PRELIMINARY PROBIOTIC POTENTIAL OF SELECTED AEROCOCCUS SPP ., ENTEROCOCCUS SPP., AND

current study as target


INTRODUCTION
Lactic acid bacteria (LAB) are ubiquitous, unicellular gram positive, nonsporulating, catalase-negative, facultative anaerobic microorganisms.LAB are frequently found in food and traditional ones.As well, they live in a bacterial ecosystem such as the vagina and gastrointestinal tract (Quinto et al., 2014).They are heterotrophic that generate energy from carbon substrates fermentation to produce organic acids such as lactic acid as one of the main fermentation products (Quinto et al., 2014;Carr et al., 2002).LAB are generally recognized as safe "GRAS" by the agency of the Food and Drug Administration (FDA) or qualified presumption of safety "QPS" by the European agency of the Food Safety Authority (EFSA).These friendly human microbes are essentially utilized as food, food supplements, food ingredients, medical food, recently, as drugs, for therapeutic purposes to enhance human health and treatment of some gastrointestinal microbial disorders.This is due to their ability to inhibit harmful bacteria (Mokoena, 2017; Urdaci et al., 2004).This inhibition is related to the production of organic acids and various other metabolites; bacteriocins mainly (Izuchukwu, 2017;Hor and Liong, 2014;Lairini et al., 2014;Guarner et al.,2011).The International Scientific Association for Probiotics and Prebiotics (ISAPP) defined probiotics as "a live organism, which provides a benefit to the host when provided in adequate quantities" (de Oliveira, 2018; Gasbarrini et al., 2016).Health benefits of probiotics include beneficial effects in prevention of diarrhea, inflammatory bowel disease, urogenital infections, lactose intolerance, lowering serum cholesterol level, colon cancer, regulation of microbial balance of intestinal microbiota, prevention of allergies, enhancement of the immune system, nutrient bioavailability, and calcium absorption (Angmo et al., 2016 ; Tokatlı et al., 2015).
To ensure that the probiotic candidate strains exert their beneficial effect on human health, several required characteristics main currently examined, through in vitro tests as recommended by FAO/WHO guidelines (Morelli and Capurso, 2012; FAO/WHO, 2002).Taxonomic identification must be achieved using reliable phenotypic and molecular techniques (Reid, 2005).The origin of the strain must be human origin, preferably from the same site to obtain maximum efficiency and effectiveness.Those probiotic strains must be assessed by clinical trials on animals and on humans to avoid side effect.In addition, they must display an antimicrobial activity and resist to the gastrointestinal environment (Zhang et al., 2018;Ashraf and Smith, 2016).A high percentage to adhere to the intestinal cell epithelium or mucosa is an essential property.It may eventually stimulate the immune system through mucin production and prevent the implantation of pathogens.Antibiotic susceptibility profile is the most important parameter for the LAB candidates, to exclude transferability of antibiotic resistance to commensal or pathogen bacteria present inside the gut (Zhang et al., 2018;Sanders, 2011;Foligne et al., 2013).We carried out this study aiming to screen, through a series of tests, functional, technological, and safety proprieties of ten (10) autochthonous LAB.Those LAB were previously isolated from an ethnic Algerian salted and dried meat (Guedid), characterized at the genus level by phenotypic tests and selected for possessing an important antibacterial activity.

Laboratory bacterial strains and culture media
The four gram-positive potentially pathogenic bacteria used in the current study as target strains were: Listeria ivanovii CECT148, Bacillus cereus ATCC11778, Staphylococcus aureus ATCC25923, and Clostridium perfringens CECT486.
Working target strains were activated in TSB broth and/or agar (Trypticase Soy broth; Condalab) for 24 hours at 37°C.The ten autochthonous LAB used in the current study as test cultures: Aerococcus spp.(Lbm3,10,17,18 and 19),Enterococcus spp. (Lbm46,47,49 and 50) and Weisella sp.Lbm22, were obtained from Guedid prepared from lamb meat by salting and sun-drying (Gagaoua, and Boudechicha, 2018;Boudechicha et al., 2017).Samples from homemade preparation were collected in sterile bags.LAB were isolated on elective growth MRS agar (De Man, Rogosa, &Sharpe; Condalab) and phenotypically identified (Axelsson 2004; Doyle et al., 2006).Working LAB cultures were activated in MRS broth and/or agar for 24 to 72 hours at 30°C under anaerobic atmosphere.Pathogens and LAB were regularly checked out by catalase reaction test and gram staining with microscopic observation according to standard procedures (Goldman and Green, 2015).Their stock cultures were maintained in their respective media; as refrigerate stock in agar slants at 4°C and as frozen stock in 20% (v/v) glycerol-supplemented broth at -20°C (Spencer, 2001).

Antagonistic activity against pathogenic bacteria
Antibacterial activity was screened using two methods: agar spot test in dual culture and agar well diffusion assay.The first assay was employed for the direct detection of antibacterial activity as described by Fleming et al. (1975) and Schillinger and Lücke (1989).An overnight LAB cultures (30°C on MRS agar) were spotted on the surface of solidified MRS base agar (1.5% agar) seeded with 200 µl (approximately 10 8 CFU/mL) of the target pathogenic bacteria standardized at 0.5 McFarland from overnight cultures (37°C in TSB broth).The plates were incubated under anaerobic atmosphere at 30°C for 24 hours.The second assay was used for the indirect detection to determine the cell-free supernatants (CFS) antibacterial activity as described by Schillinger and Lücke (1989).Culture supernatants (6000 rpm for 15 minutes at 4°C) from overnight cultures (MRS broth inoculated with 0.2% of LAB fresh cultures and incubated at 30°C for 48 hours) were treated at 80°C to eliminate vegetative forms for 10 min and then refrigerated at 4°C.Ten (10) mL of solidified TSA (Trypticase Soy agar; Condalab) base agar (1.5% agar) were overlaid with 10 mL TSA soft agar (0.75% agar) inoculated with 200 µl (approximately 10 8 CFU/mL) of the target pathogenic bacteria standardized at 0.5 McFarland from overnight cultures (37°C in TSB broth).Wells (7 mm in diameter) were bored at equal distance on the solidified TSA agar and filled with 40 µl of culture supernatants.The central well, as control, received 40 µl of sterile MRS broth.The plates were stored at room temperature (20°C for 2 hours) until the culture supernatant was absorbed.LAB were tested in duplicate and incubated under anaerobic atmosphere at 30°C for 24 hours.Anaerobic conditions were ensured to bypass catalase inhibition as reported by Vermeiren et al., 2004 and a buffered TSA agar, at pH 7.0 and 0.2 M, was utilized to avoid acid inhibition.The buffered TSA agar was prepared by dissolving 19.5 parts of K2HPO4 at 0.1 M with 30.5 parts KH2PO4 at 0.1 M in a total of 100 parts of distilled water.The presence of inhibition zone (inhibition halo), surrounding the test culture spot or the culture supernatant higher than 0.5 mm, was considered as positive antagonistic activity (Fleming et al., 1975).Both assays were performed in duplicate.

Acidity, growth kinetics and production of bacteriocin-like substances
Determination of acidity, growth kinetics and production of bacteriocin-like substances were carried out during the same experiment.From overnight cultures of LAB, MRS broth was inoculated with 0.2% and incubated at 30°C with shaking (100 rpm/min), Samples were collected after 0, 2, 6, 12, 18, 24, and 48 hours.Acidity was measured by pH-meter, optical density by spectrophotometer at 620 nm, and bacteriocin-like substances production was investigated by agar well diffusion assay on buffered TSA agar as noted above.The experiment was performed in duplicate (Musikasang et al., 2012;Musikasang et al., 2009).

Heat and enzymes sensitivities of bacteriocin-like substances
Samples of CFS obtained by centrifugation (6000 rpm for 15 minutes at 4°C) from an overnight test cultures in MRS broth inoculated with 0.2% of fresh LAB incubated at 30°C for 48 hours.Samples were treated at 100°C for 10, 30, and 60 min and at 120°C for 15 min, then were cooled at 4°C.Other samples were exposed to the action of two proteolytic enzymes (pepsin and α chymotrypsin at a final concentration of 1 mg/mL) (Sigma-Aldrich).Resistance or sensitivity of CFS containing bacteriocin-like substances after these treatments were determined by the fact of the presence or absence of antibacterial activity by agar well diffusion assay applied as noted above.All tests were performed in duplicate (Hanchi et al., 2014;Monteagudo-Mera et al., 2011).

Exopolysaccharides and acetoin production
Capacity to produce exopolysaccharides (EPS) was determined on modified MRS agar with 10% (w/v) sucrose (Sigma-Aldrich).Overnight LAB cultures (MRS agar at 30°C for 24 hours) were streaked on the surface of the agar.After incubation at 30°C for 24 to 48 hours, was considered as positive result streaks with viscous colonies (Angmo et al., 2016).For the capacity to produce acetoin, 1 mL from the overnight LAB cultures (MRS broth at 30°C for 24 hours) was inoculated in Clark Lubs broth (Condalab).After incubation at 30°C for 24 hours, 0.5 mL of both VP1 and VP2 reagents were added.Ten (10) minutes of rest at room temperature, acetoin production is indicated by pink ring and/or the diffusion of the pink color on the surface of the tube.Each experiment was performed in triplicate.

Assessment of proteolytic, lipolytic, amylolytic, and bile salts hydrolase
Assessment of proteolytic, lipolytic, amylolytic, and bile salts hydrolase activities was performed on modified MRS agar containing skimmed milk (1%, 2%, and 10% w/v), tween 80 (at 1% and 3% v/v), soluble starch (2% w/v), and bile salts (0.5% w/v with 0.037% of CaCl2 w/v) (for all reagents Sigma-Aldrich), respectively.Overnight LAB cultures (MRS agar at 30°C for 24 hours) were spotted on the surface of media.After incubation at 30°C for 24 to 48 hours, a positive activity was indicated by higher than 1 mm clear zone around the spot.The starch agar plate must be flooded with iodine solution and examined 15-30 min later.The zone diameters were then measured.

Surviving in acidity conditions, bile salts, simulated gastric, and intestinal juice
The capacity of the LAB to survive in acidity conditions was tested in phosphatebuffering saline (PBS) adjusted to pH 2 and 3 (0.9% NaCl, 0.9% K2HPO4, 0.15% KH2PO4).The capacity to survive in gastric and intestinal conditions was also tested in a saline solution (0.85% NaCl) supplemented with 3 mg/mL of pepsin adjusted to pH 3 and with 3 mg/mL of bile salts adjusted to pH 8 (for all reagents Sigma-Aldrich), respectively.These broths were inoculated by aliquots from overnight LAB cultures (MRS broth at 30°C for 24 hours) to attend 10 8 CFU/mL.These preparations were examined before and after 4 hours of incubation at 37°C and survival LAB were then calculated using single plate-serial dilution spotting (SP-SDS) on MRS agar in duplicate.Percentages of survival LAB were calculated as follow: % survival = (log CFU of viable cells survived / log CFU of initial viable cells inoculated

Cholesterol assimilation
Cholesterol assimilation was assayed in MRS Broth supplemented with 50 g/mL of cholesterol (Quimicaclinica) and inoculated with 1% of overnight LAB cultures (MRS broth at 30°C for 24 hours) standardized at 10 8 CFU/mL.After incubation at 30°C for 24 hours, CFS (6000 rpm for 15 min at 4°C) and inoculated control (sterile MRS broth) were assayed for their cholesterol content by spectrophotometry at 505 nm.The difference in cholesterol content between the control and the CFS was considered as a percentage of assimilated cholesterol (Dubey and Jeevaratnam, 2015; Tokatlı et al., 2015).

Statistical analysis
Statistical analysis was performed using originPro v9.5.Probiotic and technological potential was calculated as (observed score / maximum score) x 100.Indication of score was: (0) lytic / no producer or (1) no lytic / producer for technological properties, and (0) sensitivity or (1) resistance for functional properties.

Antimicrobial activity assessment
Potential bacteria candidates require minimum criteria in order to display probiotic status and exert their beneficial effect on human health.Antimicrobial activity represents the essential criteria due to inhibition of growth and attachment of pathogens in the intestine (FAO/WHO, 2002).In this study, we pointed out that the ten tested LAB inhibit pathogenic bacteria as follow: in the spot MRS agar test with 2 to 3 mm of diameter, two Aerococcus spp.

Technological properties assessment
Approaches

Functional properties assessment
The selection criteria for potential probiotic candidates include also abilities to survive gut transit (Giraffa et al., 2010), as well as their abilities to resist gastric acidity and bile salts in the small intestine (
Lbm50 that displayed the highest potential probiotic score (up to 55%).In addition, Aerococcus sp.Lbm18, Enterococcus sp.Lbm46, and Enterococcus sp.Lbm49, revealed the best technological properties.Finally, those promising candidates should be better characterized throughout a molecular assessment; and to be tested thorough ex vivo and in vivo to confirming their human or veterinary utilization as probiotics.

Figure 1 Figure 2
Figure 1 Antagonistic activity against St. aureus ATCC25923 of candidates LAB in the spot MRS agar (a) and in the agar well diffusion assay (b)

Mechai et al., 2014; Musikasang et al., 2012; Musikasang et al., 2012; Musikasang et al., 2009 ; Essid et al., 2009).
for improving probiotic status include but not limited to the selection of technological advantageous properties.All LAB tested exhibited protein digesting on modified MRS with skimmed milk at 2% and 10% with 17 to 22 mm for Aerococcus spp.(Lbm3, 10, 17, 18), Enterococcus sp.Lbm50 and Weisella sp.Lbm22, with 10 to 15 mm for Aerococcus sp.Lbm19 Enterococcus spp.(Lbm46, 47, 49).In comparison, Essid et al. (2009) found that L. plantarum have a proteolytic activity up to 5 mm on 10% skimmed milk.However, digesting capacity on modified MRS was not exhibited for protein at 1%, starch at 3%, and lipids at 1% and 3% of tween 80. Numerous probiotic studies with various strains showed negative results for amylolytic and lipolytic activities (In fact, meat substrate, muscle proteins and fat, should be more suitable to assess lipolytic and proteolytic activities than commonly used substrates such as powdered milk, gelatin, and Tween 80 (

Lähteinen et al., 2010).
The pH of the stomach ranges from 2.5 to 3.5 and bile salts concentration reaches to 0.3%, reason why they are considered as standard to assess gastric and intestinal tolerance (

Ashraf and Smith, 2016; Tokatlı et al., 2015; Leite et al., 2015; Dubey and Jeevaratnam, 2015).
For the capacity to survive gut transit, the ten LAB were found to resist pH 2 less than pH 3 with percentages ranging 82.83% to 92.56% for Aerococcus spp.and from 93.16% to 97.09% for Enterococcus spp.and with 76.58% for Weisella sp.Lbm22 (Table1).The decrease in cell viability was approximately 1-2 log CFU/mL (in the two pH values tested).In comparison, different probiotic LAB displayed survival ability at pH 2.5 up to 85% (Tokatlı et al., 2015) and reductions in counts at least 2 logarithmic units compared with controls (Leite et al., 2015 ).For others, the maximum 4.8 log CFU/mL decline in viability at pH 2 (Angmo et al., 2016) and 3.3 log at pH 3 (

Table 2
Capacity to survive gut transit Gastric

in saline solution suspending with 3 mg/ml of pepsin adjusted to pH 3 Intestinal in saline solution suspending with 3 mg/ml of bile salts adjusted to pH 8
t0 : initial time, t4 : after 4h of incubation, % :percentage of survival LAB (log CFU) Values represented as mean ± standard deviation Furthermore, LAB tested were able to survive after passing through the gastric, for 4 hours, by 96.35% to 99% for Aerococcus spp.and from 93.91% to 96.97% for Enterococcus spp.and with 98.36 % for Weisella sp.Lbm22.As for intestinal environments, survival variability ranged from 92.15% to 99.48% for Aerococcus

Ashraf and Smith, 2016; Angmo et al., 2016; Quinto et al., 2014).
spp. and from 92.43% to 97.91% for Enterococcus spp.and was 93.62 % for Weisella sp.Lbm22 (Table2).They were most tolerant to gastrointestinal stress factors with similar behavior and decreased by approximately 1-2 log CFU/mL in cell viability.This finding is in accordance with previous studies, that found minor reduction illustrating the high resistance to gastrointestinal passing (Hanchi et al., 2014) and those reporting a decrease of approximately 3 log CFU/ml in simulated gut juices.As it is recommended that 6 log at 7 log CFU/mL (10 6 at 10 7 CFU) constitutes a minimum number of each viable probiotic strain at the end of their shelf life for the exhibition of health benefit (Enterococcus sp.Lbm50 and Weisella sp.Lbm22.LAB producing α-or γ-hemolysis were considered non-hemolytic (

Rather et al., 2015; Maragkoudakis et al., 2009).
All LAB showed no gelatinase activity.In most cases, probiotic bacteria candidates demonstrated no presence of this virulence factors(Angmo et

al., 2016; Leite et al., 2015).
Such no presence of this virulence factors indicates the safety in the use of such LAB as probiotic cultures (

Monteagudo-Mera et al., 2011; FAO/WHO, 2002).
All LAB were found to resist to vancomycin and sulfadiazin, whereas Enterococcus spp.resisted to oxytetracyclin and ceftiofur, except Enterococcus sp.Lbm50 resisted to only ceftiofur.Inversely, they were all sensitive for ampicillin, amoxicillin, tylosin, florfinicol, erythromycin and gentamicin.Consequently, resistance coefficient was ranging from 0.28 to 0.10 and sensitivity coefficient was ranging 0.72 to 0.90.Resistance for tetracycline, erythromycin, chloramphenicol, streptomycin and vancomycin is clearly reviewed (Quinto et al., 2014).These results are consistent with many previous tests reporting a vancomycin resistance (Angmo et al., 2016; Leite et al., 2015; Dubey and Jeevaratnam, 2015), and were weakly antibiotic resistant to response at the requirement to avoid transfer genes inside the gut microbiota (Mathur & Singh, 2005) and thus were safe to use as potential probiotics (