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 antimi- crobial 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 sam- ples 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.