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 and and the nature of the inhibitory compound produced by the organisms. Eight strains each of Streptococcus thermophilus, 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 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 , 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 , 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 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. was removed from the incubator and cells were removed

Plates were then incubated as described previously and by centrifugation (4,500 rpm, 15 min, 4•Ž) using a zones of inhibition measured. Beckman Centrifuge CS-15R. The cell-free superna-

Sensitivity to proteolytic enzymes. Cell free super- tant was adjusted to pH 6.0 and filter-sterilised to re-

natant from the producer organisms that showed zones move any remaining cells. The supernatant was stored of inhibition after neutralising (pH 6.0) and reacting at 4°C and used as the starting material for purification.

with catalase was tested for sensitivity to proteolytic The neutralized extract containing active ST-1 bac-

enzymes, chymotrypsin, papain, and lipase (final con- teriocin was concentrated ten-fold using an Ultrasart centrations 1.0 mg/ml) (Sigma Chemical Co., St. Louis, Cell 50 unit (Sartorius AG, Germany) and 5, 10, and

MO, USA). The samples were incubated at 37•Ž for 2 20 kDa Sartorius membranes. The supernatant and hr after which the remaining activity was determined retentate were assayed for the presence of inhibitory

by the agar diffusion technique. The experiments were activity using the agar diffusion technique. The retentate carried out in duplicate. containing bacteriocin and other proteins concentrated

Characterisation and purification of the bacteriocin using 20 kDa membrane was fractionated with ammo- produced by S. thermophilus. The 32 isolates obtained nium sulfate at 50% saturation level (291 el, at 0•Ž) to

from the commercial yogurts were screened against precipitate the bacteriocin. After the mixture was stirred each of the eight isolates of bifidobacteria (BB) and L. for 3 hr in an ice bath, the precipitate was collected by

acidophilus (LA) as indicator organisms. This was per- centrifugation (4,800 rpm, 30 min, 0•Ž). The resulting formed to determine the inhibitory activity of yogurt suspension and the liquid supernatant containing am-

organisms in commercial yogurt preparations against monium sulfate were dialysed using a dialysis tubing probiotic bacteria. Only one isolate of S. thermophilus against citrate buffer (0.001 M, pH 6.0) for 24 hr at 4•Ž. (ST-1) produced proteinaceous antimicrobial substance After every fractionation process, the solutions were (which will be referred to as ST-1 bacteriocin) against assayed for their remaining inhibitory activity using the an isolate of Bifidobacterium spp. (BB-2). The S. agar diffusion technique.

thermophilus (ST-1) bacteriocin against BB-2 was fur- The partially purified protein was subjected to SDS- ther studied. The latter two organisms were isolated PAGE analysis using the Bio-Rad mini protein II dual

from the same product. Slabcell (Bio-Rad Laboratories, Hercules, CA, USA). Effect of temperature on stability of bacteriocin pro- A 200 ƒÊl sample was first boiled for 5 min at 100•Ž in duced by S. thermophilus. Cell-free supernatant of an 50 ƒÊl sample buffer containing 2-mercaptoethanol and

overnight culture was obtained by centrifuging (4,500 SDS. All materials required for the preparation of the

rpm, 12 min, 4•Ž) and pH adjusted to 6.0. Aliquots of gel followed the procedures of Walker (19), unless oth- the supernatant were incubated at 50, 60, 70, 80, 90 erwise stated. and 100•Ž for 5 and 30 min and at 121°C for 15 min. After the heat treatments, the tubes were immedi- RESULTS AND DISCUSSION

ately cooled in an ice bath and the supernatant was as- Eight isolates each of S. thermophilus, L. delbrueckii sayed for antimicrobial activity against BB-2. Similarly subsp. bulgaricus, L. acidophilus and Bifidobacterium

treated sterile MRS broth was used as a control. spp. were isolated successfully using the selective me- Effect of pH on stability of bacteriocin produced by dia protocols suggested by Dave and Shah (2). Rybka

S. thermophilus. Samples of the crude bacteriocin were and Kailasapathy (10) suggested that in order to pro- adjusted to pH 1 to 10 using sterile 10% or duce therapeutic benefits, the number of probiotic

4 M NaOH. After 4 hr of incubation in a 37°C water counts should be 106 viable cells per gram of a product. bath, the remaining antimicrobial activity was measured Although the counts of L. acidophilus and bifidobacteria

by the agar diffusion method, using BB-2 as the target were found to be almost two log cycles lower than those organism. of S. thermophilus and L. delbrueckii ssp. bulgaricus,

Partial purification and estimation of molecular the average viable counts in most of the products was weight of ST-1 bacteriocin. Sterile ST broth (500 ml) observed to be between the suggested 105-106 thresh-

was inoculated with 1% of the active strain S. ther- old. However, the count of bifidobacteria in one of the

mophilus-1 and incubated at 37•Ž. The initial pH of products was lower (data not shown). 128 N.P. SHAH and L. Dr

Table 1. Preliminary screening of yogurt and probiotic bacteria isolates for inhibitory activity against probiotic organisms.

11mm, (+++) = 11-13mm, (++++) = >13mm, (-) = no zone. Zone size is inclusive of well diameter. Inhibitory activity was not observed by LA-1, LA-7, and LA-8. Inhibition of LA-6 to LA-8 was not observed.

The 32 isolates were screened for the presence of L. acidophilus producer strains posed a greater in- inhibitory activity against the eight strains each of L. hibitory effect on bifidobacteria isolates than S. acidophilus and Bifidobacterium spp. Table 1 summa- thermophilus, L. delbrueckii subsp. bulgaricus and rizes the results from the preliminary screening of sen- bifidobacteria producer strains. An exception was the sitive organisms to antagonistic activity from the pro- ST-1 producer strain, which distinctively targeted the ducer organisms using the spot on the lawn method. BB-2 and BB-3 strains, producing zones of inhibition The results showed that bifidobacteria strains were more greater than 13 mm. vulnerable to inhibitory activity as evidenced by much As the inhibitory substance may be due to organic larger zones of inhibition compared to L. acidophilus acid, or bacteriocin, the agar diffu- strains. Inhibition of several bifidobacterial strains was sion assay was performed for the confirmation of the observed by a majority of producer strains of L. acido- type of antagonistic compound. Table 2 shows the in- philus, two strains of S. thermophilus and three strains hibitory activity of the producer organisms after neu- of L. delbrueckii subsp. bulgaricus. However, only tralization and treatment with catalase of the cell-free minimal inhibition zones were observed by two L. aci- supernatant in order to eliminate the effects of acid and dophilus strains, three L delbrueckii subsp. bulgaricus, hydrogen peroxide. S. thermophilus-1 was the only and two strains of Bifidobacterium spp. against five of strain found to be active against its target organisms, the indicator L. acidophilus strains. while all the other strains lost their inhibitory activity. BACTERIOCIN PRODUCED BY BACTERIA 129

Table 2. Agar diffusion screening of organisms possessing antimicrobial activity.

Zones of inhibition: + = zones <9mm, ++ = 10-13mm, - = no zone.

ST1 producer organism was the only organism found to have ƒÕantibacterial activity against BB2

and BB3 organisms.

Treatment of the supernatant to eliminate possible in- motrypsin and papain, while the activity was unaffected hibitory effects of organic acids (neutral pH) and hy- after treatment with lipase. These results agree with the drogen peroxide (reaction with catalase) confirms the study of Aktypis et al. (I) which noted the same effect inhibitory compound of ST-1 to be a bacteriocin (re- of thermophilin T after the treatments with chymo- ferred to as ST-1 bacteriocin), as shown in Table 2. trypsin, papain, and lipase. From the plate count results, the viable counts of Bacteriocins possess a number of properties that al- bifidobacteria in yogurts were found to be lower, espe- low them to be identified according to their molecular cially in the commercial product from which ST-1 was weight, susceptibility to enzymes, heat tolerance, and isolated (data not shown). This may suggest that the resistance to low pH. Some characteristics of ST-1 bac- survival of bifidobacteria may be related to the inhibi- teriocin was studied including sensitivity to various tem- tory compound produced by ST-1. Dave and Shah (3) peratures and pH, and the molecular weight of the bac- reported that a strain of bifidobacteria lost its viability teriocin was determined using sodium SDS-PAGE (so- in yogurts made from commercial starter cultures that dium dodecyl sulphate-polyacrylamide gel electro- contained yogurt and probiotic bacteria. Their investi- phoresis) analysis. gation could be related to the production of antimicro- Table 3 shows the effect of temperature on the crude bial substances by yogurt organisms present in yogurt. ST-1 bacteriocin and Table 4 the effect of pH on stabil- The inhibitory activity of the ST-1 broth was lost ity of ST-1 bacteriocin. The bacteriocin retained full after treatment with proteolytic enzymes including chy- activity after various heat treatments including auto- 130 N.P. SHAH and L. LY

Table 3. Effect of temperature on the stability Table 4. Effect of pH on stability of ST-1 bacteriocin. of the ST-1 bacteriocin.

Fig. 1. SDS-PAGE of purified ST-1 bacteriocin.

claving at 121°C for 15 min and was active over a wide only displayed inhibitory activity. Thus the molecular range of pH. The bacteriocin was found to be optimally weight of the bacteriocin appeared to be greater than active at pH between 6 to 10, and some inactivation 20 kDa. The concentrated retentate (concentration ra- occurred at pH between 3 to 5. However, total loss of tio 10) was precipitated using ammonium sulfate (50% activity was observed below pH 2.0. These findings saturation). Some activity still remained after this frac-

agree with the results of Dave and Shah (3) and Aktypis tionation stage, as determined by the agar diffusion as- et al. (I). `Thermophilin T,' a bacteriocin produced by say. However, at this point, the bacteriocin activity was S. thermophilus ACA-DC 0040 was found to be stable lost to a considerable extent (data not shown).

at pH between 1 to 9, and inactivation of the bacterio- After dialysis, the protein concentration was deter- cin occurred only at pH 10 to 12. The bacteriocin was mined using modification of Lowry assay (3). The pro-

heat stable at 121°C for 30 min without any loss of tein remaining in the last fractionate was found to be activity. approximately 3.6 ƒÊg/ƒÊl. SDS-PAGE analysis of the

A series of purification steps were employed to par- purified bacteriocin showed a molecular weight of ap-

tially purify the bacteriocin and to determine the mo- proximately 80 kDa. Figure 1 shows the band of the lecular weight of the putative bacteriocin compound. bacteriocin obtained from SDS-PAGE. The compound was concentrated by passing the super- natant through 5, 10, and 20 kDa membrane filters (Sar- CONCLUSIONS torius). After screening for inhibitory activity of the In this study, eight commercial yogurts sold in the

retentate and permeate fractions, the retentate fraction Australian market were assessed for the inhibitory ac- BACTERIOCIN PRODUCED BY BACTERIA 131 tion of bacteriocins released by yogurt bacteria against yoghurt bacteria and probiotic bacteria isolated from com probiotic bacteria. Of the 32 isolates obtained, 12 strains mercial starter cultures, commercial yoghurts and a (consisting of L. acidophilus 2-6, S. thermophilus 1 probiotic capsule. Food Aust 50: 20-23. and 5, L. delbrueckii ssp. bulgaricus 1,4, and 5, and (7) Kandler 0, Weiss N. 1986. Lactobacillus. In Bergey's Manual of Systematic Bacteriology, Vol 2, Sneath bifidobacteria 4 and 6) were identified to release in- PH, Mair NS, Sharpe ME, Holt JG (eds), The William and hibitory activity against the eight isolates of Bifidobac- Wilkins, Baltimore. terium spp. and five strains of L. acidophilus. How- (8) Kim HS. 1988. Characterisation of lactobacilli and Bifido- ever, only one strain of yogurt bacteria, S. thermophilus bacteria as applied to dietary adjuncts. Cult Dairy Prod J (ST-1) was found to be a bacteriocin-producing organ- 24: 6-9. ism. The bacteriocin compound was proteinaceous in (9) Klaenhammer TR. 1993. Genetics of bacteriocins produced nature as confirmed by treatment to various proteolytic by . FEMS Microbiol Rev 12: 39-86. enzymes. (10) Rybka S, Kailasapathy K. 1995. The survival of culture bacteria in fresh and freeze-dried AB yoghurts. Aust J Dairy The ST-1 strain was found to target the Bifidobac- Tech 50: 51-57. terium sp.-2 (BB-2) and Bifidobacterium sp.-3 (BB-3). (11) Saloff-Coste CJ. 1997. Nutritional and health benefits of The bacteriocin was heat stable, even after autoclaving yoghurt and fermented . Danone World Newsletter, for 121°C for 15 min. Further, the inhibitory compound November, Issue 16. was found to be active at a pH range of 6 to 10. (12) Samona A, Robinson RK. 1994. Effect of yoghurt cultures Purification of the bacteriocin was carried out by on the survival of bifidobacteria in fermented milks. J Soc ultrafiltration, precipitation with ammonium sulphate Dairy Tech 47: 58-60. and dialysis. SDS-PAGE of the bacteriocin showed that (13) Scardovi V. 1986. Genus Bifidobacterium. In Bergey's Manual of Systematic Bacteriology, Vol 2, Sneath PH, Mair it had a molecular mass of approximately 80 kDa. NS, Sharpe ME, Holt JG (eds), The William and Wilkins, Baltimore, p. 1418-1434. REFERENCES (14) Shah NP, Ali JF, Ravula RR. 1999. Populations of Lacto- (1) AktypisA, KalantzopoulosG, Huisin't VeldJHJ, ten Brink bacillus acidophilus, Bifidobacterium spp. and Lactobacil- B. 1998.Purification and characterizationof thermophilin lus casei in commercial . Bio- T, a novel bacteriocinproduced by Streptococcusther- science Microflora (in press). mophilusACA-DC 0400. J Appl Microbiol84: 568-576. (15) Shah NP, Lankaputhra WEV. 1997. Improving viability of (2) DaveRI, ShahNP. 1996.Evaluation of mediafor selective Lactobacillus acidophilus and Bifidobacterium spp. in yo- enumerationof Streptococcusthermophilus, Lactobacillus ghurt. Int Dairy J 7: 349-356. delbrueckiispp. bulgaricus, Lactobacillus acidophilus and (16) Shah N, Lankaputhra WEV, Britz M, Kyle WSA. 1995. bifidobacteria.J DairySci 79: 1529-1536. Survival of L. acidophilus and (3) DaveRI, Shah NP. 1997.Viability of yoghurtand probiotic commercial yogurt during refrigerated storage. Int Dairy J bacteriain yoghurtsmade from commercial starter cultures. 5: 515-521. Int DairyJ 7: 31-41. (17) Tagg RJ, Dajani SA, Wannamaker WL. 1976. Bacterio- (4) Gilliland SE. 1990.Health and nutritionalbenefits from cins of gram-positive bacteria. Bact Rev 40 (3): 722-756. lactic acid bacteria.FEMS Microbiol Rev 87: 175-188. (18) Tagg RJ, McGiven AR. 1971. Assay system for bacterio- (5) HardieJM. 1986.Other Streptococci.In Bergey'sManual cins. Appl Microbiol 21: 943. of SystematicBacteriology, Vol 2, SneathPH, Mair NS, (19) Walker JM. 1996. SDS polyacrylamide gel electrophore- SharpeME, Holt JG (eds),The Williamand Wilkins,Bal- sis of proteins. In The Proteins Protocol Handbook, Walker timore,p. 1068-1071. JM (ed), Hamana Press, New Jersey, USA, p. 55-61. (6) JosephJP, DaveIR, ShahNP. 1998.Antagonism between