Department of Biological Science, Ubon Ratchathani University, Warin Chamrap, Ubon Ratchathani 34190, Thailand

Transcription

1 1937 Journal of Food Protection, Vol. 69, No. 8, 2006, Pages Copyright, International Association for Food Protection Isolation and Preliminary Characterization of a Bacteriocin Produced by Lactobacillus plantarum N014 Isolated from Nham, a Traditional Thai Fermented Pork PONGSAK RATTANACHAIKUNSOPON* AND PARICHAT PHUMKHACHORN Department of Biological Science, Ubon Ratchathani University, Warin Chamrap, Ubon Ratchathani 34190, Thailand MS : Received 11 March 2006/Accepted 14 April 2006 ABSTRACT Lactobacillus plantarum N014 was isolated from nham, a traditional Thai fermented pork, and exhibited antimicrobial activity against Listeria monocytogenes. Its bacteriocin had a broad inhibitory spectrum toward both gram-positive and gramnegative bacteria. The bacteriocin activity was sensitive to all proteolytic enzymes used in this study, including papain, pepsin, pronase E, proteinase K, and trypsin, but was resistant to the other enzymes, such as -amylase, lipase A, and lysozyme. Furthermore, activity was stable over various heat treatments and ph values. The bacteriocin exerted a bacteriolytic mode of action. It was produced during the exponential growth phase and reached its highest level as producer cells entered the stationary phase. Adsorption of the bacteriocin onto producer cells was ph-dependent. No bacteriocin adsorption was detected at ph 1 to 3, whereas 100% bacteriocin adsorption was found at ph 7. Plasmid isolation revealed that L. plantarum N014 contained no plasmids. From Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis and growth inhibition testing against L. monocytogenes, the estimated molecular mass of L. plantarum N014 bacteriocin was 8 kda. Lactic acid bacteria play important roles in the production of fermented dairy products, fermented meat products, fermented fruits and vegetables, and fermented beverages (10, 12, 30, 46). They produce many substances required for the development of flavor, aroma, color, and texture of fermented foods. Those substances are organic acids, diacetyl, exopolysaccharides, L-alanine, and acetaldehyde (33). Lactic acid bacteria can also produce antimicrobial substances, including organic acids, acetoin, hydrogen peroxide, reuterin, and bacteriocins (8, 19, 24). Among the antimicrobial substances produced by lactic acid bacteria, bacteriocins have become substances of extensive studies in recent years because of their unique characteristics and their potential use as natural food preservatives. Bacteriocins are proteinaceous in nature and are produced by various strains of bacteria, most importantly lactic acid bacteria. They usually exhibit antagonistic activity against microorganisms closely related to the bacteriocinproducing organism. Several of these bacteriocins are bacteriocidal against pathogenic and food spoilage bacteria (10, 15, 18, 27, 37, 40, 42). In the production of fermented foods, fermentation can be mediated by naturally occurring bacteria or by intentionally added starter cultures. By the latter type of fermentation, food manufacturers are able to increase process control and product consistency. Several lactic acid bacteria have been used as starter cultures in food production (9, 22, 33, 34, 45), while many others are being intensely studied for future use in the food industry. Furthermore, in re- * Author for correspondence. Tel: 66(45)288380; Fax: 66(45)288380; pongsak@sci.ubu.ac.th. cent years, there have been several reports showing the potential use of bacteriocin-producing lactic acid bacteria as starter cultures for improving food safety and quality (33, 41). Nham, an indigenous fermented pork product of Thailand, is made from ground pork meat and chopped pork skin mixed with cooked rice and seasonings, including salt, pepper, chili, and garlic. It can be consumed in the raw state as a condiment, served cooked on its own, or consumed as part of a main meal. Since the production of nham in Thailand still relies on natural fermentation, final products usually exhibit quality inconsistency and vary in microbial composition. Occasionally, pathogenic bacteria, food spoilage bacteria, or both have been recovered from nham in the market (55). To improve quality consistency and food safety, the use of suitable bacteriocin-producing lactic acid bacteria as starter cultures is now being considered for nham fermentation. This study aimed to screen lactic acid bacteria isolated from nham for the production of bacteriocins against some pathogenic bacteria and to determine some characteristics of the peptide. Bacteriocin-producing lactic acid bacteria obtained from this study will ultimately be developed as starter cultures in the production of nham. MATERIALS AND METHODS Bacterial strains and culture conditions. Lactic acid bacteria screened for bacteriocin production and bacteriocin-producing strain Lactobacillus plantarum N014 were isolated from nham. Bacterial strains used as test organisms are shown in Table 1. All lactic acid bacteria were grown in deman Rogosa Sharpe (MRS) broth and agar at 37C. The remaining bacterial strains

2 1938 RATTANACHAIKUNSOPON AND PHUMKHACHORN J. Food Prot., Vol. 69, No. 8 TABLE 1. Antimicrobial activity of L. plantarum N014 against various strains of bacteria examined by the swab paper disc technique Tested organism Antimicrobial activity a Bacillus cereus ATCC B. subtilis TISTR 008 Enterococcus faecalis TISTR 927 E. hirae TISTR 928 Escherichia coli ATCC E. coli O157:H7 ATCC Klebsiella pneumoniae ATCC Lactobacillus acidophilus ATCC 4356 L. delbrueckii subsp. bulgaricus ATCC L. casei ATCC 334 L. curvatus ATCC L. fermentum ATCC 9338 L. helveticus ATCC L. plantarum N014 L. reuteri DSM L. sake TISTR 911 Lactococcus lactis subsp. cremoris DSM L. lactis subsp. lactis DSM Leuconostoc mesenteroides TISTR 473 Listeria monocytogenes DSM L. innocua DSM Pediococcus pentosaceus TISTR 374 Proteus mirabilis ATCC P. hauseri ATCC Pseudomonas aeruginosa ATCC Salmonella enterica serovar Paratyphi A ATCC 9150 S. enterica serovar Typhi ATCC 6539 Serratia liquefaciens ATCC Shigella dysenteriae ATCC Staphylococcus aureus subsp. aureus ATCC Streptococcus pneumoniae ATCC S. pyogenes ATCC a, have no antimicrobial activity;, have antimicrobial activity (size of inhibition zone less than 10 mm);, have antimicrobial activity (size of inhibition zone between 10 and 12 mm);, have antimicrobial activity (size of inhibition zone more than 12 mm). were cultured in brain heart infusion (BHI) broth and agar, unless stated otherwise. Bacterial stock cultures were stored as frozen cultures at 200C in appropriate culture media containing 20% glycerol (vol/vol). Screening of bacteriocin-producing lactic acid bacteria. Ten grams of nham was added to 90 ml of sterile phosphate buffer (ph 7) and then thoroughly homogenized for 1 min in a Laboratory Blender Stomacher 400 (Seward, London, UK). One hundred microliters of appropriate serial dilutions was spread on 0.2% glucose MRS agar plates. After anaerobic incubation at 37C overnight, the plates that provided separated bacterial colonies were overlaid with 10 ml of 0.2% glucose MRS soft agar (0.7% agar) containing approximately 106 cells of Listeria monocytogenes DSM The plates were anaerobically incubated overnight at 37C and then checked for inhibition zones around colonies of isolated lactic acid bacteria. Spectrum of antimicrobial activity. In this study, the swab paper disc technique (43) was used to detect the antimicrobial activity of L. plantarum N014 against a wide range of test organisms. Each test organism was spread with a sterile swab on appropriate agar plates. Sterile 6-mm-diameter filter paper discs (Schleicher & Schuell, Inc., Keene, N.H.) were placed on the agar plates containing the test strain. Twenty-five microliters of culture supernatant from L. plantarum N014 was pipetted onto the paper discs. The plates were incubated overnight at 37C and then checked for inhibition zones around the paper discs. To prepare culture supernatants of L. plantarum N014, the bacterial culture was anaerobically grown in 0.2% glucose MRS broth at 37C overnight and then centrifuged at 15,000 g for 10 min. The culture supernatant was adjusted to ph 7, treated with catalase (Sigma, St. Louis, Mo.) at a final concentration of 1 mg/ml, and passed through a 0.22-m sterile filter (Sigma). Heat, enzyme, and ph sensitivity. The L. plantarum N014 culture supernatant was treated with heat (100C for 10, 20, and 30 min or autoclaved at 121C for 15 min) or exposed for 1 h at 37C to various enzymes (all from Sigma), including papain, pepsin, pronase E, proteinase K, trypsin, -amylase, lipase A, or lysozyme, each at a final concentration of 1 mg/ml. After heating or enzyme exposure, the remaining antimicrobial activity of the L. plantarum N014 culture supernatant was tested by the swab paper disc technique against L. monocytogenes DSM The culture supernatant of L. plantarum N014 also was adjusted with sterile 5 mm NaOH or 5 mm HCl to different ph values between 1 and 14. Each of the treated culture supernatants was incubated at 25C for 12 h, adjusted to ph 7, and then assayed for antimicrobial activity. Mode of action. Cells of L. monocytogenes DSM cultured at 37C for 18 h were harvested, washed, and suspended in sterile 50 mm phosphate buffer (ph 7) to a final concentration of 10 8 CFU/ml. Fifteen milliliters of a freshly prepared cell suspension of L. monocytogenes DSM was mixed with 1 ml of L. plantarum N014 culture supernatant and incubated at 37C. The optical density at 660 nm (OD 660 ) and the number of viable cells of L. monocytogenes DSM were then determined at 30-min intervals over 2 h. This experiment was performed together with a control experiment that used 0.2% glucose MRS broth instead of the L. plantarum N014 culture supernatant. Bacteriocin production. To examine bacteriocin production by L. plantarum N014 at different growth stages, 2 ml of an overnight L. plantarum N014 culture was inoculated into 100 ml of fresh 0.2% glucose MRS broth. Samples were taken hourly over 12 h for determination of OD 660 and bacteriocin activity. For determining bacteriocin activity, dilutions of the bacterial culture supernatant were tested for antimicrobial activity against L. monocytogenes DSM by the swab paper disc technique. Bacteriocin activity expressed as arbitrary units per milliliter (AU/ml) was defined as the reciprocal of the highest dilution showing definite inhibition of the test strain. Identification of bacteriocin-producing lactic acid bacteria. Bacteriocin-producing lactic acid bacteria were identified on the basis of Gram staining, catalase reaction, and other identification tests described by Schillinger and Lucke (49). The sugar fermentation pattern was obtained by the API 50CHL system as

3 J. Food Prot., Vol. 69, No. 8 BACTERIOCIN PRODUCED BY L. PLANTARUM N specified by the manufacturer (biomérieux, Marcy l Etoile, France). Plasmid DNA isolation. L. plantarum N014 was subjected to plasmid DNA isolation by the method of Anderson and McKay (1). Electrophoresis of isolated DNA was performed on 0.8% (wt/ vol) agarose gels in Tris-acetate buffer at 60 V. The DNA was visualized on a Hoefer MacroVue UVis-20 transilluminator (Amersham Biosciences, Buckinghamshire, UK) after staining with ethidium bromide solution (0.5 g/ml; Sigma). Lactococcus SK 2.1, a lactic acid bacterium isolated in our laboratory that harbors a 3.5-kb plasmid, was used as a positive control. Influence of ph on bacteriocin adsorption by producing cells. In this experiment, L. plantarum N014 was grown in 50 ml of MRS broth containing 0.2% glucose at 37C for 18 h. Thereafter, the cells were harvested by centrifugation at 15,000 g for 10 min, washed with 10 ml of 5 mm sodium phosphate (ph 7), and resuspended in 10 ml of 1 M NaCl (ph 2). After stirring at 4C for 1 h, the cell suspension was centrifuged at 15,000 g for 10 min and resuspended in 1 liter of sterile deionized water. The L. plantarum N014 culture supernatant was prepared from 250 ml of the 18-h-old culture and then concentrated to a final volume of 1 ml. To study the influence of ph on bacteriocin adsorption by producing cells, 0.1 ml of freshly prepared L. plantarum N014 cell suspension and 0.1 ml of culture supernatant were added to 1.8 ml of 5 mm sodium phosphate adjusted to ph 1 to 10. This mixture was incubated at 4C for 30 min and then centrifuged at 15,000 g for 10 min. The supernatant was collected for determining bacteriocin activity. In this experiment, two sets of controls were also used. The first control consisted of 0.1 ml of deionized water instead of culture supernatant, whereas the second control contained 0.1 ml of deionized water instead of L. plantarum N014 cell suspension. The percentage of bacteriocin adsorbed was calculated as 100 {1 [(AU/ml in supernatant AU/ml in control I)/(AU/ml in control II)]}. Purification of bacteriocin. Purification of the bacteriocin produced by L. plantarum N014 was performed according to the method described by Yang et al. (57) with some minor modifications. L. plantarum N014 was grown to early stationary phase (approximately 10 8 cells per ml) in 1 liter of MRS broth containing 0.2% glucose at 37C. The bacterial culture was adjusted to ph 7, heated to 80C for 30 min, pelleted by centrifugation at 15,000 g for 10 min, washed with 5 mm sodium phosphate buffer (ph 7), and resuspended in 50 ml of 100 mm NaCl (ph 2). After 1 h of stirring at 4C, the cell suspension was centrifuged at 15,000 g for 10 min. The supernatant was then dialyzed in a 1,000-molecular-weight-cutoff dialysis bag (Sigma) against distilled water at 4C for 24 h and freeze-dried. Determination of molecular weight of bacteriocin. The purified bacteriocin was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by the Tris-Tricine system (5, 6, 48). The stacking and separating gels used in this study were 10 and 16.5%, respectively. Sample preparation, application of samples, and gel division after electrophoresis were performed as reported by Bhunia et al. (5). After electrophoresis, each gel containing duplicate samples was sliced into two pieces. One half of the gel was stained with Coomassie brilliant blue, destained, and photographed, and the remaining half was used to detect antimicrobial activity of the purified L. plantarum N014 bacteriocin. The gel was washed in deionized water for 2 h at room temperature, placed on a BHI agar plate, and overlaid with BHI soft agar seeded with L. monocytogenes DSM The plate was incubated at 37C overnight, examined for the location of the zone of growth inhibition, and photographed. RESULTS Lactic acid bacteria isolated from nham were initially screened for bacteriocin production against L. monocytogenes DSM Five lactic acid bacteria isolates, designated N003, N014, N049, N087, and N132, inhibited the test strain. When the swab paper disc technique was used to assess the ability of the culture supernatants to inhibit L. monocytogenes DSM 20600, only three of the five isolates (N014, N049, and N132) still exhibited antimicrobial activity. Bacterial isolate N014 was selected for further study because it produced the largest inhibition zone against the test strain. Bacterial isolate N014 was identified as L. plantarum N014 on the basis of cell morphology, Gram staining, catalase activity, other identification tests described by Schillinger and Lucke (49), and sugar fermentation patterns. The L. plantarum N014 culture supernatant was inhibited by various bacteria by the swab paper disc technique (Table 1), with a wide spectrum of activity seen toward both gram-positive and gram-negative bacteria. It inhibited most gram-positive bacteria, except for the organism itself; two of eight strains of Lactobacilli (L. helveticus and L. reuteri); and all strains of streptococci (Streptococcus pneumoniae and Streptococcus pyogenes) in addition to four strains of gram-negative bacteria, including Escherichia coli ATCC 27736, E. coli O157:H7 ATCC 35150, Pseudomonas aeruginosa ATCC 27853, and Shigella dysenteriae ATCC The degree of inhibition against sensitive organisms varied from strain to strain. To confirm that inhibition of all sensitive strains was due to a bacteriocin, the culture supernatant was treated with proteinase K (final concentration of 1 mg/ml) and then tested for antimicrobial activity against all sensitive strains. It was found that the treated culture supernatant was unable to inhibit the test strains. The effects of enzymes and heat on antimicrobial activity of the L. plantarum N014 culture supernatant were determined by the swab paper disc technique against L. monocytogenes DSM Antimicrobial activity of the L. plantarum N014 culture supernatant was not observed after treatment with proteases, including papain, pepsin, pronase E, proteinase K, and trypsin. However, activity was unaffected by -amylase, lipase A, and lysozyme (Table 2) or by 10, 20, or 30 min of boiling. The same result was found with the autoclaved culture supernatant (Table 2). In addition to these findings, no change in antimicrobial activity was detected when the culture supernatant was exposed to ph 2 to 10. However, exposing the culture supernatant to ph 1 and ph 11 to 14 eliminated antimicrobial activity (Table 2). Culture supernatant of L. plantarum N014 decreased the number of viable cells of L. monocytogenes DSM from 10 8 CFU/ml to about CFU/ml within 2 h of exposure, with the OD 660 decreasing from 0.65 to For the control set of experiments, no change was detected in the number of viable cells or the OD 660 of the cell suspension of the sensitive strain (Fig. 1).

4 1940 RATTANACHAIKUNSOPON AND PHUMKHACHORN J. Food Prot., Vol. 69, No. 8 TABLE 2. Effects of heat and enzymes on antimicrobial activity of L. plantarum N014 culture supernatant against L. monocytogenes DSM a Treatment Antimicrobial activity No treatment Heat treatments 100C, 10 min 100C, 10 min 100C, 10 min Autoclaving (121C, 15 min) Enzyme treatments Papain Pepsin Pronase E Proteinase K Trypsin -Amylase Lipase A Lysozyme ph a, have no antimicrobial activity;, have antimicrobial activity (size of inhibition zone more than 12 mm). Bacteriocin activity of L. plantarum N014 was not detectable until the L. plantarum N014 culture entered exponential phase, after which bacteriocin activity continuously increased to a maximum in stationary phase. However, after reaching the highest level, bacteriocin activity decreased very rapidly (Fig. 2). The effect of ph on self-adsorption of bacteriocin produced by L. plantarum N014 was also studied. No bacteriocin adsorption was detected at ph 1 to 3. While the percentage of bacteriocin adsorption increased from 50 to FIGURE 2. Growth and bacteriocin production of L. plantarum N % as the ph increased from 4 to 7, at ph values above 7, adsorption decreased with increasing ph (Fig. 3). L. plantarum N014 was subjected to plasmid isolation by the method of Anderson and McKay (1). After agarose gel electrophoresis, no plasmids were detected in the agarose gels stained with ethidium bromide and observed on a UV transilluminator. Bacteriocin produced by L. plantarum N014 was purified by the method of Yang et al. (57) with some modifications. The purified bacteriocin was analyzed by Tris- Tricine SDS-PAGE. A Coomassie brilliant blue stained SDS-PAGE gel revealed a single protein band with a molecular mass of about 8 kda. In the SDS-PAGE gel overlaid with BHI soft agar containing L. monocytogenes DSM 20600, an inhibition zone was detected at the same position as the protein band in the stained SDS-PAGE gel (Fig. 4). DISCUSSION Among lactic acid bacteria isolated from nham, L. plantarum N014 produced a bacteriocin with a broad spec- FIGURE 1. Effects of L. plantarum N014 culture supernatant on viable counts and absorbance of L. monocytogenes DSM cell suspension. FIGURE 3. Influence of ph on bacteriocin adsorption by producing cells.

5 J. Food Prot., Vol. 69, No. 8 BACTERIOCIN PRODUCED BY L. PLANTARUM N FIGURE 4. Tricine-SDS-PAGE analysis and detection of antimicrobial activity of the purified L. plantarum N014 bacteriocin. (A) Gel stained with Coomassie brilliant blue; lane 1, protein standard M12 (Invitrogen, Paisley, UK); lane 2, purified L. plantarum N014 bacteriocin. (B) Gel overlaid with cells of L. monocytogenes DSM inoculated in BHI soft agar. trum of antimicrobial activity against both gram-positive and gram-negative bacteria. To our knowledge, this is the first report of antimicrobial activity against gram-negative bacteria from a bacteriocin produced by lactic acid bacteria isolated from nham. Antimicrobial activity against gramnegative bacteria is uncommon among bacteriocins produced by lactic acid bacteria, except for species of Lactobacillus, including L. acidophilus (3), L. brevis (38), L. fermentum (3, 47), L. paracasei (11), L. plantarum (3, 16, 35, 38, 52, 54), and L. reuteri (14). Besides Lactobacillus spp., Lactococcus (32), Leuconostoc (17, 47), and Streptococcus (26) were found to exhibit this unusual characteristic. Antimicrobial activity of neutralized catalase-treated culture supernatant from L. plantarum N014 was inactivated by all proteolytic enzymes used in this study but not by -amylase, lipase A, lysozyme, and heat. These results suggest that the substance in L. plantarum N014 culture supernatant responsible for antimicrobial activity is a heatstable peptide, not hydrogen peroxide or an organic acid, and that its activity was not dependent on the presence of either carbohydrate moiety or lipid moiety. Furthermore, antimicrobial activity of L. plantarum N014 culture supernatant was stable over ph 2 to 10. This heat and ph stability may be useful if the bacteriocin is to be used as an antimicrobial agent in fermented foods or thermally processed foods. Bacteriocins characterized to be heat-stable peptides and active over a wide range of ph values are produced by many strains of Lactobacillus, including L. acidophilus 30SC (39), L. brevis OG1 (38), L. brevis SB27 (4), L. curvatus IFPL105 (13), L. plantarum BFE 905 (21), L. plantarum F1 (38), and L. plantarum KW 30 (31). The bacteriocin produced by L. plantarum N014 exerted a bacteriolytic mode of action because it significantly reduced the number of viable cells of L. monocytogenes DSM and the OD 660 of the L. monocytogenes cell suspension. Among known bacteriocins produced by Lactobacillus, some were shown to be bacteriolytic, including plantaricin C (23), plantaricin LP84 (52), and plantaricin SIK-83 (2), while others were shown to have a bacteriocidal effect without cell lysis on sensitive cells, such as acidocin J 1229 (53), gaserricin A (25), plantaricin KW 30 (31), and bacteriocin produced by Lactobacillus lactis subsp. lactis (44). The bacteriocin from L. plantarum N014 was produced during exponential growth and reached the highest level as the producer cells entered stationary phase. Growth beyond stationary phase resulted in a decrease in bacteriocin activity. This decrease could be due to the activity of an extracellular endogenous proteinase induced during this growth phase, with confirmation of this hypothesis requiring further study. Adsorption of L. plantarum N014 bacteriocin onto producing cells was strongly influenced by the ph of the suspending environment. The bacteriocin was completely adsorbed at ph 7, while at ph 3 or below, its adsorption could not be detected. Similar results were found in studies that used pediocin AcH, nisin, sakacin A, and leuconocin Lcm1. The highest percentage of bacteriocin adsorption onto producing cells occurred at ph 6 and 6.5 for pediocin AcH, 6.5 for nisin, 5.5 and above for sakacin A, and 5.5 for leuconocin Lcm1, while no bacteriocin adsorption was detected at ph below 1.5 for pediocin AcH, below 3 for nisin, below 2 for sakacin A, and below 3 and above 8 for leuconocin Lcm1 (57). Genetic determinants for bacteriocin production of Lactobacillus can be either plasmid or chromosomally encoded. Lactobacillus spp. that produce plasmid-linked bacteriocins include L. acidophilus M46 (56), L. acidophilus TK8912 (29), L. brevis SB27 (4), L. curvatus IFPL105 (13), L. sake L45 (36), and L. sake LB706 (50), whereas helveticin 51 (7), helveticin J (28), plantaricin A (20), plantaricin D (21), plantaricin KW 30 (31), and plantaricin S (51) are chromosomally encoded. To determine whether the bacteriocin gene of L. plantarum N014 was plasmid or chromosomally encoded, plasmid isolation was performed, with no plasmids recovered from L. plantarum N014. These results suggest that bacteriocin of L. plantarum N014 was encoded on a chromosome. On the basis of Tricine-SDS-PAGE and growth inhibition testing against L. monocytogenes DSM as an indicator, the bacteriocin produced by L. plantarum N014 was a peptide with a molecular mass of approximately 8 kda. Further investigations are currently in progress to identify the amino acid composition, structure, and classification of the peptide. Major problems in nham production in Thailand are low consistency in product quality and high risk of pathogen contamination, which is due to dependence on naturally occurring bacteria for the fermentation process. Use of bacteriocin-producing lactic acid bacteria as starter cultures in nham production may be potentially beneficial. Since L. plantarum N014 was isolated from nham with its heat- and ph-stable bacteriocin able to kill several strains of pathogens, it may be a suitable microorganism for use in nham production. However, use of L. plantarum N014

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