LACTOBACILLUS STRAINS FROM APPLE AND FERMENTED MILK AND THEIR PROBIOTIC PROPERTIES

ABSTRACT


INTRODUCTION
It is noteworthy that, in the process of evolution, humans have made radical changes in their diet; in particular, a new trend of the XXI century has emerged in the form of probiotics-based foods and beverages (Biswal et al., 2021), which are primarily intended for lactose intolerant and vegetarian consumers (Samedi and Charles, 2019).Probiotic and prebiotic non-dairy products have a great marketing future, as recent studies have shown that strains have adapted well to alternative matrices when incorporated into juices (Aspri et al., 2020).In addition, 65-70% of the world's population faces to lactose intolerance challenge (Bayless et al., 2017).Finally, based on the amount of production, the top three most popular fruits in the world include apples, which underlines their importance from an economic point of view (Shahbandeh, 2021).Lactic acid bacteria (LAB), especially Lactobacilli, are considered to be plantassociated bacteria (Lamont et al., 2017), and LAB are often isolated from fruits and vegetables (Yu et al., 2019).One of the species of lactic acid bacteria, Lactiplantibacillus (Lpb.)plantarum (previously named Lactobacillus plantarum; Zheng et al., 2020) can be used as an interesting probiotic candidate (Fidanza et al., 2021).For instance, in one study, L. plantarum and L. casei were isolated from fruit residues such as banana leaf and stem, pineapple peel, and papaya peel (Yang et al., 2016).There has also been increased interest in autochthonous lactic acid bacteria in table olives, with 197 reports having been published worldwide over the last two decades.Among the LAB, Lactobacillus is the most common genus to exist in olives (Portilha-Cunha et al., 2020).Besides, Leuconostoc mesenteroides subsp.mesenteroides LB7 was found on the surface of the apple (Ngea et al., 2021).In addition, several studies have been conducted on the isolation and characterization of Lactobacillus strains from plant leaf surfaces (Samedi and Charles, 2019).Three new species of Lactobacilli (Lactobacillus micheneri, Lactobacillus timberlakei, and Lactobacillus quenuiae) were isolated from flowers (Abutilon sp.) and wild bees (McFrederick et al., 2017;2018).These bacteria are involved in food digestion, stimulate the immune system, and have the ability to resist pathogenic microorganisms in the gastrointestinal tract (Lorizzo et al., 2020).In general, a common characteristic of both plant-associated and other LABs is the synthesis of large amounts of lactic acid and acetic acid via fermentation (Tyler et al., 2016).From a biochemical point of view, it is important to consider these aspects, as the selection of LAB for the enrichment of apple juice can be based on technological, sensory, or nutritional criteria (Di Cagno et al., 2013Cagno et al., , 2015)).The survival of LAB while moving through the digestive tract plays an essential role in revealing their probiotic properties (Yang et al., 2019).The probiotic strain should be resistant to small intestinal bile salts at a concentration of approximately 0.3%, as well as resistant to the gastric acid environment, especially its low pH (1.0-3.0)(Mainville et al., 2005).Among the selected criteria of probiotic properties, antimicrobial activity against pathogens is also important (De Vries et al., 2006).It should be noted that Lactiplantibacillus (Lpb.)plantarum is especially distinguished by these properties (Garcia-Gonzalez et al., 2021).According to a study by Tao et al. (2021), the use of a plant substrate fermented with Lactobacillus plantarum instead of antibiotics may have potential against foodborne infections caused by pathogens.The dependence of lactic acid bacteria on antibiotics is an area of interest as well.Moreover, the significant characteristics of starter or probiotic cultures depend on the strains themselves.Therefore, studies have aimed to discover a new starter culture, which, in turn, is based on screening LAB strains from various sources (Barbosa et al., 2015;

Rzepkowska et al., 2017).
There is a great diversity of plant species and cultivars, including the fruits in eastern and western parts of Georgia, suggesting them as an interesting and rich source of LAB.The aim of this study was to isolate Lactobacillus sp. from the fruits of different apple varieties from these regions of Georgia to obtain pure cultures, conducting molecular identification, and identify potential probiotic properties.All isolated Lactobacillus sp. were selected according to their probiotic characteristics such as resistance to low pH and various concentrations of bile salts; moreover, they were tested based on several parameters, including susceptibility/resistance to antibiotics and antibacterial activity against food pathogens.The selected strains of lactic acid bacteria will be used in the future as starter cultures for the fermentation of apple juices.An important link in this process is the fact that most probiotic foods are based on dairy products because of historical and technological reasons, as well as the nutritional value of milk (Nguyen et al., 2019), but the final product, apple juice enriched with probiotics, will follow the recommendations of the health authority on the one hand focus on plant-based foods, on the other hand, will meet the needs of consumers, in terms of healthy food intake and dietary needs, such as vegetarianism and production of lactose-free beverages (Pontonio et al., 2020).

Isolation of lactic acid bacteria
Lactic acid bacteria were isolated from the apples using a serial dilution method.The peels of three to four fruits of each variety (collected from one tree) were crushed.Then, 10 g of the grinded skin of the sample was homogenized in a mortar, transferred into a sterile bag (Whirl-Pak Stand-Up Bags), and was suspended in 90 ml buffered peptone water (Oxoid, Milan, Italy).After thorough mixing under aseptic conditions, 1 mL of the 10 -1 dilution was serially diluted using nine 9 mL of pre-sterilized physiological saline and this was continued until the required dilution.Additionally, 0.1mL aliquots from each dilution were used as an inoculum and plated onto sterile MRS agar (De Man et al., 1960) containing cysteine hydrochloride (0.05 g/100 mL) to inhibit fungi, yeast, and Gram-negative microorganisms, also to keep low the redox potential of the medium (Hartemink et al., 1997).The Petri plates were incubated anaerobically using a candle jar for 24 h at 37 o C (De et al., 2016).The cultures were sub-cultured on MRS agar to obtain pure isolates.The first isolate identification was made based on meeting the characteristic of LAB on agar: a white/cream or yellow color (Theingi et al., 2019) and round shape.Further Gram staining was done, and the cell morphology of the presumptive Lactobacillus sp. was observed using a light microscope and catalase reaction.For these studies, the isolates were grown on MRS agar under appropriate conditions.The Gram staining was conducted using a single colony of each LAB isolate, following the standard protocol for Gram staining while catalase reaction was performed on a sterile microscope glass slide with drops of 3% hydrogen peroxide onto the selected colony, as mentioned by Nanasombat et al. (2012).Only Gram-positive bacterial isolates (cocci or rods) that were catalase-negative were presumptively identified as LAB.The pure cultures were maintained at -20°C in glycerol stocks for further studies.Except the lactic acid bacteria isolated from apples, in the study were also included the samples previously isolated from the fermented milk for comparison: Levilactobacillus brevis (10,15,18,49,51) Lactiplantibacillus pentosus (40,57,63,85,88,92), and Lactobacillus fermentum (44) (Bokulich et al., 2015).

The determination of optimal growth temperature
The selected bacterial isolates were grown in MRS broth at 30, 37, and 40 0 C for 48-72 hrs.Then, 0.1ml inoculum (10 8 CFU/mL) was transferred to MRS plates by pour plate method and incubated at 37 0 C for 48hrs.The growth of apple and fermented milk origin LAB on MRS agar plates was used to designate isolates as temperature tolerant (Tambekar and Bhutada, 2010).

Glucose Fermentation
Phenol red broth was used as the medium in this test.Then, 1% of sugar substrate, such as glucose was added into each phenol red broth tubes.Durham tubes were added into each phenol red tubes for the purpose of gas bubbles detection.Sterilized phenol red broth tubes were inoculated (10 8 CFU/mL) with active LAB isolates of apple and fermented milk origin (uninoculated tubes was kept as control) and incubated for 24 hours at 37℃.Changes of phenol red broth from red to yellow indicated positive reaction (Rhaiem et al., 2016).

Molecular Identification of LAB Isolates
DNA was extracted from an overnight culture in 500 µl MRS broth at 37 °C using the OxGEn Gram-positive DNA Purification Kit according to the manufacturer's instructions.DNA concentration and purity were determined via absorbance using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) with 260 nm/280 nm absorbance ratios.In the present study, a Lactobacillus genusspecific primer was used to amplify the 16S rRNA gene region of Lactobacilli DNA: IDL04F 5'-AGGGTGAAGTCGTAACAAGTAGCC -3', and IDL03R 5'-CCACCTTCCTCCGGTTTGTCA -3' ( Kwon et al., 2004).All the reaction mixtures were amplified in a GenePro thermocycler PCR system (Bioer, China).The reaction mixture 25 µl 1X Taq Master Mix contained 17 µl of deionized water, 2.5 μL of 10X PCR buffer, 2.5 µl MgCl2, 0.5 µl of primer, 0.5 µl dNTP, 1 U/µl Taq DNA polymerase (Solis BioDyne, Estonia), and an extract containing an equal amount of DNA in 1 μl.The same amount of Levilactobacillus brevis 15 DNA isolated from fermented milk was used for positive control (Bokulich et al., 2015); the negative control contained only 1 μl deionized water.The cycling conditions were as follows: an initial heating of 95 °C for five minutes, followed by 35 reaction cycles of denaturation at 95 °C for 30 s, annealing at 48 °C for one minute, extension at 72 °C for two minutes, a 10 min final incubation at 72 °C, and a 4 o C hold.PCR fragment analysis was performed in 1% agarose gel after staining with ethidium bromide in 1 x TAE buffer; visualization was performed with a transilluminator (Vural and Ozgun, 2011).Initial species identification of the potential probiotic bacteria was done by intact cell MALDI-TOF mass spectrometry (ICMS).ICMS was done by standard procedures recommended for the MALDI Biotyper system (Bruker Daltonics, Bremen, Germany).For analysis, 600 spectra from 2-20 kDa were gathered in 100-shots steps on an Autoflex III system and added up.Results with MALDI Biotyper identification score values ≥2.000 were considered correct.After the ICMS results which yielded only a group of microbial species, 16S rDNA sequencing was additionally performed.For amplification of a high conserved 899 bp region of the 16SrRNA gene, the following primers were used: 27f: 5'-AGAGTTTGATCMTGGCTCAG-3' and 926r: 5'-CCGTCAATTCCT TTRAGTTT-3'.DNA isolated from samples 52, 53, 74, 76 were used as a matrix for PCR amplification of fragments (The method of DNA extraction is described above).Amplicon purification was performed using the ExoSAP-IT™ PCR Product Cleanup Reagent (Applied Biosystems, Waltham, Massachusetts, USA).Asymmetric PCR was performed using the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Waltham, Massachusetts, USA) and amplicon purification was performed using the DyeEx 2.0 Spin Kit (Qiagen, Hilden, Germany) followed by sequencing in a 3130xl Genetic Analyzer (Applied Biosystems, Waltham, Massachusetts, USA).The sequences obtained were analyzed using NCBI BLAST Search.

Resistance to Low pH
Resistance to pH was examined using the method described by Tambekar and Bhutada (2010) and Liong et al. (2005) with certain modifications.The method is used to evaluate the viability of the cells under acidic stress.For this purpose, in sterile tubes, inoculation of MRS broth acidified with 1N HCl to pH 2.0 was performed with 16-18 hour cultures of lactic acid bacteria.After 0-, 30-, 60-, and 90-min incubation periods, 100 μl of each culture from the broth was spread on MRS agar and incubated at 37 °C under anaerobic conditions for 24 h to determine the viable cell.The growth of LAB on MRS agar was used to designate isolates as pH tolerant (Tambekar and Bhutada, 2010; Liong et al., 2005).MRS broth without acidification was used as a positive control.pH of MRS broth was 5.7.

Bile Salt Tolerance
The ability of the isolates to tolerate bile salts was studied according to the method described by Maragkoudakis et al. (2006) and Tambekar and Bhutada (2010), but with additional incubation time periods.16-18 h grown cultures were added to the MRS broth with varying concentrations of bile salts (0.3%; 0.5%; 1% and 1.5%), and incubation was carried out for 2 h, 4 h, 24 h, and 48 h.Then, 0.1ml inoculums was transferred to MRS agar via the pour plate method and incubated at 37 °C for 24 h under anaerobic conditions.The bile salt tolerance of selected isolates was determined according to LAB culture growth on agar plates.Bile saltfree MRS broth was used as a positive control for this experiment (Tambekar and Bhutada, 2010; Maragkoudakis et al., 2006).

Antimicrobial Activity
The antimicrobial activity of the screened LAB isolates was tested against Salmonella enterica ATCC 14028; Klebsiella pneumoniae ATCC 13833; Bacillus cereus ATCC 10876; Proteus mirabilis ATCC 12453; Streptococcus pyogenes ATCC 21059; Enterococcus faecalis ATCC 29212; Pseudomonas aeruginosa ATCC 27853; Staphylococcus aureus ATCC 25923; Shigella flexneri ATCC 12022; and Escherichia coli ATCC 25922 using the agar diffusion method (the so-called method of agar blocks) (Egorov, 1965).Mueller-Hinton agar (Biolife, Italy) plates were swabbed with the pathogenic cultures.Freshly prepared pure cultures of 100 μl LAB isolates were first incubated on MRS agar for 24 h at 37 °C under anaerobic conditions.From the grown isolates, agar blocks were cut and transferred to Mueller-Hinton agar Petri dishes containing the above-mentioned target cultures.The diameter of the agar blocks was 7 mm.The plates were aerobically incubated at 37 °C for 24 h.The inhibition activity of LAB isolates was evaluated by measuring inhibition zone around their blocks.Isolates with an inhibition zone greater than 10 mm are considered to have antimicrobial activity (Menberu et al., 2021).The paper disk soaked with sterile distilled water was used as the negative control.In this case, bacteria grow on the agar without any inhibition.

Statistical Analyses
Results were expressed as mean ± standard deviation (SD) of three independent experiments.Statistical analysis was carried out using one-way ANOVA and Tukey's HSD tests.One-way analysis of variance (ANOVA) was done to analyze the variation of the means between the experimental samples.Tukey's HSD test was used to differentiate between the mean values.All the analyses were done using XLSTAT (free trial version 2022, Addinsoft, Inc., Brooklyn, NY, USA).p value < 0.05 was considered statistically significant.

Collection of apple varieties
In the present study, lactic acid bacteria were isolated from both ripe and unripe apple samples collected from two regions (eastern and western) of eco-biodiverse Georgia featuring different soil-climatic zones.Table 1 provides a list of the apple samples collected, and obtained LAB isolates, used in the study.(Aleksidze, 2015) no isolate Georgian Sinap (Local) (Goginava and Khidesheli, 2019) 53 ( 1 Isolates obtained from the aforementioned apple varieties throughout Georgia had both cocci and rod shapes upon microscopy.Four of them with milk isolates were Gram-positive, catalase-negative, and had an optimal growth at 37 °C, indicating that they were lactic acid bacteria.From apple samples, only four isolates (cell shapeshort bacilli) had small (1-3 mm) round, white or yellowish colonies on the Petri plates.Along with the general characteristics of the LAB, they expressed the feature of facultative anaerobes with respect to oxygen.According to glucose fermentation, four isolates of apple origin (Lpb.plantarum 52, Lpb.plantarum 53, Lpb.plantarum 74, and Lpb.plantarum 76) represent homo fermenter cultures since only lactic acid was produced from glucose and no gas or ethanol production was observed.In the case of milk isolates, all of them were heterofermentative.

Identification of Isolates of Lactic Acid Bacteria
Pure isolates obtained from apple peels were identified by genus-specific PCR.A PCR amplicons electropherogram is represented in Fig. 1.
As is seen in all four isolates, as in the positive control, the 1500 bp fragment was amplified, which indicates that all four experimental isolates belong to the genus Lactobacillus.Intact cell MALDI-TOF mass spectrometry (ICMS) analysis showed that three Lactobacillus isolates (52, 53, and 76) belonged to Lactiplantibacillus pentosus or Lpb.plantarum or Lpb.paraplantarum and Lactobacillus isolate 74 is identified as Levilactobacillus brevis.For specification of the results, 16S rDNA sequencing was additionally done.Based on sequence analysis of the purified amplicons, all of these isolates appeared to be Lactiplantibacillus plantarum, which was confirmed by BLAST analysis of the sequence results.As the Table 2 shows, the isolates are 99.88% -100%, identical to the Lactiplantibacillus plantarum strain in the database (Table 2).

Tolerance of Lactic Acid Bacteria to Acidic Conditions and to Bile Salt Concentrations
In present research, almost all selected LAB isolates were able to grow at pH 2.0 except for two strains of milk origin, L. brevis 10 and L. brevis 51.L. brevis 10 completely lost its resistance after 60-90 min incubation time while L. brevis 51 did not show acid tolerance during any of the incubation periods (please see table 3).All of the isolates were also able to survive in 0.3, 0.5, 1.0, and 1.5% bile salt after 4h incubation time, while after 24h no growth of any isolate was observed in 0.5-1-1.5% bile salt as shown in Table 3.

Antibiotic Resistance / Susceptibility
Antibiotic Resistance results of the selected Lactobacillus sp.isolates are shown in Table 4.All of them were found to be resistant to ciprofloxacin and streptomycin.In the case of erythromycin, all selected Lactobacillus strains showed susceptibility except the Lpb.pentosus 57, isolated from the fermented milk.

DISCUSSION
In the present study, lactic acid bacteria were isolated from both ripe and unripe apple samples collected from two regions (eastern and western) of eco-biodiverse Georgia featuring different soil-climatic zones (Table 1).Although the apple orchards were 150-200 km apart, similar cultures were isolated from the same apple species, namely Lactiplantibacillus plantarum 52 from Lechkhumuri Sinapi (Village Akhalsopeli, Ambrolauri Municipality), Lactiplantibacillus plantarum 53 from Georgian Sinap (Village Rekha, Gori Municipality) Besides, Lactiplantibacillus plantarum 74, 76 were obtained from Kekhura (Village Karaleti).LAB are a natural resident on the surface of fruits; therefore, they represent an important source of probiotics.In recent years, many scientists have studied fruits in this direction.According to Until now, there was no available information regarding the microbiota on the surface of apple varieties in Georgia.The main objective of this study was to characterize the important parameters of lactic acid bacteria isolated from apple samples and to select Lactobacillus sp. as a starter culture with the purpose of juice enrichment.Di Cagno et al. (2013) suggested using autochthonous starter cultures of fruits and vegetables, which mainly include lactic acid bacteria.This may be due to the fact that they have the ability to extend the shelf life, as well as to improve the nutritional (synthesis of exo-polysaccharides, increased antioxidant activity) and sensory characteristics (synthesis of aromatic compounds).Due to the great health benefits of probiotics, their use as a way to improve the functional properties of food has an increasing trend (Afrin et al., 2021).It should also be noted that a good nutritional rate in turn plays an important role in maintaining the normal functioning of the body and preventing dysfunction caused by internal or external factors (Muscaritoli, 2021).
In the present study, lactic acid bacteria were isolated from both ripe and unripe apple samples collected from two regions (eastern and western) of eco-biodiverse Georgia featuring different soil-climatic zones (Table 1).Although the apple orchards were 150-200 km apart, similar cultures were isolated from the same apple species.According to the characteristics described by Holt et al. (1994), lactic acid bacteria belonging to the genus Lactobacillus must be Gram-positive, rod-shaped, and catalase negative as well as have the ability to break down sugars and produce acid.LAB should also be tolerant of pH, temperature, and bile salts.
Based on these parameters, as well as on the amplification fragment of 1500 bp detected by gel electrophoresis, and comparison with the positive control (Levilactobacillus brevis) we can summarize that four of the 20 isolates belong to the genus Lactobacillus.Moreover, based on sequence analysis of the purified amplicons, all of these isolates appeared to be Lactiplantibacillus plantarum.This indicates the presence of Lpb.plantarum on the surface of apple varieties cultivated in Georgia.
First, an important step in the selection of probiotics is the ability to survive in the gastrointestinal tract, as gastric juice is generally considered to be one of the strongest barriers to probiotics, followed by bile acids in the small intestine (Piano et al., 2011), after which probiotics reach their destination and are able to be metabolized in the human body and have a beneficial effect on the "host" (Charteris et al., 1998;Argyri et al., 2013).It should be noted, however, that not all probiotic strains can survive in sufficient numbers to reach the gut microbiome, and even if they do, they can leave the body soon after (Hughes, 2020).
Cell viability at different concentrations of pH and bile salts was determined by increasing the intensity of growth on the MRS agar during a 24-hour incubation period at 37 °C (De et al., 2016).According to the results, all Lactobacillus isolates except for L. brevis 10, and 51, were found to have growth at pH 2 (Table 3).Milk and apple Lactobacillus samples were distinguished based on their tolerance to bile salts.All isolates were found to have the ability to grow on MRS agar containing a 0.3% concentration of bile salts during 4 h incubation period.Lactobacillus sp. were considered resistant to this concentration.As Table 3 shows, according to these two parameters, isolates obtained from similar species had similar results, in the example of Lactiplantibacillus plantarum 52 from Lechkhumuri Sinapi and Lactiplantibacillus plantarum 53 from Georgian Sinap.This characteristic play one of the most important roles in probiotic properties since it is known that the retention time of food in the small intestine is four hours and the value of intestinal bile concentration on average is considered to be 0.3% (0.3 kg per 100 ml) (Prasad et al., 1998).It is also noteworthy that similar findings have been obtained by other researchers.Abouloifa et al. (2020) studied Lactobacillus strains isolated from naturally fermented Moroccan green olive brine.All of them showed high resistance up to 2% of bile salts.
In addition to pH and bile salt tolerance, in this work, we also studied various characteristics of probiotic bacteria, in particular resistance to antibiotics and antimicrobial activity.The safety of using strains as probiotics should be evaluated according to their antibiotic susceptibility profile and antibiotic resistance.The resistance of probiotic strains to some antibiotics can be used for both prophylactic and therapeutic purposes in the control of intestinal infections.Moreover, resistance of the Lactobacillus sp. to antibiotics underscores their potential to reduce the negative effects of antibiotic therapy on the bacterial ecosystem of the host organism (El-Naggar, 2004).
A study of antibiotic dependence showed a diverse picture.

CONCLUSION
The current study aimed to isolate, identify, and study some probiotic properties of lactic acid bacteria from the surface of both local and imported apple varieties throughout Georgia.Of the 20 isolates, four met the selected characteristics of probiotics.A comparison of the cultures isolated from the apple and fermented milk samples showed a similar picture.In particular, Lpb.plantarum 52, Lpb.plantarum 53, Lpb.plantarum 74, and Lpb.plantarum 76 and all milk isolates except for L. brevis 10, and L. brevis 51 were distinguished by their ability to withstand the pH 2 of the gastric acid environment.All samples were also characterized by their tolerance to bile salts.In our study of antibiotic resistance/susceptibility, all Lactobacillus sp. were found to be susceptible to ciprofloxacin and streptomycin, and approximately the same results could be seen in regard to antimicrobial activity.In particular, the ability to inhibit the growth of Salmonella enterica ATCC 14028 was found in both milk L. brevis 51 and Lactiplantibacillus plantarum 52 of apple.Almost none of the isolated Lactobacillus sp. were characterized by the inhibition of growth against Proteus mirabilis ATCC 12453 and Shigella flexneri ATCC 12022-except for Lactiplantibacillus plantarum 76.Thus, LAB autochthonous apple strains, like the cultures obtained from the milk samples, were distinguished by their tolerance to pH and bile salts and also had a well-defined resistance to selected antibiotics.They can be characterized by their different activity in regard to food pathogens.Therefore, it can be concluded that apples can be considered as source of lactic acid bacteria, suggesting significant technological potential for the production of apple juices with probiotic properties.The selected LAB isolates will be used for future studies, which include investigating the relationship of the apple juice fermentation at different periods and LAB isolates' cell viability.Future research should focus also on the optimization of probiotic cell use, considering key factors such as the effect of fermentation on total phenolic content (TPC), and the total antioxidant capacity, observation of the metabolism of sugars, and organic acids during fermentation.pH and titratable acidity should be studied before and after fermentation as well.
Evaluating these properties will be used for future industrial applications of the selected probiotic microorganisms.
Fessard et al. (2019), three of the 77 isolates of lactic acid bacteria isolated from fruits and vegetables belonged to Lactobacillus plantarum.In addition, Lactobacillus brevis was obtained from the mango fruit by Liao et al. (2016); a strain of Lactobacillus plantarum CM-3 was isolated from strawberry fruit (Chen et al., 2020); Choi et al. (2018) isolated strains of Lactobacillus plantarum from black raspberries and Tosin and Temitope (2018) from banana, orange, and watermelon; Verón et al. (2017) managed to obtain it from Mexican fruits, namely Opuntia ficus-indica.A new strain of lactic acid bacterium, Lactobacillus musae sp., was obtained from banana fruit (Chen et al., 2017).Moreover, a source of Lactobacilli is various pickles, with Prakash et al. (2020) reported in their work about a source of L. fermentum in lemon pickles.On the other hand, Barache et al. (2020) studied blackberries (Rubus sp.), Fresh figs (Ficus carica), and pickled pears (Opuntia ficus-indica), for isolating of lactic acid bacteria such as Lactobacillus plantarum and Lactobacillus paracasei.Also noteworthy is the fact that endophytic LABs are promising resources for the biocontrol of post-harvest spoilage caused by fungus (Chen et al., 2020).In addition, LAB strains have the ability to inhibit several pathogens, including L. monocytogenes, Salmonella, and E. coli O157: H7 (Ayala et al., 2019), which is important in mixed (bacterial) diseases.They can also be used in food production as far as probiotics are non-pathogenic microorganisms, they meet the recommendations of the World Health Organization and EFSA (Fernández-Pacheco et al., 2021) and generally are recognized as safe (GRAS) microorganisms (Stiles and Holzapfel, 1997).

Table 2
Results of a BLAST analysis of 16S rRNA gene sequences from four isolates

Table 3
Tolerance of lactic acid bacteria isolates to pH 2.0 for different incubation periods and viability of Lactobacillus sp. in the presence of different concentrations of bile salts Legend: "+" Growth and "-" No growth/ No survival

Table 4
Resistance of Lactobacillus sp.isolates to antibiotics Values are presented as mean ± SD.Legend: S-susceptible (≥14 mm),I-intermediate (11-13 mm), and R-resistant (≤10 mm)Antagonistic Activity of LAB against Food-Borne Pathogenic BacteriaThe antibacterial activity shown by the selected Lactobacillus sp.isolates is presented in Table5.According to the inhibitory action on pathogens, the results showed that only two Lactobacillus sp.isolates, in particular, fermented milk L. brevis 51 and apple Lactiplantibacillus plantarum 52, had inhibitory activity against Salmonella enterica ATCC 14028.Growth of Proteus mirabilis ATCC 12453 and Shigella flexneri ATCC 12022 were inhibited only by Lpb.plantarum 76, isolated from apple sample.L. brevis 51 and Lpb.plantarum 52, Lpb.plantarum 53, Lpb.plantarum 74 showed low antibacterial activity against Klebsiella pneumoniae ATCC 13833.Only L. brevis 18 was able to inhibit Staphylococcus aureus ATCC 25923, while together with Lpb.plantarum 76 it was moderately active against Klebsiella pneumoniae ATCC 13833.The Lactiplantibacillus plantarum 76 also showed moderate activity as compared to others against Streptococcus pyogenes ATCC 21059 and Escherichia coli ATCC 25922.Almost all Lactobacillus strains showed significant inhibition against Bacillus cereus ATCC 10876, Enterococcus faecalis ATCC 29212, and Pseudomonas aeruginosa ATCC 27853, including 2 Lactobacillus strains (L.brevis 10, L. brevis 15) with strong inhibitory activity.

Table 5
Antimicrobial activity of Lactobacillus sp.isolates

Strain Pathogen Salmonella enterica ATCC 14028 Klebsiella pneumoniae ATCC 13833 Bacillus cereus ATCC 10876 Proteus mirabilis ATCC 12453 Streptococcus pyogenes ATCC 21059 Enterococcus faecalis ATCC 29212
Legend: Means ± standard deviation (SD) in the table with different alphabet letters indicate the significant difference at p < 0.05.