Full Paper Bioscience Microflora Vol. 18 (2), 125-131, 1999 Bacteriocin Produced by Streptococcus thermophilus against Bifidobacterium Species Nagendra P. SHAH* and Linh LY School of Life Sciences and Technology, Victoria University of Technology, Werribee Campus, PO Box 14428 , Melbourne City Mail Centre, VIC 8001, Australia Received April 26, 1999; Accepted for publication, August 24, 1999 This study was carried out to evaluate the antagonistic relationship between yogurt and probiotic bacteria and the nature of the inhibitory compound produced by the organisms. Eight strains each of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus and bifidobacteria were isolated from eight commercial AB (L. acidophilus and Bifidobacterium spp.) products containing these four groups of bacteria. The isolates were screened for the production of bacteriocins against each of the 8 isolates of L. acidophilus and Bifidobacterium spp. Twelve strains showed inhibitory activity against all the 8 strains of Bifidobacterium spp. and 5 L. acidophilus isolates with the 'spot on lawn' assay. Of these, only one yogurt bacterium, S. thermophilus was identified to be a bacteriocin- producing organism. The S. thermophilus strain was found to specifically target 2 strains of bifidobacteria. The crude antimicrobial compound was found to be heat stable, resistant over a wide range of pH, and sensitive to proteolytic enzymes, but it retained activity after treatment with lipase. The compound was purified using ultrafiltration, precipitation with ammonium sulfate and dialysis. The bacteriocin-fractionate was also subjected to SDS-PAGE analysis and the molecular weight of the bacteriocin was estimated to be approximately 80 kDa. Key words: yogurt bacteria; probiotic bacteria; antagonism; viability Bacteriocins are defined as 'proteinaceous com- INTRODUCTION pounds that show antibacterial activity against closely The benefits derived from the consumption of related species' (17). While the definition holds true probiotics such as Lactobacillus acidophilus and for majority of bacteriocins, it is now evident that bac- Bifidobacterium spp. (known as AB products) are well teriocins may act beyond closely related species or those documented (4, 8, 11, 15). Probiotic bacteria grow confined within the same ecological niche (9). The pres- slowly in milk, so the usual practice is to add yogurt ence of bacteriocin producing organisms can influence bacteria, Streptococcus thermophilus, and Lactobacil- or alter the stability of a culture. Although bacteriocins lus delbrueckii subsp. bulgaricus to enhance the fer- have been studied for many years, much of work has mentation process to obtain a milk product of 'excel- focused on evaluating the performance of bacteriocins lent therapeutic value' (10). Presently, over 90 prod- as inhibitors for pathogens for food preservation. A re- ucts containing probiotics are available in the market cent study by Joseph et al. (6) reported that the viabil- worldwide. To achieve health benefits, the suggested ity of probiotic organisms was related to antagonism minimum level of probiotic bacteria is 106 viable cells between yogurt and probiotic bacteria. per gram of a product (3). Despite the importance of The viability of probiotic bacteria has been a serious the viability of these beneficial bacteria, various stud- problem. Several factors have appear to be responsible ies have shown poor survival of probiotic organisms, for the viability of probiotic bacteria including acid pro- especially bifidobacteria in fermented foods (12, 14- duced during fermentation and storage, dissolved oxy- 16). The exact cause for loss of viability is uncertain; gen and antimicrobial substances produced by yogurt however, bacteriocin produced by yogurt bacteria bacteria against probiotic bacteria. Dave and Shah (3) against probiotic bacteria is likely to be one of the fac- reported that a strain of bifidobacteria lost its viability tors responsible for the loss of viability of probiotic in yogurt made from starter culture containing S. bacteria. thermophilus, and bifidobacteria. This inhibition was presumed to be due to production of antimicrobial sub- *Corresponding author . Mailing address: School of Life Sciences and Tech- stances produced by S. thermophilus against bifidobac- nology, Victoria University of Technology, Werribee Campus, PO Box 14428, Melbourne City Mail Centre, VIC 8001, Australia. Phone: +61-3-9216-8289. teria. Fax: +61-3-9216-8284. The aims of this study were to (i) determine antago- 125 126 N.P. SHAH and L. LY nism between yogurt bacteria and probiotic bacteria in tine bacterial culturing, ST broth was used for strepto- commercial yogurts, and (ii) characterise the antimi- cocci, MRS broth for lactobacilli and MRS broth crobial substance produced by yogurt organism(s) supplemented with 0.05% L-cysteine hydrochloride against probiotic organism(s). (MRS-C) was used for Bifidobacterium spp. (2), un- less otherwise stated. MATERIALS AND METHODS Detection of inhibitory activity. The spot on lawn Commercial products. S. thermophilus, L. method devised by Tagg et al. (17) was used with some delbrueckii subsp. bulgaricus, L. acidophilus and modification for the preliminary detection of inhibitory bifidobacteria were isolated from eight commercial activity produced by the yogurt and probiotic organ- probiotic AB yogurts: Jalna (Jalna Dairy Foods Pty. isms. Twenty-five millilitres of 1.0% agar was poured Ltd., Thomastown, VIC), Bulla (Regal Cream Prod- into sterile petri plates and left to solidify. Wells were ucts Pty. Ltd., N. Melbourne, VIC), Yoplus (National then cut into the agar using a sterile 7 mm borer and the Foods Ltd., Morwell, VIC), Vaalia (QUF Industries cut portion discarded. The bottom of the wells was Ltd., S. Brisbane, QLD), Ski (Aust. Co-operative Foods sealed with 0.9% sterile agar followed by the addition Ltd., Lidcom, NSW), So Natural (Aust. Natural Foods, of 50 ƒÊl of fresh overnight culture of the producer or- Tarenpoint, NSW), Eve Balance (Dairy Vale Foods ganism into the wells. Plates were then left at room Ltd., Cla. Gardens, NSW), and Nestle (Nestle Dairy temperature for 2 hr to allow migration of the cultures Products, Mulgrave, VIC). through the agar. The wells were then sealed over the Isolation and identification of bacterial cultures. layer of producer organisms and plates incubated at One gram of representative sample was appropriately 37°C for further 3 hr. Finally, the spotted plates were diluted in sterile peptone and water medium (0.15%) overlaid with 10 ml of 0.9% agar seeded with 1.0% of followed by pour plating. The selective media used for indicator strains. Plates were left to solidify and incu- the isolation of S. thermophilus, L. delbrueckii ssp. bated for 72 hr at 37°C, aerobically for S. thermophilus bulgaricus, L. acidophilus and bifidobacteria were ac- and anaerobically for other organisms. All inhibition cording to Dave and Shah (2). The isolates were iden- assays were carried out in duplicates. tified by Gram staining and catalase test and their bio- After the incubation, the plates were examined for chemical characteristics were confirmed by comparing zones of inhibition around the wells. For inhibition as- their carbohydrate fermentation patterns according to says, ST agar devoid of bromocresol purple was used Bergey's Manual (5, 7, 13). for S. thermophilus and MRS agar was used for the The isolates were designated as follows: ST for S. other group of organisms (2). thermophilus, LB for L. delbrueckii subsp. bulgaricus, Producer organisms that tested positive in the initial LA for L. acidophilus, and BB for Bifidobacterium spe- screening were used in this study to determine the na- cies and were numbered 1-8 according to the product ture of the inhibitory substance produced by the organ- used (Table 1). To protect the reputation of the com- isms. The agar well diffusion technique in liquid me- pany, the brand names do not necessarily correlate with dia described by Tagg and McGiven (18) was employed the designated codes. to screen for the presence of bacteriocin activity. Maintenance of bacterial cultures. Bacterial cul- Elimination of effects of organic acids and hydrogen tures were maintained in sterile reconstituted (12%) peroxide. MRS agar (0.9%) held at 45°C was seeded skim milk (RSM). L-Cysteine hydrochloride (0.05%) with 1.0% overnight culture of the indicator organism. was additionally incorporated into RSM for bifidobac- Approximately 25 ml of the agar were poured in sterile teria isolates to lower the oxidation-reduction potential petri plates and left to set and wells were cut into the of the medium and to enhance the growth of anaerobic solidified agar as previously described. Cell-free su- bifidobacteria. Sterile RSM was inoculated with 1.0% pernatant from the broth was collected by centrifuga- of each isolate and incubated at 37°C for approximately tion (4,500 rpm, 15 min, 4•Ž) using a Beckman CS- 18 hr. The coagulated media were then transferred into 15R centrifuge (Beckman Instruments, Palo Alto, CA, 2 ml cryogenic vials (Iwaki Glass, Canada) and stored USA). The crude extract was divided into three por- at 20•Ž as frozen stock cultures. tions: (1) untreated, (2) neutralised to pH 6.0 using 2 All cultures were propagated twice in broth before NaOH and, (3) neutralised to pH 6.0 and treated with use, and subcultured into RSM on a weekly basis for a catalase (0.05-0.1 mg/ml). All the three portions were maximum of 10 subcultures before a new working cul- filter sterilised by passing through a 0.45 ƒÊm membrane ture was made from the frozen stock cultures. For rou- filter and dispensed into sterile Eppendorf tubes. The BACTERIOCIN PRODUCED BY BACTERIA 127 tubes were incubated for 2 hr in a 37•Ž water bath to the medium was adjusted to 5.0-5.5 with concentrated allow for the enzyme reaction. Wells were then filled HC1 and this pH was maintained by adjusting the pH with 200 ,u1 of each sample and left to stand for 2 hr at every 6 hr. After 12 hr incubation, the broth culture room temperature for diffusion of the test material.