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Please cite this article as: Piedra V, Usaga J, Redondo-Solano M, et al. Inhibiting potential of selected lactic acid bacteria isolated from Costa Rican agro-industrial waste against Salmonella sp. in yogurt. Ital J Food Saf doi:10.4081/ijfs.2024.12494 Submitted: 23-03-2024 Accepted: 09-09-2024 Note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries should be directed to the corresponding author for the article. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. Italian Journal of Food Safety https://www.pagepressjournals.org/index.php/ijfs/index Inhibiting potential of selected lactic acid bacteria isolated from Costa Rican agro-industrial waste against Salmonella sp. in yogurt Valeria Piedra,1 Jessie Usaga,2 Mauricio Redondo-Solano,3 Lidieth Uribe-Lorío,4 Carol Valenzuela-Martínez,2,3 Natalia Barboza1,2 1Food Technology Department, University of Costa Rica, San Pedro; 2National Center of Food Science and Technology, University of Costa Rica, San Pedro; 3Research Center for Tropical Diseases and Food Microbiology Research and Training Laboratory, College of Microbiology, University of Costa Rica, San Pedro; 4Agronomic Research Center, Agronomy Department, University of Costa Rica, San Pedro, Costa Rica. Correspondence: Natalia Barboza, Food Technology Department, University of Costa Rica, San Pedro, Costa Rica. Tel.: +(506) 2511-7222 E-mail: natalia.barboza@ucr.ac.cr Key words: bioprotective culture, food pathogens, in vitro assays. Contributions: approved the final version of the manuscript, and agreed to be accountable for all aspects of the manuscript, and agreed to be accountable for all aspects of the work. Conflict of interest: the authors declare no conflict of interest. Ethics approval and consent to participate: not applicable. Funding: this research was sponsored by the Vicerrectoría de Investigación of the University of Costa Rica, Project number 735-B9-457. Availability of data and materials: data used to support the findings of this study are included within the article. Acknowledgements: this work was supported by the technical assistance of Marcelo Jiménez at the Food Microbiology Laboratory in CITA, UCR. Abstract This study aimed to characterize lactic acid bacteria (LAB) isolated from Costa Rican agro-industrial waste and explore their bioprotective potential against Salmonella in yogurt. A total of 43 LAB isolates were identified using the 16S rRNA region. In vitro inhibition of Salmonella, Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli was determined. 15 of the 43 isolates showed a good to strong antimicrobial effect against at least two pathogens. 14 selected isolates were evaluated for antibiotic resistance, gelatinase, and hemolytic activity. The bioprotective effect of the most promising strain, Lactiplantibacillus pentosus, was assessed against Salmonella sp. during yogurt fermentation. All the isolates were resistant to vancomycin and showed variable degrees of susceptibility to other antibiotics. All of the isolates were negative for gelatinase, and 5 isolates had no hemolytic activity. A significant inhibitory effect of L. pentosus_58(6)-2I (p<0.05) against Salmonella during fermentation was found, but pathogen reduction was limited to 0.611 log CFU/mL. Introduction In Costa Rica, more than 6.3 billion tons of organic waste are generated in the primary economic sector alone (Miranda-Durán et al., 2020), underscoring the urgent need for innovative waste management strategies. Agro-industrial waste is any substance or object that the holder discards or intends or is required to discard, considering residue as everything that is not the final (main) product of the process (Okino Delgado et al., 2015). The isolation of lactic acid bacteria (LAB) from agro-industrial waste could be a feasible option to obtain microorganisms adapted to local conditions. Recent research (de Melo Pereira et al., 2018; Todorov et al., 2023) has shown that bacteria isolated from sources other than the gastrointestinal tract such as strains from local foods, traditional drinks, fruits, fermented process, and agroindustry waste (Santos et al., 2016; Wu et al., 2021; Amenu et al., 2024; Prihanto et al., 2024; Taboada et al., 2024) could be an option to obtain isolates with probiotic potential. Bioprotective potential is a desired feature that can offer an additional safety barrier against pathogenic microorganisms in addition to the thermal treatments used by the food industry. The use of LAB in fermented products can contribute to preventing foodborne diseases (Martin- Garcia et al., 2023). Some members of the genera Lactobacillus and Bifidobacterium are characterized by the production of organic acids, specifically lactic and acetic acids, and some strains have been studied to prevent the growth of pathogenic bacteria such as Salmonella (Motahari et al., 2017). Salmonella spp. is one of the most frequent bacterial etiological agents of foodborne diseases in the European Union and the United States (EFSA and ECDC, 2021; Williams et al., 2023). It is transmitted by the fecal-oral route, either directly or through food. Milk and milk derivative products have been implicated in the transmission of Salmonella, mostly due to the use of raw or inadequately pasteurized milk or contaminated after pasteurization (Olsen et al., 2004; Singh et al., 2018). A survival of S. Typhimurium for 23 days and S. Typhi during 16 days in a refrigerated (4°C) Egyptian yogurt has been previously reported (El-Gazzar and Marth, 1992). Yogurt presents unfavorable conditions for the growth of Salmonella; however, a research study shows a maximum specific growth rate of Salmonella during yogurt fermentation that ranged from 0.26 to 0.38 for S. Enteritidis and from 0.50 to 0.56 log CFU/g/h for S. Typhimurium (Savran et al., 2018a). Moreover, it has been confirmed that S. Enteriditis is able to survive longer during yogurt storage when temperatures are low (e.g., 304 h at 4 °C, 60 h at 25 °C) (Savran et al., 2018b). The use of contaminated raw milk or the incorrect application of hygiene practices could be a source of contamination (Kumbhar et al., 2009). Despite only one outbreak of salmonellosis in yogurt has been reported associated with cross contamination due to an open, blood-stained yogurt pot stored beneath a rack of raw lamb (Evans et al., 1995), recent data show the presence of Salmonella in raw milk, yogurt, and other dairy products (Asfaw et al., 2023). This research aimed to characterize LAB isolated from Costa Rican agro-industrial residuals and explore the bioprotective potential of a selected one against Salmonella during yogurt fermentation. Materials and Methods Isolation of lactic acid bacteria from agro-industrial waste Agro-industrial wastes were collected from Costa Rican companies that produce value-added products from coffee, pineapple, orange, coffee, cocoa, and carrot (Table 1 and Supplementary Table 1). The colonies of LAB for each agro-industrial waste were obtained according to Wu et al. (2021). Selected colonies were identified by Gram staining and morphology. The cultures were preserved as glycerol stocks (20% v/v) at -80°C until examination. DNA extraction and polymerase chain reaction amplification LAB were grown in De Man, Rogosa, and Sharpe agar (MRS) culture medium (Thermo Scientific™ Oxoid™, MA, USA) for 22 ± 2 h at 35.0 ± 0.5 °C. Using a miniprep extraction protocol (Birnboim and Doly, 1979), total nucleic acids were extracted from each isolate. The primer pair 27F/1492R was used to obtain a 1.5-kb fragment of the 16S rRNA gene (Edwards et al., 1989). Polymerase chain reaction was conducted according to Wu et al. (2021). Antimicrobial activity of lactic acid bacteria isolates against foodborne pathogens A modified methodology of the overlay assay (Hütt et al., 2006; Soleimani et al., 2010) was used to evaluate the in vitro antagonistic capacity of the LAB isolates against Salmonella, L. monocytogenes, S. aureus, and E. coli. Microorganisms used in the study included five L. monocytogenes strains (ATCC 19116 and wild strains isolated from meat products), five Salmonella isolates (Salmonella Typhimurium, Salmonella Typhi, and three wild isolates of an undefined serotype); all of them isolated from clinical samples, E. coli ATCC 25922 and S. aureus ATCC 25923. Pathogens were provided by the bacteriology collection of the Food Microbiology Research and Training Laboratory from the Faculty of Microbiology at the University of Costa Rica. In the case of Salmonella or L. monocytogenes, a cocktail suspension was used to inoculate. Before the experiments, the plates were incubated at 35.0±0.5°C for 24±2 h in MRS (Thermo Scientific™ Oxoid™, MA, USA) or Tryptic Soy Broth (TSB) (Thermo Scientific™ Oxoid™, MA, USA), respectively. After incubation, each LAB isolate was inoculated in a straight line 7 cm long and 0.5 cm from the edge, using MRS agar. The plates were incubated under capnophilic conditions at 35.0±0.5°C for 24±2 h. Before, 5 mL of Brain Heart Infusion agar (Thermo Scientific™ Oxoid™, MA, USA) was added. After solidification, a cocktail suspension prepared with the overnight cultures of each pathogen was added. The Petri dishes were incubated at 35.0±0.5°C for 24±2 h under aerobic conditions and they were examined for the presence of an antagonistic interaction between each LAB isolate and the pathogens. Antagonistic effect was visualized as a clear inhibition zone around the line of each LAB. Clear zones were measured (Pan et al., 2009; Wu et al., 2021; Duche et al., 2023) and the isolates were classified according to the size of the inhibition zone. The 14 isolates showing the strongest inhibition halo against the pathogens were selected for safety testing. Safety assays Antibiotic resistance Antibiotic resistance of the LAB isolates was evaluated. A total of nine antibiotics of the main classes were used (Table 2). Each isolate was grown in MRS broth (Thermo Scientific™ Oxoid™, MA, USA) incubated at 35.0±0.5°C for 24±2 h and they were swabbed on Mueller-Hinton agar (Thermo Scientific™ Oxoid™, MA, USA) using a sterile cotton swab. Disks, impregnated with each antibiotic, were placed on the agar plates that were incubated at 35.0±0.5ºC for 24±2 h in capnophilic conditions. Diameter of the inhibition zones was measured after incubation and interpreted according to the standards established by the Clinical and Laboratory Standard Institute (Sharma et al., 2016). Experiments were performed in duplicate. Gelatinase and hemolytic activity The LAB isolates were grown on MRS agar (Thermo Scientific™ Oxoid™, MA, USA) at 35.0±0.5ºC for 48±2 h. For the gelatinase test, one colony from each isolate was inoculated into nutritive gelatin tubes and incubated for 7 days at 35.0±0.5 ºC. Every 48±2 h, tubes were placed in an ice bath for 15±2 min and observed for gelatin hydrolysis. Isolates were considered gelatinase negative if the gel remained solid after 7 days of incubation, or positive if there was hydrolysis (Klamm, 2019). The experiment was performed in duplicate. S. aureus ATCC 25923 and E. coli ATCC 25922 were used as positive controls, and an uninoculated tube as a negative control. For the hemolysis test, 0.5 McFarland standard suspension was prepared for each isolate in 0.1% sterile peptone water. The suspensions were streaked as pure cultures on Columbia agar with 7% sheep blood and incubated at 35°C for 48 h (Maasjost et al., 2019). Color changes in zones on the blood agar indicated hemolytic activity: green zone (α-hemolysis), light zone (β-hemolysis), and no color change (γ- hemolysis). Two replicates were performed. S. aureus ATCC 25923 was used as a positive control (Aziz et al., 2021). Bioprotective effect of Lactiplantibacillus pentosus against Salmonella sp. in yogurt Pathogen inoculation Five Salmonella strains (S. enterica serovar Typhimurium 93, S. enterica 750, S. enterica 80, and Salmonella DA36) were grown individually in TSB at 35.0±0.5ºC for 24±2 h. Stationary phase cultures were then mixed in equal proportions. From the initial Salmonella sp. cocktail decimal dilutions were made to obtain a population of 7 log CFU/mL. Finally, 1 mL was inoculated into 1 L of yogurt targeting an initial pathogen population of 4 log CFU/mL. Lactiplantibacillus pentosus inoculation L. pentosus_58(6)-2I was grown for 24 h in MRS broth at 35±0.5ºC. The inoculum was added to the yogurt (20 mL for 2 L of product) for an initial population of 6-7 log CFU/mL. Yogurt manufacture and inoculation A formulation of 95% skim milk and 5% skim milk powder was used. The mixture was pasteurized at 90±2ºC for 10 min and then cooled to 43-44 ºC. The commercial culture Yo-Fle (CHRHANSEN, Hørsholm, Denmark), (Streptococcus thermophilus and Lactobacillus bulgaricus), was added in the amount recommended by the supplier. The yogurt was divided into four portions and used in the following treatments: i) uninoculated yogurt; ii) yogurt inoculated with the Salmonella sp. Cocktail; iii) yogurt supplemented with 6 log CFU/mL of L. pentosus_58(6)-2I; and iv) yogurt supplemented with 6 log CFU/mL of L. pentosus_58(6)-2I and Salmonella sp. All treatments were incubated at 41 ºC in 8 oz containers until a pH of approximately 4.5 was reached. The assay was performed in triplicate. Microbiological analysis Treatments were sampled hourly during 6 h of fermentation. Decimal dilutions of each sample were made in 0.1% phosphate saline solution (PSA). LAB counts [including L. pentosus_58(6)-2I] were performed with the 3M Petrifilm method whereas Salmonella sp. was quantified on xylose- lysine/deoxycholate agar (Thermo Scientific™ Oxoid™, MA, USA) using the spread plate technique. Plates were incubated at 35 ºC for 48±3 h. The yogurt pH was monitored in the four treatments to observe the effects of Salmonella sp. or L. pentosus_58(6)-2I on the acidification curve during fermentation. The pH of the uninoculated yogurt, and of the yogurt with added L. pentosus was measured every 30 min using a HI2002-01 edge pH meter (Hanna Instruments, Woonsocket, RI, USA) equipped with a HI10530 electrode (Hanna Instruments, Woonsocket, RI, USA). The pH of the treatments inoculated with Salmonella spp. was measured every hour. The uninoculated yogurt’s moisture, ash, protein and sodium contents were determined using standard AOAC International methods (AOAC International, 2012). Fat content was determined in yogurt as previously described (Carpenter et al., 1993), and carbohydrate content was determined by calculation. Statistical analysis An analysis of variance (ANOVA) was performed to determine differences between the growth or death curves (log CFU/mL) at time 0 and 6 h of L. pentosus_58(6)-2I and Salmonella sp. in the yogurt treatments. ANOVA was also performed for the acidification curves (pH values at time 0 and 6 h). A significance level of 5% was established, with values of p<0.05 considered significant. When significant differences were identified, an honestly significant difference (HSD)-Tukey multiple comparison of means test was performed to determine the difference between treatments. Results At least 10 different species of LAB were identified from the agro-industrial wastes (Table 1 and Supplementary Table 1). Out of 43 isolates obtained from culture, 17 showed some degree of antagonistic activity against at least one of the tested pathogens. However, just 14 isolates were selected for further trials based on their antimicrobial effect against at least two pathogens (inhibition diameter larger than 6 mm). The only exception was L. argentoratensis_79(4)-2C (Table 1). For antibiotic resistance, all the selected LABs were resistant to vancomycin. L. paracasei subsp. tolerans strains were susceptible to tetracycline but they were resistant to streptomycin, chloramphenicol, erythromycin and penicillin whereas the isolates L. plantarum_17-(4D), L. plantarum subsp. plantarum_71-6(2F), L. argentoratensis_57(7)-1H, and L. argentoratensis_79(4)- 2C were resistant to ciprofloxacin (Table 2). In the case of hemolytic and gelatinase activity, L. paracasei subsp. tolerans (2A2-B, IA2P, II-CI-C Y 11-C1-B) and L. casei ATCC 393 did not produce beta hemolysis and were negative for gelatinase activity (Table 3). Biopreservative effect of Lactobacillus pentosus during yogurt processing Based on the previous results, L. pentosus_58(6)-2I was selected as a potential biopreservative for yogurt. The nutritional profile of the yogurt used is summarized in Table 4. Supplementary Figure 1 shows the pH of the four yogurt treatments during fermentation. The acidification curves were consistent with the profile provided by the reference starter culture after 6 h of fermentation at 41°C. There were no significant differences among treatments (p=0.338). Total LAB counts differed significantly (p=0.010) among three of the treatments (yogurt inoculated with L. pentosus and Salmonella sp., yogurt inoculated with Salmonella spp., and uninoculated yogurt) after 6 h of fermentation. Specifically, there were differences in bacterial counts after 6 h of fermentation, between yogurt inoculated with L. pentosus and Salmonella sp. and yogurt with Salmonella sp. (p=0.008) (Supplementary Figure 2). However, these two treatments did not differ from the control (uninoculated sample) (p=0.538 and p=0.108, respectively). As expected, the initial LAB population was higher in yogurt inoculated with L. pentosus and Salmonella sp. than in yogurt inoculated with Salmonella. However, the LAB population stabilized after 2 h of fermentation and remained constant until the end of the process. This was consistent with the acidification curves since the pH values did not change with the addition of L. pentosus (Supplementary Figure 1). Salmonella sp. survival in yogurt inoculated with L. pentosus_58(6)-2I was significantly lower (p=0.019) compared to the control (Supplementary Figure 3). However, the HSD-Tukey test did not show differences between pathogen populations at times 0 and 6 in either of the treatments (p=0.331 and p=1.00, respectively), and differences between the two treatments at time 6 h were not significant (p<0.05). There was a pathogen reduction of 0.611 log CFU/g in the treatment with L. pentosus_58(6)-2I and 0.017 log CFU/g in the negative control. The Salmonella population increased during the initial stage of fermentation (after 2 h of fermentation in the positive control and after 3 h in the negative control). However, after a longer period of fermentation, this population decreased, especially in the presence of L. pentosus_58(6)-2I. Discussion and Conclusions L. paracasei frequently exhibits broad-spectrum antimicrobial activity with simultaneous inhibitory effects against L. monocytogenes, E. coli, S. aureus, and Salmonella (Akpinar and Yerkliyaka, 2021), that is related with the production of antimicrobial compounds such as organic acids, bacteriocins and exopolysaccharides (Amini et al., 2022). The antagonistic activity of L. argentoratensis is closely related to L. plantarum and it was recently classified as a new species (McFrederick et al., 2018). Literature about the antimicrobial capacity of this species is relatively scarce; however, some studies have confirmed the antimicrobial capacity of some isolates against Gram positive and Gram negative bacteria (Siangpro et al., 2023). Recent advances in whole genome sequencing of L. argentoratensis are providing insights about the potential of this species as a biocontrol agent (Syrokou et al., 2021). Vancomycin resistance found in this research was similar to previous reports (Guo, 2017). This resistance is intrinsic in nature and is given by the vanX gene which codes for the dipeptide ligase enzyme (Ddi) (Guo, 2017; Zhang et al., 2018), and transfer to foodborne pathogens is not expected (Álvarez and Poce, 2018). LAB normally have more than 70% resistance to amynoglicosides (gentamicin and streptomycin) and ciprofloxacin, and low resistance to penicillin, tetracycline and chloramphenicol. Variability in antibiotic resistance among species may be related to intrinsic traits. For example, more than 68% of Lactobacillus species are resistant to ciprofloxacin due to the gyrA gene. The tet(M) and erm(B) genes of L. paracasei confer resistance to tetracycline and erythromycin (Guo et al., 2017). Bacteria that produce total hemolysis in agar may contribute to anemia, inflammation, and edema, mostly due to decreased iron availability (Rastogi et al., 2021). Therefore, non-hemolytic LAB strains are considered safer for food applications. Some isolates from this study were classified as partial- hemolytic strains; however, this trait is normal in Lactobacillus and it is attributed to the generation of hydrogen peroxide (Aziz et al., 2021). Also, no gelatinase activity was found in Lactobacillus (Aziz et al., 2021) due to its low capacity to hydrolyze tissue components. This feature supports that LAB strains are safe for food applications (Hashem et al., 2020). The pathogen reduction observed in this study in the presence of L. pentosus_58(6)-2I was greater than the decrease in Salmonella sp. due to the effect of low pH reported by Savran et al. (2018a). Other mechanisms not studied here that may explain the pathogen reduction include the synthesis of biosurfactant compounds, bacteriocins, and hydrogen peroxide, which have in vitro inhibitory effects against Salmonella sp. (Liu et al., 2018). Moreover, the bioprotective effect of L. pentosus against Salmonella sp. was demonstrated by Motahari et al. (2017) using another L. pentosus strain. Further analyses are required to elucidate the causes behind the greater decrease in Salmonella spp. in the presence of L. pentosus_58(6)-2I. The effect of a higher load of L. pentosus_58(6)-2I on Salmonella sp. survival during yogurt fermentation should be tested. If effective, this approach may be suitable if the sensorial properties of yogurt and acidification curves are not affected, and consumer acceptance is not compromised. L. pentosus_58(6)-2I should also be evaluated in other foods such as dairy products and fermented meats. 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Zhang S, Oh J, Alexander L, Ozcam M, van Pijkeren J, 2018. d-Alanyl-d-alanine ligase as a broad- host-range counterselection marker in vancomycin-resistant lactic acid bacteria. J Bacteriol 200:e00607-17. Online supplementary material Supplementary Table 1. Inhibition halo of Salmonella enterica, Listeria monocytogenes, Staphylococcus aureus 29213 and Escherichia coli 25922 grown on culture media pre-inoculated with different lactic acid bacteria strains isolated from agro-industrial waste. Supplementary Figure 1. pH values during fermentation of yogurt subjected to different inoculation treatments (means, error bars show the standard deviation for n=3). Supplementary Figure 2. Lactic acid bacteria count during fermentation of yogurt subjected to different inoculation treatments (means, error bars show the standard deviation for n=3). Supplementary Figure 3. Salmonella sp. counts during fermentation of yogurt subjected to different inoculation treatments (means, error bars show the standard deviation for n=3). Table 1. Inhibition halo of Salmonella enterica, Listeria monocytogenes, Staphylococcus aureus 29213 and Escherichia coli 25922 grown on culture media pre-inoculated with selected LAB strains isolated from agro-industrial waste. 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 ++ ++ ++ + + Inhibition zone 0- 3 mm in diameter (weak), ++ inhibition zone 3- 6 mm in diameter (good), +++ inhibition zone larger than 6 mm in diameter (strong). MFC=microbial fuel cells. Table 2. Antibiotic resistance/susceptibility of selected isolates from agro-industrial waste. Isolate Antibiotic (concentration) A m ox ic ill in w ith cl av ul an ic a ci d (3 0 μg ) St re pt om yc in (1 5 μg ) C hl or am ph en ic ol (3 0 μg ) G en ta m ic in (1 0 μg ) Er yt hr om yc in (1 5 μg ) Te tra cy cl in e (3 0 μg ) C ip ro flo xa ci n (5 μg ) V an co m yc in ( 30 μg ) Pe ni ci lli n (1 0 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 S, susceptible; R, resistant; I, intermediate. Table 3. Results of hemolytic activity and gelatinase activity to evaluate the probiotic profile of selected lactic acid bacteria isolated from agroindustrial waste. 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). Table 4. Nutritional composition of yogurt. 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.