Isolation and identification of probiotic lactic acid bacteria from curd and in vitro evaluation of its growth inhibition activities against pathogenic bacteria

The aim of this study was to isolate and identify probiotic lactic acid bacteria from curd and evaluate in vitro, its growth inhibition activities against pathogenic bacteria. A total of nine strains of Lactobacillus were isolated from curd and identification of strains was done by biochemical and physiological tests and Lactobacillus leichmannii, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus coagulans, Lactobacillus acidophilus, Lactobacillus lactis and Lactobacillus rhamnosus strains were identified from curd. Lactobacillus strains survival were also assessed under conditions simulating human GI tract. Therefore, resistance to antibiotics, resistance to low pH, resistance to bile salt and bile salt hydrolysis was performed. Results showed that all tested isolates were able to grow at low pH 3.0, and at 0.3% bile concentration. L. casei, L. delbrueckii and L. brevis showed more resistance to antibiotics. According to haemolytic activity, all examined strains did not exhibit β-haemolytic activity when grown in Columbia human blood agar. Regarding the bile salt hydrolysis, L. casei and L. delbruekii showed partial bile salt hydrolysis activity and colony morphology was recorded as differentiated in comparison with the control MRS agar plates. Finally, antimicrobial activities of lactobacillus isolates were tested against five pathogenic bacteria (Staphylococcus sp., Bacillus sp., Klebsiella sp., Pseudomonas sp. and E. coli sp.) at pH 6.5 by disc diffusion method. All the tested isolates showed in vitro inhibitory zone against pathogenic bacteria. L. casei and L. delbrueckii showed maximum inhibition zone. In conclusion, the present study showed that L. casei and L. delbrueckii can be used as potential probiotic lactic acid bacteria.


INTRODUCTION
The term probiotic, literally meaning "for life", was first addressed by Lilly and Stillwell (1965) and was used to describe substances produced by protozoa to stimulate the growth of other organisms.Nowadays, the term refers to viable, nonpathogenic microorganisms (bacteria or yeasts) nonpathogenic microorganisms (bacteria or E-mail: estifanos_hawaz@yahoo.com.Tel.+251932214923. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License yeasts) that, when ingested, are able to reach the intestines in sufficient numbers to confer health benefits to the host (Schrezenmeir and De Vrese, 2001).Commonly used bacterial probiotics include various species of Lactobacillus, Bifidobacterium and Streptococcus, as well as Lactococcuslactis and some Enterococcus species (Morrow et al., 2012).It is well established that probiotics confer a number of beneficial health effects to humans and animals.Intake of probiotics stimulates the growth of beneficial microorganisms and reduces the amount of pathogens, thus improving the intestinal microbial balance of the host and lowering the risk of gastro-intestinal diseases (Fuller, 1989;Cross, 2002;Chiang and Pan, 2012).Their benefits also include the alleviation of certain intolerances (such as lactose intolerance), the enhancement of nutrients bioavailability, and prevention or reduction of the prevalence of allergies in susceptible individuals (Isolauri, 2001;Chiang and Pan, 2012).Probiotics are reported to also have antimutagenic, anticarcinogenic, hypocholesterolemic, antihypertensive, anti-osteoporosis and immuno-modulatory effects (Chiang and Pan, 2012).They relieve the symptoms of inflammatory bowel diseases, irritable bowel syndrome, colitis, alcoholic liver disease, constipation and reduce the risk for colon, liver and breast cancers (Prado et al., 2008).
Lactic acid bacteria are Gram positive organisms which are safely applied in medical and veterinary functions (Holzapfel and Schillinger, 2002).Isolation and screening of lactic acid bacteria from naturally occurring processes have always been the most powerful means for obtaining useful cultures for scientific and commercial purposes and the proper selection and balance of lactic acid bacteria used for starter culture is critical for the manufacture of diary fermented food products with their desirable texture and flavor (Sanders, 2000).Mankind exploited these bacteria for thousands of years for the production of traditionally fermented dairy products because of ability to produce desirable changes in taste, flavor, texture and extending the shelf life of food products (Dave and Shah, 1996).Many studies have reported that the best matrices to deliver probiotic lactic acid bacteria are dairy fermented products such as yogurt, cheese and other fermented milk products (Salminen, 1996).Their unique presence in intestinal epithelium and human gastrointestinal tract, and their traditional use in fermented foods and dairy products without remarkable problems prove their safety (Pangallo et al., 2008).The aim of this study was to isolate and identify probiotic lactic acid bacteria from curd and evaluate in vitro, its growth inhibition activities against pathogenic bacteria.

Isolation of lactic acid bacteria from curd
Lactic acid bacteria were isolated from curd obtained from different sources of dairy products.Samples were collected using sterile bottles and stored in an icebox until delivery to the laboratory for analysis.The curd samples were suspended appropriately and diluted in sterile saline and spread plated into selective medium: De Mann, Rogosa Sharpe (MRS) (Dave and Shah, 1996) and incubated at 37°C for 24 h anaerobically for isolation.The selected colonies were purified by streak plate technique.The purified bacterial strains were stored at -80°C in sterile reconstituted skim milk 12.5% (w/v) supplemented with 15% glycerol.

Physiological and biochemical characterization of lactic acid bacteria
Growth of lactic acid bacteria (LAB) isolates was examined in MRS broth at temperature 15, 37 and 45°C by incubating for 24 h anaerobically (Briggs, 1953).CO 2 and lactic acid production from glucose was tested in citrate lacking MRS broths media containing inverted Durham tubes.Salt tolerance was tested in MRS broth by incorporating at 2, 4 and 6.5% (w/v) sodium chloride, incubated at 37°C was carried out according to method described by Briggs (1953).Catalase test was performed by adding 3% of hydrogen per oxide (H 2 O 2 ) in a test tube containing an overnight culture of lactic acid bacteria.Ammonia production in MRS broth containing 0.3% arginine and 0.2% sodium citrate instead of ammonia citrate and the production of ammonia was detected using Nessler's reagent (Briggs, 1953).Sugar fermentation test was performed using eight different sugars (mellibiose, cellobiose, lactose, sucrose, raffinose, gluconate, salicine and arabinose) as reported by Cullimore (2008).Gram positive, catalase negative and bacilli colonies were taken as lactic acid bacteria and stored in glycerol culture and kept at -80°C for further investigation.

Survival of lactic acid bacteria isolates under conditions simulating human GI tract
Major selection criteria (antibiotic resistance, resistance to low pH, tolerance against bile salt, bile salt hydrolysis and antimicrobial activity) were used for the determination of probiotic properties of lactic acid bacteria isolates (Bassyouni et al., 2012).

Antibiotic resistance
Lactic acid bacteria strains were assessed their antibiotic resistance by disc diffusion method using antibiotics discs.One milliliter of actively growing cultures was mixed with 10 ml of MRS agar and poured into a petriplates.After solidification, the antibiotic disks were placed on the solidified agar surface, and the plates were left over for 30 min at 4°C for diffusion of antibiotics and then anaerobically incubated at 37°C for 48 h.Resistance was defined according to the disc diffusion method by using antibiotic discs of streptomycin, gentamycin, chloramphenicol, ciprofloxacin, norfloxacin and tetracycline (Hi Media Laboratories Pvt. Ltd.Mumbai, India.)(Felten et al., 1999).The zone of inhibition was measured by calipers in millimeters (Lleó et al., 1998;Mir-hoseini, 2004).

Resistance to low pH
Lactic acid bacteria isolates obtained from overnight culture were harvested by centrifugation for 10 min at 5000 rpm and 4°C, washed twice with PBS buffer (pH 7.2), and adjusted to pH 2.5.Resistance were assessed in triplicates in terms of viable colony counts and enumerated on MRS agar after incubation at 37°C for 0, 1, 2 and 3 h, indicating the time spent by food in the stomach as described by Maragkoudakisa et al. (2006) and Zoumpopoulou et al. (2008).Also, growth was monitored at OD620 (Bassyouni et al., 2012).

Resistance to bile salt
The tolerance against bile was carried out based on the intestinal bile concentration, 0.3% (w/v) and the staying time of food in small intestine is 4 h (Kumar and Murugalatha, 2012).MRS medium containing 0.3% (w/v) bile concentration was inoculated with overnight culture of lactic acid bacteria.Viable colonies were counted for every hour of incubation time on MRS agar and also growth was monitored at OD620 (Bassyouni et al., 2012).

Bile salt hydrolysis
Bacterial cultures were grown overnight in MRS broth at 37°C with 5% CO 2 and streaked in triplicates on MRS agar containing 0.5% (w/v) taurodeoxycholic acid.The hydrolysis effect was indicated by different colony morphology (partial hydrolysis) from the control MRS plates, after 48 h of anaerobic incubation at 37°C.

Antimicrobial activity against bacterial pathogens
The lactic acid bacteria isolates were tested for antagonistic activity against five bacterial pathogens (Staphylococcus sp., Bacillus sp., Klebsiella sp., Pseudomonas sp. and E. coli sp.) (Mir-hoseini, 2004) was obtained from Haramaya University, Microbiology Laboratory.The lactic acid bacteria strains were inoculated in MRS broth for 24 h at 37°C and centrifuging the culture at 5000 rpm for 10 min at 4°C, and the bacterial cells were removed.The pH values of supernatants were neutralized to 6.5 by the addition of 0.1 N NaOH, and filtered through a sterilized cellulose acetate filter and stored at 4°C.Supernatants were directly used for antagonistic test.Pellet was diluted with sterile distilled water and used for antimicrobial activity test.

Antimicrobial assay
The antimicrobial activity of the lactic acid bacteria isolates was determined by using the disc diffusion method as described by Sandra et al. (2012).These assays were performed in triplicate.The plates were poured with 20 ml Nutrient Agar.The pathogenic strains (Staphylococcus sp., Bacillus sp., Klebsiella sp., Pseudomonas sp. and E. coli sp.) were spread on the surface of nutrient agar.The agar plates inoculated with test organism were incubated for 1 h before placing the extract impregnated paper discs on the plates.Five sterile papers blank disks of 6 mm diameter were placed on the surface of agar plate which was inoculated by indicator strains of 5, 10 and 15 μl of the filtered supernatant and pellets of Lactobacilli were applied for screening of antimicrobial activity.The plates were kept at 4°C for 30 min to permit diffusion on the assay material, and incubated at 37°C for 24 h.Discs dipped in sterile water served as control and the antibiotics Chloramphenicol, Penicillin G and Streptomycin were used as positive control.Zones of inhibition were measured by calipers in millimeters (Lleó et al., 1998;Mir-hoseini, 2004).

Haemolytic activity
Fresh overnight bacterial cultures was streaked in triplicates on Columbia agar plates, containing human blood and incubated at 30°C for 48 h.Haemolytic activities of the bacterial culture were examined for signs of β-haemolysis (clear zones around colonies), α-haemolysis (green zones around colonies) or γ-haemolysis (no clear zones around colonies) on human blood agar plates (Hargrove and Alford, 1978).

Isolation of lactic acid bacteria (LAB)
Nine Lactobacillus strains were isolated from curd and based on their physiological and biochemical characterristics isolates were designated as: A, B, C, D, E, F, G, H and I.

Physiological and biochemical identification
According to biochemical characterization, all tested isolates were grown at 37, 45°C where as isolate B, D, F, and I were grown at 15°C.All tested isolates did not produce ammonia from arginine and did not produce gas from glucose.All isolates tolerated 2, 4 and 6.5% of NaCl concentration and was catalase negative.2012) who isolated L. acidophilus from cheese and found that L.l actis and L. casei were considered as the predominated species in fermented dairy products (Tables 1 and 2).

Survival of lactic acid bacteria isolates under conditions simulating human GI tract
In order to use lactic acid bacteria as a probiotic, the bacteria must be able to survive the acidic conditions in the stomach and resist bile acids concentration (Holzapfel et al., 1998;Klaenhammer and Kullen, 1999) and possess antagonistic effect against pathogenic bacteria (Mirhoseini, 2004).

Resistance to low pH
Being resistant to low pH is one of the major selection criteria for probiotic strains (Çakır, 2003).To reach    the small intestine they have to pass through stressful conditions of the stomach (Çakir et al, 2003).Although in the stomach, pH can be as low as 1.0, in most in vitro assays pH 3.0 has been preferred.Due to the fact that a significant decrease in the viability of strains is often observed at pH 2.0 and below (Prasad et al., 1998), for selection of the strains resistant to low pH, PBS pH was adjusted to 3.0.The time taken during the digestion in the stomach is 3 h.So, all the isolates were detected whether they were resistant to pH 3.0 during 3 h or not.All the tested isolates were able to survive at pH 3.0 during three hours of incubation.L. casei and L. delbrueckii were more resistant to low pH than other strains.This correlated with the previous report by Argyri et al. (2013) were nine strains of Lactobacillus showed very high resistance to low pH (L.plantarum L. pentosus, L. paracasei subsp.paracasei)with final populations exceeding 8 log cfu/ml, whereas 12 strains showed high resistance to low pH (L.plantarum, L. pentosus) which along with the reference strains L. casei and L. rhamnosus GG showed final populations within 6-8 log cfu/ml and according to Bassyouni et al. (2012), eight of eleven tested lactobacillus isolates were resistance to pH 3.0 during three hours.All isolates that survive in pH 3.0 were taken to the next step for further investigation (Table 3).

Resistance to bile salt and bile salt hydrolysis
The strains, resistant to low pH, were screened for their ability to tolerate the bile salt.Although the bile concentration of the human gastro intestinal tract varies, the mean intestinal bile concentration is believed to be 0.3% (w/v) and the staying time is suggested to be 4 h (Prasad et al., 1998).Strains were detected in 0.3% during 4 h of stay.Results showed that all isolates after four of exposure retaining their viability with negligible reduction in viable counts to 0.3% (w/v) bile concentration and among all of tested isolates, L. casei and L. delbruekii were more tolerant than others.This in agreement with Jensen et al.
(2012) report that Lactobacillus species tolerate gastric juice well with no reduction in viability.Similarly observation by Abriouel et al. (2012), showed all lactic acid bacteria isolated from fermented olive were able to grow and survive at 0.3% (w/v) bile salt.The survival at bile salt condition is one of the main criteria for in vitro selection of potentially probiotic bacteria and critical points for the microbes.Regarding the bile salt hydrolysis, L. casei and L. delbruekii show partial bile salt hydrolysis activity and colony morphology was recorded as differentiated in comparison with the control MRS agar plates (Table 4).

Haemolytic activity
Non-haemolytic activity and antibiotic resistance strains are considered as a safety prerequisite for the selection of a probiotic strain.Results showed that all examined strains did not exhibited β-haemolytic activity when grown in Columbia human blood agar.Most of the strains (7 strains) were γ-haemolytic (non-haemolysis), while two strains exhibited α-haemolysis.These were L. coagulans, L. rhamnosus.Isolate L. casei, L. delbruekii and L. lactis showed γ-haemolysis (Maragkoudakisa et al., 2006).

Antimicrobial activity
The cell free supernatants and cell masses of different lactobacillus strains were tested for antimicrobial activity against five pathogenic bacterial strains (Staphylococcus sp., Bacillus sp., Klebsiella sp., Pseudomonas sp. and E. coli sp.) by using disc diffusion method.The production of antimicrobial compounds such as organicacids, short chain fatty acids and bacteriocins is one of the functional properties used to characterize probiotics (Fuller, 1989).The capacity to produce different antimicrobial compounds may be one of the critical characteristics for effective competitive exclusion of pathogen survival in the intestine and expression of a probiotic effect for the host (Salminen et al., 1998).In this study, the antimicrobial activities of supernatants and cell masses were conducted at three different concentrations of 5, 10 and 15 μl/discs were inoculated and compared with those of positive control such as tetracycline, penicillin G and streptomycin and distilled water as negative control.The data obtained from the disc diffusion method indicated that the extract displayed a variable degree of antimicrobial activity on different tested strains.The inhibitory effect was increased with the increase of the extract concentration from 5 to 15 μl.The diameters of inhibition zones were measured and compared.Result showed that L. casei and L. delbrueckii showed maximum inhibition zone against bacterial pathogens among all the Lactobacillus isolates.This in agreement with Bassyouni et al. (2012) that Lactobacillus strains tested against Salmonella thyphimurium, E. coli, and Staphylococcus spp.and all of tested isolates have antibacterial effect against the pathogenic bacteria (Tables 5 and 6).

Resistance to antibiotics
Resistance was defined according to the standard disc diffusion method by using antibiotic discs of Streptomycin, Gentamycin, Chloramphenicol, Ciprofloxacin, Norfloxacin and Tetracycline (Hi Media Laboratories Pvt. Ltd.Mumbai, India.).The zone of inhibition was measured by calipers in millimeters (Lleó et al., 1998;Mir-hoseini, 2004).Results showed that L. casei, L. delbruekii and L. brevis were resistant to all antibiotics and L. fermentum, L. lactis and L. rhamnosus were sensitive to all antibiotics except Streptomycin.L. leichmannii, L. acidophilus and L. coagulans were sensitive to all antibiotics.L. casei, L. delbruekii, L. lactis, L. rhamnosus and L. brevis were taken to the next step for further investigation (Table 7).

Conclusions
The Lactobacillus cultures were isolated and identified according to their physiological and biochemical characteristics.examined strains did not exhibited β-haemolytic activity when grown in Columbia human blood agar.Most of the strains (7 strains) were γ-haemolytic (non-haemolysis), while two strains exhibited α-haemolysis.These were L. coagulans, L. rhamnosus.Isolate L. casei, L. delbruekii and L. lactis showed γ-haemolysis.Regarding the bile salt hydrolysis, L. casei and L. Delbruekii show partial bile salt hydrolysis activity and colony morphology was recorded as differentiated in comparison with the control MRS agar plates.Finally, antimicrobial activities of Lactobacillus isolates were tested against five pathogenic bacteria (Staphylococcus spp., Bacillus spp., Klebsiella spp., Pseudomonas spp., and E. coli spp.) at pH 6.5 by disc diffusion method.All the tested isolates showed in vitro inhibitory zone against pathogenic bacteria at pH 6.5.Among the Lactobacillus strains, L. casei, and L. delbrueckii showed maximum inhibition zone.In conclusion, the present study showed that from all the isolated Lactobacillus strains, L. casei and L. delbrueckii can be used as potential probiotic lactic acid bacteria.

Table 1 .
Morphological and cultural characteristics of Lactobacillus isolates.

Table 2 .
Carbohydrate fermentation of lactic acid bacteria.

Table 5 .
Antimicrobial activity of lactic acid bacteria cell mass.

Table 6 .
Antimicrobial activity of lactic acid bacterial supernatant.

Table 7 .
Drug resistance of Lactobacillus strains.