pentosus_58(6)-2I α Neg Lactiplantibacillus pentosus_58(6)-1I α Neg Lactiplantibacillus argentoratensis_79(4)-2C α Neg Lacticaseibacillus paracasei ATCC 393 γ Neg Lacticaseibacillus paracasei_6714 α Neg Absence of hemolysis (γ), partial hemolysis (α), negative (neg). 518 519 520 521 522 26 523 Table 4. Nutritional composition of yogurt. 524 Analysis Percentage (%) ± SD Moisture 85.57 ± 0.29 Fat <0.20 ± 0.00 Protein 4.95 ± 0.14 Ash 1.12 ± 0.10 Carbohydrates 7.32 ± 0.17 Sodium 74.30 ± 4.98 Total energy value 218.67 ± 3.21 Energy value 85.57 ± 0.00 Mean values ± standard deviation, n = 3. 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 27 Supplementary Table 1. Inhibition halo of Salmonella enterica, Listeria monocytogenes, Staphylococcus aureus 29213 and Escherichia coli 25922 543 grown on culture media pre-inoculated with different LAB strains isolated from agro-industrial waste. 544 Halo LAB strain GenBank code Isolation source Salmonella L. monocytogenes S. aureus E. coli Weissella soli_29-5(1) ON763313 Carrot waste residues + + + + Weissella soli_30-6(3) ON763314 Carrot waste residues + + + + Weissella soli_31-2(9B) ON763315 Carrot waste residues + + + + Lactiplantibacillus pentosus_16-6(1C) ON763300 Coffee brush + + + + Leuconostoc pseudomesenteroides_18-(1B) ON763310 Coffee brush ++ + + + Lactobacillus pentosus_19-(3A) ON763312 Coffee brush + + + + Lactobacillus pentosus_19-(5A) ON763302 Coffee brush + + + + Leuconostoc_66-2(4A) ON763311 Orange waste residuals + + + + Levilactobacillus brevis_68-6(1C) ON763329 Orange waste residuals + + + + Lactobacillus plantarum_69-2(3D) ON763306 Orange waste residuals + + + + Lactobacillus pentosus_70-6(1E) ON763307 Orange waste residuals + + + + Lactobacillus plantarum subsp. plantarum_70-6(13E) ON763327 Orange waste residuals + + + + Lacticaseibacillus paracasei_P2 ON763288 MFC of coffee effluent + + + + Lacticaseibacillus paracasei_P4 ON763289 MFC of coffee effluent + + + + Lacticaseibacillus paracasei_P6 ON763290 MFC of coffee effluent + + + + 28 Lacticaseibacillus paracasei_P8 ON763291 MFC of coffee effluent + + + + Lacticaseibacillus paracasei_P9 ON763292 MFC of coffee effluent + + + + Lacticaseibacillus paracasei_P10 ON763293 MFC of coffee effluent + + + + Lacticaseibacillus paracasei_P13 ON763294 MFC of coffee effluent + + + + Limosilactobacillus fermentum_56(6)-2F ON763317 Trinitario cocoa +++ + + + Limosilactobacillus fermentum_56(6)-1F ON763318 Trinitario cocoa + + + + Limosilactobacillus fermentum_56(7)-1G ON763319 Trinitario cocoa + + + + Limosilactobacillus fermentum_57(7)-2H ON763324 Trinitario cocoa ++ + + + Limosilactobacillus fermentum_58(7)-1J ON763325 Trinitario cocoa + + + ++ Limosilactobacillus fermentum_78(6)-1A ON763321 Trinitario cocoa + + + + Pediococcus acidilactici_78(6)-3A ON763330 Trinitario cocoa ++ ++ + + Limosilactobacillus fermentum_78(6)-2A ON763322 Trinitario cocoa + + + + Limosilactobacillusfermentum_79(6)-1D ON763323 Trinitario cocoa + + + + Weissellaghanensis_80(6)-1E ON763316 Trinitario cocoa + + + + + Inhibition zone 0- 3 mm in diameter (weak), ++ inhibition zone 3- 6 mm in diameter (good), +++ inhibition zone larger than 6 mm in diameter 545 (strong). MFC=microbial fuel cells. 546 29 547 List of figures 548 Figure 1. pH values during fermentation of yogurt subjected to different inoculation 549 treatments (means, error bars show the standard deviation for n = 3). 550 Figure 2. Lactic acid bacteria count during fermentation of yogurt subjected to different 551 inoculation treatments (means, error bars show the standard deviation for n = 3). 552 Figure 3. Salmonella s p. counts during fermentation of yogurt subjected to different 553 inoculation treatments (means, error bars show the standard deviation for n = 3).554 1 555