In vitro evaluation of probiotic potential of Lactobacillus paracasei BIOTECH 10363 strain isolated from Tapuy

Fermented foods can be a source of potentially probiotic microorganisms as they contain different types of lactic acid bacteria. In the Philippines, there are a variety of traditional fermented food products that may harbor probiotics. This study determined the probiotic properties of Lactobacillus paracasei BIOTECH 10363 isolated from Tapuy, an indigenous alcoholic beverage made from fermented glutinous white rice. The acid and bile resistance, antibacterial, cell surface hydrophobicity, auto - aggregation, NaCl tolerance, hemolytic, and antibiotic sensitivity properties of L. paracasei BIOTECH 10363 were evaluated in vitro . The isolate displayed resistance to acid (pH 3 to 5) and bile (0.1 to 0.5%). Its cell - free supernatant exhibited strong inhibitory activity against Escherichia coli . Higher affinity to xylene and chloroform was observed in L. paracasei BIOTECH 10363. This suggests that the cell surface of the isolate has hydrophobic and electron donor (basic) properties. Moreover, the isolate had a strong auto - aggregating phenotype. Cell surface hydrophobicity and strong auto - aggregation activity could enable the isolate to adhere, colonize and survive in the gastrointestinal tract. The isolate could withstand MRS broth with 9% NaCl. It also displayed γ - hemolytic activity in blood agar plates indicating the non - hemolytic nature of the isolate. L. paracasei BIOTECH 10363 expressed sensitivity against various cell wall synthesis inhibitors (amoxicillin, ampicillin, augmentin, penicillin, and cefaclor) and protein synthesis inhibitors (streptomycin, chloramphenicol, clindamycin, erythromycin, and tetracycline). However, it displayed intrinsic resistance toward kanamycin. The findings of this study showed the promising potential of L. paracasei BIOTECH 10363 as a candidate probiotic strain as it exhibited desirable attributes in vitro .


Introduction
In recent years, fermented foods are increasingly recognized as a good source of potentially probiotic microorganisms because they normally contain a wide range of lactic acid bacteria (Rezac et al., 2018).In the Philippines, there are numerous indigenous fermented food products that have been traditionally part of the local diet.These include cheese and fermented foods made from fish and fishery products, meat and sausages, vegetables and fruits, rice, cassava, sugar cane, coconut and soya (Banaay et al., 2013).Tapuy is an indigenous alcoholic beverage made from fermented glutinous white rice and produced mainly in the northern part of Luzon Island, Philippines.On its own, Tapuy has naturally occurring bioactive compounds such as phenolics, flavonoids, and tannins that act as antioxidants and play a role as free radical scavengers (Hipol and Alma-in, 2018).Furthermore, it contains various microorganisms including many beneficial bacteria (Sanico and Medina, 2020).
Probiotics are cultures of live microorganisms which confer health benefits to the host when administered in adequate amounts (Food and Agriculture Organization of the United Nations and World Health Organization (FAO/WHO), 2001).They are a diverse group of microbes that consist of beneficial bacteria, molds and yeast.Consumption of probiotics has been linked to improved health and better overall well-being.Numerous scientific works have described the beneficial role of probiotics in the treatment and prevention of acute RESEARCH PAPER infectious diarrhea, antibiotic-associated diarrhea, acute respiratory tract infections, Clostridium difficile infection, eczema, inflammatory bowel diseases, infant colic and necrotizing enterocolitis (Liu et al., 2018;Sanders et al., 2018).Moreover, many potential health benefits are currently being investigated including the effect of probiotics on depression, anxiety, psychological stress, and other brain functions (Sarkar et al., 2016).The exact mechanisms of action of probiotics have not yet been fully elucidated.However, some key mechanisms that may be involved include competitive inhibition of pathogens, reduction of gut pH, stimulation of mucosal barrier function, inhibition of bacterial adherence or translocation, increasing mucus production, secretion of antimicrobial peptides (bacteriocins/ defensins) and production of volatile fatty acids (Ng et al., 2009;Bermudez-Brito et al., 2012;Kechagia et al., 2013).
Lactic acid bacteria (LAB) are often considered one of the most important groups of probiotic microorganisms (Sadiq, 2022).The most widely used LAB with an application as probiotics belong to the two main genera of Lactobacillus and Bidifobacterium (FAO/ WHO, 2006).Although, microorganisms from other genera are also being used such as Enterococcus, Lactococcus, Leuconostoc, Pediococcus, Sporolactobacillus and Streptococcus (Kailasapathy, 2013).Today, L. paracasei is one of the most frequently utilized probiotics in food, dietary supplements and pharmaceutical products (Fijan, 2014;Stefanovic et al., 2017).Lactobacillus paracasei is a gram-positive, catalase-negative, non-spore-forming, rod-shaped bacterium.It is usually found in milk and dairy products, fermented sausages, vegetables and wine (Gobbetti and Minervini, 2014).
Many previous studies have concentrated on the isolation, identification and characterization of microorganisms in Tapuy.However, there is very limited information available on the probiotic potential of lactic acid bacteria isolated from this local rice wine.Therefore, the aim of this study was to assess the acid and bile tolerance, antibacterial against Escherichia coli, cell surface hydrophobicity, auto-aggregation, NaCl tolerance, hemolytic and antibiotic susceptibility properties of L. paracasei BIOTECH 10363 isolated from Tapuy.

Bacterial strains and culture conditions
An active culture of L. paracasei BIOTECH 10363 was acquired from the Philippine National Collection of Microorganisms (PNCM), National Institute of Molecular Biology and Biotechnology (BIOTECH), University of the Philippines Los Baños (UPLB), College, Laguna, Philippines.The pure culture of the isolate was obtained using the four-quadrant streak plate method (Sanders, 2012).Isolated colonies from the fourth quadrant were picked and subcultured into Lactobacillus de Man, Rogosa, Sharpe (MRS) agar stabs.Inoculated tubes were then incubated at 37°C for 18 to 24 hrs.After incubation, the prepared agar stabs were stored and served as stock cultures.For working stocks, the purified stock culture was activated by two sequential transfers in MRS broth before each use.
For the propagation of the indicator strain, Escherichia coli (laboratory isolate) was streaked in Brain Heart Infusion (BHI) agar slants using a sterilized inoculating loop.Inoculated tubes were then incubated at 37°C for 18 to 24 hrs.After incubation, the prepared agar slants were stored and served as stock cultures.For working stocks, E. coli was propagated (1% v/v) in BHI broth for 18 to 24 hrs at 37°C.All working stocks were kept in the refrigerator at 4°C for storage.

Standardization of inoculum density
Bacterial cells were collected from the working stock of L. paracasei BIOTECH 10363 using a laboratory bench-top refrigerated centrifuge (5,000×g, 10 mins, 4°C ).Cells were washed twice in 10 mL sterile ¼-strength Ringer's solution and then resuspended in the same solution.The resulting suspension was photometrically adjusted to that of a 0.5 McFarland standard (Kumar and Kumar, 2015).

Acid resistance assay
The acid tolerance assay was adapted from the method previously described by Kumar and Kumar (2015) with slight modifications.A volume of the working stock (1 mL) was inoculated into tubes containing modified MRS broth (10 mL) with different pH levels (1, 2, 3, 4, 5 and 7).MRS broth adjusted to pH 7 served as the control.The inoculated tubes were then incubated for 18 to 24 hrs at 37°C.After incubation, the optical density at 560 nm (OD 560nm ) was measured and the acid resistance (%) was calculated using the formula below:

Bile salt resistance assay
The bile salt tolerance of L. paracasei BIOTECH 10363 was assessed using the method previously described by Kumar and Kumar (2015) with slight modifications.A volume of the working stock (0.2 mL) was inoculated into tubes containing modified MRS broth (10 mL) with different concentrations (0.1, 0.3, 0.5, 0.7 and 0.9 % w/v) of ox gall bile salts.MRS broth without ox gall bile salts served as the control.The inoculated tubes were incubated at 37°C for 18 to 24 hrs.After incubation, the optical density at 560 nm was measured and the bile resistance (%) was expressed as the percentage of growth at OD 560nm in the presence of bile salts (0.1 to 0.9%) compared with the control.

Antibacterial activity assay (well-diffusion assay)
The antagonistic effect of L. paracasei BIOTECH 10363 against E. coli was evaluated using a standard agar well diffusion assay.Around 100 μl of the working stock of E. coli was streaked on the Mueller Hinton agar (MHA) plate surface using sterile cotton swabs.Agar wells (6 mm) were then made using sterilized pipette tips.
Cell-free supernatant (CFS) of L. paracasei BIOTECH 10363 was obtained following the method of Saadatzadeh et al. (2013) with slight modification.CFS was separated from the working stock of L. paracasei BIOTECH 10363 using a laboratory bench-top refrigerated centrifuge (4,000×g, 15 mins, 4℃).The supernatants were then filter-sterilized using a Millex ® -GP syringe filter unit with a pore size of 0.22 μm.A portion of the CFS was used to assay bacteriocin production by adjusting the pH to 6.5 while the rest was left with its initial pH.
Around 100 μl of CFS of L. paracasei BIOTECH 10363 were then loaded in each agar well.The plates were kept for 1 hr at ambient temperature for diffusion and incubated for 18 to 24 hrs at 37°C.The diameter of the zone of inhibition (ZOI) was measured and recorded in mm after incubation.The obtained ZOI was interpreted according to Akabanda et al. (2014).

Microbial adhesion to hydrocarbons assay
The microbial adhesion to hydrocarbons (MATH) assay was adopted from the modified method of Ji et al. (2015).Briefly, bacterial cells were collected from the working stock of L. paracasei BIOTECH 10363 using a laboratory bench-top refrigerated centrifuge (5,00×g, 10 mins, 4℃).Cells were washed twice in sterile PBS (phosphate-buffered saline) and then resuspended in the same buffer solution.The optical density at 580 nm (OD 580nm ) of the resulting suspension was determined and referred to as reading 1.Equal volumes of the suspension and solvents (xylene, chloroform, or ethyl acetate) were mixed for 2 mins using a vortex mixer (Uzusio VTX-3000L model, LMS Co., Ltd., Tokyo, Japan).The two phases were left to separate for 30 mins at 37℃.A portion of the aqueous phase was collected, and its optical density was measured at 580 nm (reading 2).The cell surface hydrophobicity was computed using the formula below:

Auto-aggregation assay
Auto-aggregation activity was measured following the method previously described by Melgar-Lalanne et al. (2015) with some modifications.Bacterial cells were collected from the working stock of L. paracasei BIOTECH 10363 using a laboratory bench-top refrigerated centrifuge (5,000×g, 10 mins, 4°C).Cells were washed twice in sterile PBS and then resuspended in the same buffer solution.The optical density at 600 nm (OD 600nm ) of the resulting suspension was adjusted to 0.5±0.01.Around 4 ml of the cell suspension was thoroughly mixed for 2 mins using a vortex mixer.Autoaggregation activity was assessed at 3-and 5-hr incubation at ambient temperature.An aliquot (0.1 ml) was taken from the upper suspension and pipetted onto a 3.9 mL sterile PBS solution.The absorbance of the resulting mixture was determined at 600 nm and the auto -aggregation activity of the tested strain was computed as follows: Where A t = Absorbance at time (t) = 3 or 5 hrs and A 0 = Absorbance at t = 0 hr

Halotolerance assay
A volume (1% v/v) of the working stock of L. paracasei BIOTECH 10363 was inoculated into tubes containing modified MRS broth (10 mL) with different concentrations (1 to 10%, w/v) of sodium chloride (NaCl).MRS broth without NaCl served as the control.The inoculated tubes were then incubated at 37°C for 18 to 24 hrs.After incubation, bacterial growth was examined using the turbidimetric method of Hoque et al. (2010).The growth was interpreted as follows: (-) = no growth; (+) = normal growth; and (++) = maximum growth.

Hemolytic activity assay
Hemolytic activity assay was performed following the modified method of Halder et al. (2017).Sterile and defibrinated sheep blood was procured from the Animal Disease Diagnostic Laboratory, Veterinary Teaching Hospital, College of Veterinary Medicine (CVM), UPLB.The blood was subjected to a sterility test.Briefly, sterile blood agar plates (BAP) were aseptically prepared by mixing blood agar base (infusion agar) and 5% (v/v) sheep blood.The prepared BAPs were then incubated for 48 hrs at 37°C.After incubation, BAPs One loopful of the working stock of L. paracasei BIOTECH 10363 was streaked on the sterile BAP surface.Inoculated BAPs were incubated for 72 hrs at 37°C.After incubation, the plates were then examined for the presence of clear zones (β-hemolysis) or greenhued zones (α-hemolysis) around individual colonies.The absence of such zones in the BAPs indicates γhemolysis.

Antibiotic susceptibility test
The antibiotic susceptibility pattern of Lactobacillus paracasei BIOTECH 10363 was determined semiquantitatively using a standard disc diffusion assay.Briefly, one loopful of the working stock of L. paracasei BIOTECH 10363 was streaked on the MRS agar plate surface using sterile cotton swabs.Using an aseptic technique, antibiotic discs were laid on the surface of agar plates using sterile forceps.The plates were then maintained at 40°C for 1 hr for diffusion and then incubated for 24 hrs at 37°C.After incubation, the diameter of the zone of inhibition (ZOI) around the antibiotic disc was measured and recorded in mm.The results were categorized as resistant (R), moderate susceptible (MS), or susceptible (S) according to the CLSI interpretative standards described previously by Charteris et al. (1998).

Ethical statement
The study did not include any human and/or animal experiments.

Statistical analysis
All experiments were performed in triplicate and the results were expressed as mean ± standard deviation (SD).Data on antibacterial activity, MATH assay, autoaggregation activity, NaCl tolerance assay, hemolytic activity, and antibiotic susceptibility were subjected to descriptive analysis.Data on acid and bile tolerance assays were analyzed using a one-way analysis of variance (ANOVA) in a completely randomized design (CRD) with pH levels and bile concentrations as treatments, respectively.SAS ® (Statistical Analysis System) OnDemand for Academics (SAS Institute Inc., Cary, NC) software was used to conduct all statistical analyses.Scheffe's post hoc test was employed for pairwise comparison of treatment means at a 5% level of significance.

Acid resistance of Lactobacillus paracasei BIOTECH 10363
Table 1 displays the optical density and resistance rate of L. paracasei BIOTECH 10363 in modified MRS broth with various pH levels.The isolate exhibited decreasing optical density and resistance rate with decreasing pH levels.The highest resistance rate of L. paracasei BIOTECH 10363 was observed in MRS broth adjusted to pH5.It differed significantly (P<0.05) from the resistance rate observed in MRS broth adjusted to pH 1, 2, 3 and 4. According to Kumar and Kumar (2015), Lactobacillus species are considered acid resistant when the resistance rate at pH3 is 50% or higher.In this study, L. paracasei BIOTECH 10363 can be labeled as an acidresistant strain since it had a resistance rate of 57.63% at pH 3.However, it was not resistant at pH 1 and 2, implying that the survival of the isolate was greatly reduced at pH values lower than 3. Several acid-resistant strains have also been identified in previous studies including L. paracasei KW3110 (Nishida et al., 2008), L. paracasei CT12 (Romero-Luna et al., 2020) and L. paracasei PCR140 (Akmal et al., 2022).
Low pH can cause changes in the structural and functional integrity of cellular components such as proteins.These changes can disrupt nutrient transport and energy generation, impairing microbial growth (Ray, 2004).Microorganisms employ a myriad of resistance mechanisms when exposed to acidic environments including F 0 F 1 ATPase proton pumps, macromolecule protection and repair, cell membrane modification, amino acid decarboxylation and deamination, metabolic regulation and protection from organelle (Guan and Liu, 2020).In lactobacilli, the acid resistance is mostly attributed to their ability to regulate and maintain a steady gradient between the intracellular (cytoplasmic) and extracellular pH through the extrusion of a proton by F 0 F 1 ATPase proton pumps (Corcoran et al., 2005)

Bile resistance of Lactobacillus paracasei BIOTECH 10363
Table 2 shows the optical density and resistance rate of L. paracasei BIOTECH 10363 in modified MRS broth with different bile salt concentrations.The isolate displayed decreasing optical density and resistance rate with increasing bile concentration.The highest resistance rate of L. paracasei BIOTECH 10363 was observed in MRS broth with 0.1% (w/v) bile salts.It differed significantly (P<0.05) from the resistance rate observed in MRS broth supplemented with 0.5%, 0.7% and 0.9% (w/v) bile salts.However, it was not significantly different from the resistance rate obtained in MRS broth with 0.3% (w/v) bile salts.According to Kumar and Kumar (2015), Lactobacillus species are considered bile resistant when the resistance rate at 0.3% bile salt concentration is 50% or higher.In this study, L. paracasei BIOTECH 10363 can be labeled as a bileresistant strain since it displayed a resistance rate of 88.56% at 0.3% (w/v) bile salts.However, the isolate was not resistant at bile salt concentrations higher than 0.5%.
Bile is extremely toxic to microorganisms, especially to those that are not adapted to intestinal conditions.It causes structural changes in the cell membrane by disturbing the lipid packaging and disrupting the proton motive force, ultimately leading to cell death (Kurdi et al., 2006).Microorganisms use a multitude of bile resistance mechanisms such as the bile-efflux systems, cell membrane and cell wall modification, bile salt hydrolysis, general stress response, protection against oxidative damage and global glycolytic reorganizations (Ruiz et al., 2013).Generally, the bile resistance of lactic acid bacteria is attributed to the active extrusion of the bile acids or salts that accumulate in the cytoplasm using bile-efflux systems.A number of these efflux pumps or transporters have been identified and characterized in lactobacilli (Whitehead et al., 2005;Pfeiler and Klaenhammer, 2009).This system would permit microorganisms to survive and persist in the intestinal tract where they can exert their health benefits to the host.

Antibacterial activity of Lactobacillus paracasei BIOTECH 10363
The inhibitory capacity of L. paracasei BIOTECH 10363 against an indicator microorganism is shown in Table 3.When the cell-free supernatant (CFS) was used, the isolate demonstrated strong inhibitory activity against Escherichia coli.However, this activity was not detected when the CFS was neutralized to pH 6.5.This finding implies that the CFS of the isolate lacks bacteriocin and bacteriocin-like activities and the observed inhibition could be due to the action of organic acids and other antimicrobial compounds.
The antimicrobial activity of L. paracasei strains against E. coli has also been described in previous studies (Islam et al., 2012;Coman et al., 2014;Smetankova et al., 2014).However, the degree of inhibition differs between strains.The antimicrobial activity of lactic acid bacteria has been mostly attributed to the production of acids (e.g., acetic acid, benzoic acid and lactic acid) and other primary metabolites such as acetaldehyde, acetoin, carbon dioxide, diacetyl, ethanol, fatty acids, hydrogen peroxide, reuterin and reutericyclin (Šušković et al., 2010;Shokryazdan et al., 2014).Biologically active peptides with antimicrobial properties may also be produced by other strains (Todorov et al., 2011;Messaoudi et al., 2013).These antimicrobial compounds may beneficially modulate the intestinal ecology by preventing the pathogenic colonization of the gastrointestinal tract.The cell surface properties of L. paracasei BIOTECH 10363 were determined photometrically using the MATH assay (Table 4).The adhesion of microorganisms to xylene, a non-polar solvent, reflects cell surface hydrophobicity.According to Sharma and Sharma (2017), Lactobacillus strains with more than 40% affinity to non-polar solvents are generally more hydrophobic.In this study, the affinity of L. paracasei BIOTECH 10363 in xylene was 82.79%.Furthermore, the isolate had a higher affinity to chloroform (acidic solvent and electron acceptor) than ethyl acetate (basic solvent and electron donor).The results suggest that the cell surface of the isolate has hydrophobic and electron donor (basic) properties.Several highly hydrophobic strains have also been described in previous studies including L. paracasei subsp.paracasei (Dewan and Tamang, 2007) and L. paracasei subsp.paracasei NIRD H831 (Harty and Knox, 1991).The hydrophobicity of bacteria is associated with the presence of proteinaceous and hydrophobic compounds on their cell surfaces (Pelletier et al., 1997).In lactobacilli, a number of these compounds have been characterized including the surface layer (S-layer) proteins and lipoteichoic acid (Krasowska and Sigler, 2014).Cell surface hydrophobicity is an essential property of probiotics as it aids in the adhesion and colonization of these microorganisms to the intestinal mucosa (Xu et al., 2009).

Auto-aggregation activity of Lactobacillus paracasei BIOTECH 10363
The sedimentation rate of L. paracasei BIOTECH 10363 is shown in Table 5.The highest auto-aggregation activity of the isolate was observed after 5 hrs of incubation.According to Wang et al. (2010), strains with activity higher than 40% are strongly auto-aggregating.In this study, the tested strain exhibited a strong autoaggregating phenotype as it had an activity of 89.53%.This finding is in agreement with previous studies which reported the strong auto-aggregation activity of several strains of L. paracasei (Bhagat et al., 2019;Fonseca et al., 2021;Sadeghi et al., 2022).However, the observed activity varies from strain to strain, indicating that autoaggregation is a strain-specific property.
Auto-aggregation is the characteristics of the bacteria belonging to the same strain to clump or selfaggregate together to achieve high cell density (Janković et al., 2012).This property enables probiotics to reach viable counts necessary for their adhesion, colonization, and survival in the oral cavity, gastrointestinal tract, and urogenital tract (Collado et al., 2008;Nikolic et al., 2010).Moreover, it also impedes the adherence of pathogens to the host's intestinal mucosa by forming a barrier via biofilm formation (Schellenberg et al., 2006).Microorganisms intended for probiotic applications should have high cell surface hydrophobicity and strong auto-aggregation activity.

Salt (NaCl) tolerance of Lactobacillus paracasei BIOTECH 10363
Lactobacillus paracasei BIOTECH 10363 was inoculated on MRS agar at 37°C with different concentrations of salt (Table 6).The tested strain had maximum growth in modified MRS broth with 5% NaCl and could withstand up to 9% NaCl.The result agrees with previous studies which reported that many probiotic potential lactobacilli were able to tolerate up to 9% NaCl (Chowdhury et al.,2012;Rahman et al., 2016 (Wood, 2015).These solutes accumulate in the cell cytoplasm and restore the balance between the intracellular and extracellular osmotic pressures (Welsh, 2000).The most common solutes identified in lactic acid bacteria are acetylcholine, carnitine, choline, glycine betaine, choline, carnitine, dimethylsulfonioacetate, and proline (Kets, 1997;Marrec, 2011).The accumulation of these solutes is a prerequisite for the survival of probiotics during osmotic stress.

Hemolytic activity of Lactobacillus paracasei BIOTECH 10363
There were no notable zones around the colonies of L. paracasei BIOTECH 10363 when grown in sheep blood agar.This indicates that the isolate displayed gamma (γ)-hemolytic activity (i.e., no hemolytic activity).Gamma (γ)-hemolytic microorganisms do not usually produce hemolysin, a compound that destroys red blood cells (RBCs) in the blood agar plates (Sohail et al., 2021).The absence of hemolysin guarantees that opportunistic virulence will not occur in the tested strain.The finding of this study corroborates with previous reports which described the gamma (γ)-hemolytic nature of L. paracasei (Le et al., 2019;Huang et al., 2021;Sornsenee et al., 2021).This suggests the non-hemolytic nature of the tested strain.However, other virulence factors such as DNAse, gelatinase, and coagulase activity must be fully examined before the isolate can be used as probiotics in foods and pharmaceuticals.

Antibiotic susceptibility of Lactobacillus paracasei BIOTECH 10363
The antibiotic susceptibility of L. paracasei BIOTECH 10363 against 11 antibiotics was evaluated using the disc diffusion method.The average diameters (mm) of the zone of inhibition are shown in Table 7.The tested strain was susceptible or moderately susceptible against various cell wall synthesis inhibitors such as aminopenicillins (amoxicillin and ampicillin), penicillin (augmentin and penicillin G) and second-generation cephalosporin antibiotic (cefaclor).Furthermore, the isolate displayed the same sensitivity against several protein synthesis inhibitors including streptomycin, chloramphenicol, lincosamides (clindamycin), macrolides (erythromycin) and tetracycline.Generally, Lactobacillus species are generally susceptible or sensitive to cell wall synthesis inhibitors and low concentrations of chloramphenicol, lincosamides, macrolides and tetracycline (Darsanaki et al., 2013;Gueimonde et al., 2013).On the other hand, the tested strain was found to be resistant to kanamycin.This finding is in agreement with previous studies which reported the intrinsic resistance of many Lactobacillus species toward kanamycin and other aminoglycosides (Benavides et al., 2016;Štšepetova et al., 2017;Zhang et al., 2022).The observed resistance is caused by a lack of cytochrome-mediated electron transport, a system that is normally involved in the uptake of antibiotics in microorganisms (Narayanan and Narayanan, 2019).

Conclusion
Lactobacillus paracasei occurs naturally in milk and dairy products, fermented sausages, vegetables, and wine.It is one of the most common and well-known probiotic microorganisms in food, dietary supplements, and pharmaceutical preparations.However, not all strains of L. paracasei demonstrate probiotic properties. 2 Susceptibility was expressed as susceptible, moderately susceptible, and resistant (Charteris et al., 1998).
Table 7. Antibiotic susceptibility pattern of L. paracasei BIOTECH 10363 using disc diffusion assay on MRS agar.

Table 6 .
Maximum growth was indicated as double positive sign (++), normal growths as single positive sign (+) and no growth as negative sign (-) for NaCl.Salt tolerance of L. paracasei BIOTECH 10363.