Antimicrobial Compounds Produced by Probiotic Lactobacillus Brevis Isolated from Dairy Products

Ann Microbiol (2013) 63:81–90 DOI 10.1007/s13213-012-0447-2

ORIGINAL ARTICLE

Antimicrobial compounds produced by probiotic Lactobacillus brevis isolated from dairy products

Abeer Ahmed Rushdy & Eman Zakaria Gomaa

Received: 13 September 2011 /Accepted: 29 February 2012 /Published online: 17 March 2012 # Springer-Verlag and the University of Milan 2012

Abstract A total of 38 lactic acid bacteria, belonging to Keywords Lactic acid bacteria . Lactobacillus brevis . Lactobacillus, isolated from 24 samples of traditional Egyp- Antimicrobial activity. Bacteriocin-like compound . tian dairy products, were screened for antimicrobial activity Probiotics . Dairy products against different Gram-positive and Gram-negative bacteria. A strain of Lactobacillus brevis showed the best inhibitory activity when tested by well diffusion assay. The antibacte- Introduction rial activity was pronounced between early logarithmic and early stationary phases. The strain produced a heat-stable The lactic acid bacteria (LAB) family is composed of a antimicrobial compound showing no reduction in activity heterogeneous group of Gram-positive, non-spore-forming, after heat treatment from 60 to 100°C for 15 and 30 min. catalase- and cytochrome-negative, anaerobic or aerotoler- Since it was inactivated by proteolytic enzymes, it is con- ant bacteria (Axelsson 1998). LAB consist of a number of sidered to be proteinaceous in nature and, therefore, referred bacterial genera within the phylum Firmicutes. Recent tax- to as a bacteriocin-like substance. This compound was also onomic studies have suggested that the LAB group includes active over a wide pH range (pH 2–6). The antimicrobial 13 genera (Carr et al. 2002). The antimicrobial effects and compound was partially purified by 40% ammonium sulfate safety of some genera of LAB, such as Lactobacillus and precipitation. Lactobacillus brevis was tested for its in vitro Lactococcus in food preservation, are widely accepted (Sit antibiotics susceptibility, tolerance to bile salts, resistance to and Vederas 2008). low pH values, acidifying activity, proteolytic activity, and In nature, LAB have several different habitats including haemolytic activity. The results showed the potential of L. plant surfaces, decaying plant material, and the mammalian brevis strain as a probiotic culture, and hence it can be intestine, vagina and oral cavity which provide the multiple utilized in the manufacturing of pharmaceuticals and dietary nutrients required by these fastidious bacteria (Axelsson supplements. 1998). The food and feed industry widely utilizes LAB in the fermentation of vegetables, silage, and dairy and meat products. Lactobacilli are important organisms recognized for their fermentative ability as well as their health and nutritional benefits (Gilliand 1990). Lactobacilli have traditionally A. A. Rushdy (*) been used as natural biopreservatives in food and animal Botany Department, University College for Women, Arts, feed. Their preserving effect relates mainly to the formation Science and Education, Ain Shams University, Cairo, Egypt of organic acids which results in lowering of pH and hydrogen e-mail: [email protected] peroxide, competition for nutrients, and production of antimicrobial substances (such as bacteriocins) (Klaenhammer 1993; E. Z. Gomaa Stiles 1996). Biopreservation refers to extended shelf-life and Biological and Geological Sciences Department, Faculty of Education, Ain Shams University, enhanced safety of foods obtained by the natural or added Cairo, Egypt microflora or their antimicrobial products (Schnürer and 82 Ann Microbiol (2013) 63:81–90

Magnusson 2005). The preserving capacity of bacteria natu- by vortexing with saline peptone water (NaCl 8.5 g/l; bac- rally occurring in food has gained increasing interest during topeptone 1 g/l) at room temperature. Serial dilutions were the recent years, due to consumer demand for reduced use of then made in saline peptone water and aliquots were plated chemical preservatives. onto De Man, Rogosa and Sharpe (MRS) agar plates (De The bacteriocins from LAB have attracted significant atten- Man et al. 1960) containing bromocresol green (25 mg/l), tion because of their potential use as non-toxic and safe addi- based on the method of Dal Bello and Hertel (2006). Plates tives for food preservation and prevention of food spoilage by were incubated for 48–72 h at 30°C, and developed colonies food-borne Gram-positive pathogenic bacteria (Morisset et al. on the plates were then repeatedly sub-cultured on MRS 2004). Bacteriocins have considerable potential for food pres- agar medium to obtain pure isolates. ervation, as well as for human therapy, as potential supple- All isolates were Gram-stained and tested for catalase ments or replacements for currently used antibiotics (Fricourt production (Forbes et al. 1998). Gram-positive, catalase- et al. 1994; Ogunbanwo et al. 2003a). negative bacilli were identified as presumptive lactobacilli. Exploitation of LAB as preservative agents is advanta- The isolates of Lactobacillus were stored at −80°C in MRS geous not only in improving the microbial safety of food but broth containing 15% (v/v) glycerol. Before use, the strains also as probiotics in animals and humans to improve the were cultivated twice for 24 h at 30°C in MRS broth balance of microflora and to inhibit pathogenic bacteria in (Todorov et al. 1999). the intestinal tract (Soomro et al. 2002). Probiotics have been defined as “a live microbial feed supplement which benefi- Screening for antimicrobial activity cially affects the host by improving its intestinal microbial balance”. Several bacteria of the LAB family, usually repre- Strains, for testing, were cultured in MRS broth at 30°C for senting species of the genera Lactobacillus, Enterococcus or 48–72 h. Cells were removed by centrifugation (5,000 g, Bifidobacterium, have been named as probiotic bacteria, 10 min, 4°C). The supernatant fluids were adjusted to pH 6.5 which are currently used in several products intended for both with sterilized 1 M NaOH to eliminate inhibitory activity of human and animal consumption (Fuller 1989). Probiotics acid and then treated with 5 mg/ml catalase (Sigma-Aldrich) to have received increasing attention in recent years and have remove antagonistic activity of hydrogen peroxide (Daba et al. been shown to be useful to treatment of some human diseases 1991). The supernatants were filtered through a 0.22-mm filter (McFarland and Dublin 2008). Probiotics exhibit strain- (Millipore, Bedford, MA, USA) to prepare cell-free super- specific differences in acid and bile resistance, ability to natants (CFSs). These CFSs were screened for antimicrobial colonize the gastrointestinal tract, clinical efficacy, and health activity against Staphylococcus aureus NCTC-7447, Escher- benefit to the host (Dunne et al. 1999;Phametal.2008). ichia coli NCTC-10418 and Salmonella typhi NCIMB-9331 The aim of this study was to screen a number of lacto- as indicator strains. All these strains were obtained from bacilli of the genus Lactobacillus isolated from Egyptian Fermentation Biotechnology and Applied Microbiology dairy products for antagonistic activity to select the most (FERM-BAM) Centre, Al-Azhar University, Cairo, Egypt. potent bacterial strain, extract the bioactive substance, in- Antimicrobial activities of CFSs were tested by two vestigate its characteristics and evaluate the probiotic poten- methods: the agar well diffusion assay (AWDA) and spot- tial (in vitro) of the selected Lactobacillus strain. on-lawn assays as described by Ennahar et al. (1999). One milliliter of the pathogenic strains with approximately 105 CFU/ml was incorporated into soft agar (1% v/v) plates Materials and methods of Luria–Bertani (LB) medium containing (g/l): tryptone, 10; yeast extract, 10; sodium chloride, 5 (Sambrook et al. Traditional Egyptian dairy products 2001). Lactobacilli supernatant samples (50 μl) were pipet- ted into holes drilled into the agar (well diffusion assay) or Twenty-four samples of traditional Egyptian dairy products spotted (10 μl ) on the surface of the agar (spot-on-lawn were collected from the Cairo markets including 4 different assay). The plates were then incubated at 37°C for 24 h. milks, 10 different yoghurts and 10 different kinds of cheese. Antimicrobial activity was recorded as growth-free inhibi- All products were commercially prepared exhibiting no evi- tion zones around the well or the spotted area. dence of spoilage and were thus considered fit for consumption. Strain identification Isolation and culture conditions 16S rRNA sequencing and analysis Lactobacilli cultures were isolated from the collected samples of dairy products. A 25-g sample of each product was One isolate showing the largest inhibition zone was identi- mixed or ground under sterile conditions and homogenized fied by 16S rRNA sequencing according to the method of Ann Microbiol (2013) 63:81–90 83

Rochelle et al. (1995). The gene sequencing was done by at 4°C with stirring over night and later centrifuged at Macrogen (South Korea). DNA sequences were aligned 20,000 g for 30 min. The resulting pellet was mixed and using GeneMapper® v.4.1 and Data Collection v.3.1 Com- solubilized in 120 ml of 10 mM sodium phosphate buffer at munication Patch1, August 2010. To extract the genomic pH 6.5 (Kelly et al. 1996). DNA, bacterial colonies were picked up with a sterilized toothpick, and suspended in 0.5 ml of sterilized saline then Characteristics of the antimicrobial compound centrifuged at 8,000 g for 10 min. After removal of the supernatant, the pellet was suspended in 0.5 ml of InstaGene The partially purified inhibiting substances were character- Matrix (Bio-Rad, USA), incubated at 56°C for 30 min and ized with respect to pH and thermal stability, stability during then heated to 100°C for 10 min. After heating, supernatant storage susceptibility to denaturation by enzymes, treatment can be used for Polymerase Chain Reaction (PCR). with surfactant agents and final the antimicrobial activity Bacterial 16S rRNA gene was amplified using the fol- against different bacteria lowing universal primers for eubacteria: forward primer 27 F (5'-AGAGTTTGATCMTGGCTCAG-3') and reverse pH sensitivity A sample of the extract was used for these primer 1492R (5'- TACGGYTACCTTGTTACGACTT-3'). tests. The partially purified preparation was adjusted to PCR was performed using kits with AmpliTaq® DNA poly- various pH values in the range of 2–12 with sterile NaOH merase (FSenzyme) (Applied Biosystems, USA). One (3 M) or HCl (3 M) (Sigma Chemical). The pH-adjusted microlitre of template DNA was added in 20 μl of PCR samples were incubated at 37°C for 30 min then re-adjusted solution. The 27 F/1492R primers were used for bacteria. to pH 6.5 and tested for antimicrobial activities by the spot- Thirty-five amplification cycles were performed at 94°C for on-lawn method (Brink et al. 1994;Ohetal.2000). 45 s, 55°C for 60 s, and 72°C for 60 s. PCR amplicon were purified by using Montage PCR clean-up kit (Millipore). Heat resistance The extract was exposed to heat treatments The purified PCR products of approximately 1,400 bp were in a water bath at 60, 70, 80, 90, and 100°C. Aliquot sequenced by using 2 primers 518 F (5'- CCA GCA GCC volumes of each fraction were withdrawn after 15 and GCG GTA ATA Cg -3') and 800R (5'- TAC CAG GGT ATC 30 min then immediately cooled by cold water and tested TAA TCC -3'). Sequencing was performed by using Big Dye for remaining antimicrobial activity (Oh et al. 2000). terminator cycle sequencing kit (Applied BioSystems). Se- quencing products were resolved on an Applied Biosystems Stability during storage To test the stability of the extract model 3730XL automated DNA sequencing system (Applied during three freeze–thaw cycles, the semi-purified compound BioSystems). Sequence analysis was performed with sequen- was frozen at −20°C for 24 h and thawed for 20 min at 5°C ces in the National Center for Biotechnology Information and tested for antimicrobial activities (Ponce et al. 2008). (NCBI), USA database using Basic Local Alignment Search Tool for Nucleotides (BLASTN), (Altschul et al. 1997). Enzyme treatments Susceptibility to various proteases was performed by incubating the extract in the presence of Antimicrobial compound production trypsin (1 mg/ml) and pepsin (1 mg/ml) at 37°C for 2 h. After incubation, the enzymes were inactivated by heat The selected LAB (B23) was grown in Tween 80-free MRS treatment at 65°C for 30 min and tested for antimicrobial medium. One microlitre of an overnight culture was used to activity (Oh et al. 2000). inoculate 100 ml of MRS broth and incubated for 40 h in a static incubator at 30°C. At 4-h intervals, the changes in cell Effect of surfactants on antimicrobial activity The effects of density were recorded at 600 nm, and the change in pH mea- chemical surfactants on antibacterial activity were tested by sured. Antimicrobial activity was recorded after adjusting pH to adding 1% (w/v) of sodium dodecyl sulfate (SDS) (anionic), 6.5 (Nowroozi et al. 2004) by AWDA as previously described. Tween 80 and urea (non-ionic), to partially purified bacteriocin (PPB) (Todorov et al. 2007). Untreated supernatant and Extraction of antimicrobial compound surfactants at each of these concentrations were used as con- trols. All samples were incubated at 37°C for 5 h and then The selected LAB strain was propagated in 1 l MRS broth at assayed for antimicrobial activity against indicator bacteria. 30°C until the late logarithmic phase. For extraction of bacteriocin-like compound, cells were removed by centrifu- Antibacterial activity spectrum The agar well diffusion gation (10,000 g, 20 min, 4°C), and pH adjusted to 6.5 by assaymethod(AWDA)wasusedtodetecttheantibac- means of 1 M NaOH to exclude the antimicrobial effect of terial spectrum of PPB against different Gram-positive the organic acid. The CFS obtained was precipitated with and Gram-negative bacteria. Gram-positive bacteria ammonium sulphate (40% saturation). The mixture was kept were Staphylococcus aureus ATCC 29213 and 29737, 84 Ann Microbiol (2013) 63:81–90 and Bacillus subtilis NCTC 10400, while Gram- microlitres of bacterial suspension were transferred into negative bacteria were Escherichia coli ATCC 10536 another tube and 0.4 ml of n-hexadecane was added. The and NCTC 10418, Salmonella typhi NCIMB 9331, tubes were shaken for 2 min and settled for 15 min to Pseudomonas aeruginosa ATCC 10145 and 25619, separate the two phases. After that, OD of aqueous and Klebsiella pneumoniae ATCC 10031. Each indicator phase at 610 nm was measured. Hydrophobicity per- strain was inoculated into LB agar (ca. 105 CFU/ml), and centage (H%) was calculated according to the following wells were punched in the plate. The wells were filled with equation [(A0–A) / A0] x 100. Where A0 and A, are the 50 μl of extract and plates were incubated overnight at 30°C. absorbance values before and after n-hexadecane extraction, The diameter of inhibition zones (mm) around the wells was respectively (Mishra and Prasad 2005). Strains were classified measured. as Low, Middle and High according to their degree of hydrophobicity (Espeche et al. 2009).

Probiotic characteristics of Lactobacillus brevis Haemolytic activity

Testing for resistance to antibiotics For testing haemolytic activity, a fresh Lactobacillus brevis (B23) culture was streaked on agar plates, containing 5% The selected Lactobacillus brevis (B23) strain was tested for (w/v) blood, and incubated for 48 h at 30°C (Maragkoudakis resistance to 23 antibiotics produced by Oxoid, Basingstoke, et al. 2006). Hampshire, UK. The result was expressed as sensitive (S) or resistant (R). This testing was performed using the standard disc diffusion method (CLSI 2009). Results and discussion

Tolerance to acidic pH values Isolation and screening for antimicrobial compound production Lactobacillus brevis (B23) was grown in MRS broth at 30°C overnight. The culture was centrifuged at 5,000 g for 10 min at Lactic acid bacteria can produce antagonistic compounds 4°C. The pellets were washed in sterile phosphate-buffered that vary in their spectra of activity. A total of 57 bacteria saline (PBS) at pH7. These pellets were diluted (1/100) in PBS were isolated from the 24 dairy samples. Of these 57 iso- at pH 1, 2 and 3, and incubated for 1, 2 and 3 h. Bacterial cells lates, 38 were Gram-positive, catalase-negative bacilli and were then transferred to MRS broth and incubated at 37°C were identified as presumptive lactobacilli. The cell-free overnight (El-Naggar 2004). Counts of surviving cells were supernatants of the 38 isolates were tested for antimicrobial determined by plating on MRS agar. Bacterial growth was activity against Staphylococcus aureus NCTC-7447, expressed in colony-forming units per milliliter (CFU/ml). Escherichia coli NCTC-10418 and Salmonella typhi The experiment was repeated twice and each reading repre- NCIMB-9331 as indicator strains by agar well diffusion sents the mean of three observations. assay (AWDA) and spot-on-lawn assay. Their sensitivity varied greatly (data not shown). The cell-free supernatant Bile tolerance of strain B23 exhibited the highest inhibitory activity against all indicators. Bile tolerance was determined in MRS broth containing 0.3% (w/v) of oxgall (dehydrated fresh bile; Difco). Before testing Identification of the selected strain for bile tolerance, the strain was incubated at 37°C for 24 h in MRS broth without bile. After centrifugation (5,000 g for In order to confirm the identification of the selected isolate, 10 min, 4°C), the collected cells were resuspended in sterile we performed 16S rRNA gene sequence analysis. The se- saline (0.85% NaCl) and then inoculated into MRS broth quence alignment using BLASTN software for the compar- supplemented with 0.3% oxgall, and enumerated after 0, 1, 2 ison up to 1,500 bp of the analysis gave a high homology of and 4 h of incubation, reflecting the time spent by food in the 99% to Lactobacillus sp. ABRIINW.F58 (Accession num- small intestine (Maragkoudakis et al. 2006). ber HQ406830.1) and 98% homology to Lactobacillus brevis strain NM101-1 (Accession number HM218421.1). Hydrophobicity Kinetics of bacteriocin-like substance production Lactobacillus brevis (B23) was grown in 3 ml MRS broth at pH 6.5. Bacterial cells were removed by centrifugation The selected strain of Lactobacillus brevis (B23) started to (5,000 g for 20 min, 4°C) and resuspended with PBS. Two produce antimicrobial compounds after 8 h incubation in Ann Microbiol (2013) 63:81–90 85

MRS broth at 30°C (Table 1; Fig. 1). Maximum activity was reached after 16 h of incubation (the stationary phase). The maximum OD was also reached after 16 h of incubation. Afterwards, the antibacterial activity slowly decreased. These results are in accordance with those of Ogunbanwo et al. (2003b) who found that bacteriocin was produced during the pre-and early exponential growth phases and reached a maximum level at late stationary phase. Verellen et al. (1998) found that maximum production of plantaricin by a strain of Lactobacillus plantarum coincided with the onset of logarithmic phase and early stages of stationary phase when nutrients were available for metabolic activity. Growth beyond the stationary phase resulted in a decrease in bacteriocin-like substance activity. This decrease could be Fig. 1 Effect of incubation period on bacterial growth (OD 600) and due to the extracellular activity of endogenous proteinases culture pH of L. brevis (B23) induced during this growth phase. Some reports indicate that bacteriocins are produced throughout the experimental growth phase and not solely during late logarithmic or early Table 2 Effect of pH, temperature, chemical surfactants and proteo- stationary phases (Piard et al. 1990). lytic enzymes on the activity of the antimicrobial compounds produced by the strain Lactobacillus brevis (B23) against the three indicator Lactobacillus brevis (B23) also showed moderate acidi- pathogens Staphylococcus aureus, Escherichia coli and salmonella fication activity (Fig. 1). These conditions of low pH with typhi an increase in cell number have also been found to be necessary for the production of high levels of a Treatment Bacteriocin activity bacteriocins-like substance (Ayad et al. 2004). The slow S. aureus E. coli S. typhi decrease of the antibacterial activity in the later stationary growth phase might be due to the partial digestion of the pH antibacterial compound by proteolytic enzymes released pH 2.0 + + + from the cells (Gong et al. 2010) pH 4.0 + + + pH 6.0 + + + Characteristics of the antimicrobial compound pH 8.0 −−− pH 10.0 −−− Results given in Table 2 reveal that the highest antibacterial pH 12.0 + + + activity was exhibited in an acidic pH range of 2–6, while Temperature 60°C 15 min + + + Table 1 Time course of antimicrobial compounds production by L. 30 min + + + brevis (B23) during growth in MRS broth at 30°C for 40 h (means ± 70°C 15 min + + + SD) 30 min + + + Time (h) Inhibition zone (mm) 80°C 15 min + + + 30 min + + + Staphylococcus Escherichia Salmonella 90°C 15 min + + + aureus coli typhi 30 min + + + 0 –––100°C 15 min + + + 4 ––– 30 min + + + 8 7.1 ± 0.28 2.2 ± 0.3 2.0 ± 0.3 Freeze + + + 12 7.5 ± 0.45 6.3 ± 0.75 2.5 ± 0.35 Surfactants 16 11 ± 2.6 8.2 ± 0.1 7.6 ± 0.3 Tween 80 −−− 20 9 ± 0.9 8.6 ± 0.36 7.2 ± 0.34 SDS + + + 24 8.3 ± 0.32 7 ± 0.17 6.0 ± 0.75 Urea + − + 28 5.3 ± 0.62 4.6 ± 0.3 4.3 ± 0.30 Enzymes 32 4.1 ± 0.32 4 ± 0.11 2 ± 0.45 Protease −−− 36 3.2 ± 0.65 ––Lipase −−− 40 –––Trypsine −−− 86 Ann Microbiol (2013) 63:81–90 inactivation occurred at pH 8–10, then antibacterial activity Inhibitory spectrum of the antimicrobial compound was resumed at pH 12. Abdel-Bar et al. (1987) found that produced by Lactobacillus brevis bacteriocins isolated from Lactobacillus bulgaricus, were shown to have the highest activity and stability at low pH. The antimicrobial activity of the bacteriocin-like substance Bacteriocins produced by LAB are generally highly stable produced by L. brevis (B23) in this study was not due to under acidic conditions, but many of them are easily inacti- hydrogen peroxide or acidity, as activity was not lost after vated under neutral and alkaline conditions (Delves- treatment with catalase or adjustment of pH to 6.5. Broughton et al. 1996). This result could be explained According to Fricourt et al. (1994), lactic acid bacteria because, at lower pH values (pH 5 and below), the solubility synthesize bactericidal agents that vary in their spectra of is often increased, less aggregation of hydrophobic peptides activity. The antagonistic activity of the antimicrobial com- occurs, and less bounding of bacteriocins to the cell surface pound produced by L. brevis (B23) was tested against S. takes place. Also, hydrophilic compounds may have an aureus and B. subtilis as Gram-positive bacteria and P. enhanced capacity to pass through hydrophilic regions of aeruginosa, S. typhi, K. pneumoniae and E. coli as Gram- the cell surface of the sensitive target bacteria (Jack et al. negative bacteria. Some of these bacteria can produce toxins 1995). Finally, we could not find any explanation for the resulting in human illness. The results presented in Table 3 return of activity at pH 12 (the experiment was repeated show that the highest inhibitory activity was demonstrated twice with the same result). against E. coli, Staphylococcus and Bacillus while the low- The bacteriocin-like substance produced by L. brevis est activity was demonstrated against Salmonella and Pseu- (B23) was considered to be heat stable, as there was no domonas, but did not inhibit Klebsiella. reduction in activity after heat treatment from 60 to 100°C Bacteriocins produced by Lactobacillus spp. have been for 15 and 30 min (Table 2). The results suggest that the reported to have a broadly inhibitory effect against several partial purified compound is strongly heat resistant. In gen- bacteria (Ogunshe et al. 2007; Karthikeyan and Santosh eral, bacteriocins obtained from lactobacilli are relatively 2009). All the bacteriocins produced by Lactobacillus plan- heat stable with promising inhibitory spectra of antimicro- tarum F1 and L. brevis OG1 isolated from Nigerian fer- bial activities. Heat stability of bacteriocins produced by mented food products had a broad spectrum of inhibition Lactobacillus spp. has been well established (Nettles and against both pathogenic food spoilage organisms and vari- Barefoot 1993). Moreover, the bacteriocins-like substance ous lactic acid bacteria (Ogunbanwo et al. 2003a). Bacter- produced by the tested isolate remained fully stable after iocins produced by LAB are of great interest to the food storage at −20°C for 24 h and thawing at 5°C for 20 min, fermentation industry because they may inhibit the growth thus indicating that low temperature may be the most ap- of many food spoilage and pathogenic bacteria (Nowroozi et propriate preservation technique (Ogunbanwo et al. 2003a). al. 2004). Therefore, an investigation of bacteriocins-like This heat and pH stability may be useful if the bacteriocin is substance in LAB may offer potential applicability in food to be used as an antimicrobial agent in fermented foods or preservation (Khay et al. 2011). So, in addition to the broad thermally processed foods (Oh et al. 2000). inhibition spectrum, its technological properties and espe- The bacteriocins-like substance produced by L. brevis cially heat and storage stability indicate that the bacteriocin (B23) was tested for its sensitivity to various enzymes. It has potential for application as a biopreservative to control is clear from Table 2 that antimicrobial activity was lost or pathogens in processed foods (Abo-Amer 2007). unstable after treatment with all the proteolytic enzymes tested (protease and trypsin). The completely inactivated Table 3 Mean diameters ± SD of inhibition zones (mm) caused by inhibitory activity indicated that it was proteinaceous antibacterial activities produced by Lactobacillus brevis (B23) against in nature and classified as a bacteriocins-like substance different Gram-positive and Gram-negative bacteria (Vaughan et al. 2001). Olukoya et al. (1993)reported Organism Strain no. Inhibition zone (mm) that the protein nature of plantaricin K was confirmed by its sensitivity to trypsin. Similar properties have been Escherichia coli NCTC-10418 8.0 ± 1.0 reported for other bacteriocins including lactacin, lacta- ATCC-10536 7.3 ± 0.3 cin 27, acidolin, pediocin A, and pediocin PA-1 (Jack et Klebsiella pneumoniae ATCC-10031 – al. 1995). Salmonella typhi NCIMB-9331 5.5 ± 0.9 The surface-active agents SDS, urea and Tween 80 at a Pseudomonas aeruginosa ATCC-10145 6.3 ± 0.3 final concentration of 1% in the culture supernatant do not ATCC-25619 6.6 ± 0.4 affect the activity of the produced antimicrobial compound Staphylococcus aureus ATCC-29737 8.9 ± 0.9 (Table 2). For food applications, the influence of all these ATCC-29213 8.4 ± 0.9 substances on the activity of the antimicrobial compound Bacillus subtilis NCTC-10400 7.3 ± 0.3 needs to be taken into account. Ann Microbiol (2013) 63:81–90 87

Elegado et al. (2004) reported after a common observa- Table 4 Antibiotic susceptibility of L. brevis (B23) isolated from dairy tion by bacteriocin workers that resistance to bacteriocin products action is not only species- or strain-specific but also dose- Antibiotic Symbol Antibiotic susceptibility dependent. Strains that have the receptors and proper characteristics of cytoplasmic membrane for proper attachment Penicillin group and sensitivity to the bacteriocin are generally inhibited Amoxycillin AMX 25 S (Ennahar et al. 2000). Ampicillin AMP 10 S Piperacillin PRL 100 S Probiotic characteristics of Lactobacillus brevis Imipenem IPM 10 S Cephalosporin group Probiotics are live and active forms of bacteria that mimic Cefixime CFM 5 R the "friendly" micro-organisms that live in the human colon. Cefotaxime CTX 30 S Probiotics are commonly used in foods and supplements Cephradine CE 30 S because of their health benefits. Lactic acid bacteria origi- Marcolide group nally isolated from dairy products are probably the best Azithromycin AZM 15 S candidates as probiotic bacteria to improve the microbiolog- Clarithromycin CLR 15 S ical safety of these foods. They are well adapted to the Lincosamides group conditions in dairy products and should, therefore, be more Clindamycin DA 2 S competitive than bacteria from other sources. Probiotic Aminoglicosides group microorganisms need to resist the adverse factors in the Amikacin AK 30 S gastrointestinal tract, like the stomach acidity and bile salts Gentamicin CN 10 S excreted in the duodenum (Nowroozi et al. 2004; Iñiguez- Streptomycin S 10 S Palomares et al. 2007) when they pass through it. Chloromphenicol group The results presented in Table 4 indicate that L. brevis Chloromphenicol C 30 S (B23) strain was susceptible to most of the antibiotics tested Tetracycline group and resistance was observed in case of cefixime, nalidixic Tetracycline TE 30 S acid, vancomycin and oxacillin. Kacem and Karam (2006) Quinolanes group found that most strains were susceptible to the antibiotics Ciprofloxacin CIP 5 S tested, which belonged to the major classes of antibiotics Nalidixic acid NA 30 R used in human clinical therapy. The absence of antibiotic Nitrofurantoin F 300 S resistance can be considered a positive trait for bacteria used Teicoplanin TEC 30 S in probiotic food productions. This is not in accordance with Vancomycin VA 30 R other reports indicating that LAB are normally resistant to Rifampicin RD 5 S the principal antibiotics, such as penicillin G, ampicillin, Oxacillin OX 1 R vancomycin, cloramphenicol or ciprofloxacin (Halami et Septrin SXT 25 S al. 2000; Coppola et al. 2005). It is well known that vancomycin (belonging to the glycopeptide antibiotics) inhibits R Resistance, S sensitive the peptidoglycan synthesis which is an important structural component of the bacterial cell wall. In the present case, L. brevis (B23) was resistant to vancomycin. This result con- firms the finding of Salminen et al. (1998) who reported that vancomycin resistance is an intrinsic property of lactobacilli. Nelson (1999) found that all isolates (85 strains) of Leuconostoc, Lactobacillus and Pediococcus were resistant to vancomycin and teicoplanin. Before reaching the intestinal tract, probiotic bacteria must first survive transit through the stomach where the pH can be as low as 1.5–2(Dunneetal.2001). The results of tolerance to acidic pH (survival percentage of L. brevis strain at various pH values) are shown in Fig. 2). The tested strain survived incubation periods of 1–3 h at pH 2.0 and 3.0 with a decrease in survival percentage when the exposure time for strains pro- gresses. No growth occurred after incubation at pH 1 for 3 h. Fig. 2 Acidic tolerance of L. brevis (B23) strains 88 Ann Microbiol (2013) 63:81–90

Similar observations were reported by Dunne et al. (2001)and however, some strains of lactobacilli express α- El-Naggar (2004). The acid tolerance of LAB is dependent haemolysin (Baumgartner et al. 1998). The examined strain upon the pH profile of H+-ATPase and the composition of the was -haemolytic (i.e., no haemolysis) on blood agar plates. cytoplasmic membrane, which is largely dependent upon the Santini et al. (2010) found that none of the probiotic type of bacteria, type of growth media, and the incubation strains possessed haemolytic activity, in agreement with conditions (Havenaar et al. 1992). other studies showing that haemolysis is rarely present Tolerance to bile salts is considered to be a prerequisite in food LAB (Maragkoudakis et al. 2006). for colonization and metabolic activity of bacteria in the small intestine of the host (Havenaar et al. 1992). Bile salts are surface-active chemicals produced in the liver from the Conclusion catabolism of cholesterol. Therefore, when evaluating the potential of using LAB as effective probiotics, it is generally A Lactobacillus brevis strain isolated from Egyptian dairy considered necessary to evaluate their ability to resist the products had good antimicrobial activity against certain effects of bile acids (Lee and Salminen 1995). The exam- pathogenic bacteria. The partially purified inhibiting sub- ined strain is resistance to bile salts: it could survive in their stance is most active at low pH values and retains its activity presence in concentrations simulating the small intestine with an increase in temperature. This suits various food and environment (0.3% w/v) (data not shown). The 2% oxgall pharmaceutical production processes. This Lactobacillus (bile salt) represents the extreme concentration obtained in strain also possesses several good characteristics that make animal or human intestines during the first hour of digestion; it suitable for use with various probiotic products. afterwards, the normal level of bile salt in intestine is around 0.3% (Kacem and Karam 2006). Acknowledgement I warmly thank Dr. Shaimaa Sobieh Lecturer in This tolerance is important if the strain is to be used Cytology and Genetics, University College for Women (Arts, Science and Education), Ain Shams University, for her supportive attitude orally as a probiotic therapeutic, since it has to tolerate a towards our work, and for valuable comments and review on the part bile salt concentration of 0.1–0.3% within the human body. of identification. It has also been mentioned that the resistance to bile salts varies a great deal among the LAB species and even between the strains themselves. Bile resistance of some strains References is related to a specific enzyme, bile salt hydrolase (BSH), activity which helps hydrolyse conjugated bile, thus reduc- Abdel-Bar N, Harris ND, Rill RL (1987) Purification and properties of ing its toxic effect (Kacem and Karam 2006). an antimicrobial substance produced by Lactobacillus bulgaricus. – In order to complete the probiotic criteria, the hydropho- J Food Sci 52:411 415 Abo-Amer AE (2007) Characterization of a bacteriocin-like inhibitory bicity and adherence to gut properties of the selected bacte- substance produced by Lactobacillus plantarum isolated from rial strain were examined. Indeed, the probiotic ability to Egyptian home-made yogurt. 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