World Journal of Microbiology & Biotechnology (2005) 21: 351358 DOI 10.

1007/s11274-004-2610-9

Springer 2005

Characteristics of the bacteriocin produced by Lactococcus lactis subsp. cremoris CTC 204 and the eect of this compound on the mesophilic bacteria associated with raw beef
R. Bromberg*, I. Moreno, R.R. Delboni, H.C. Cintra and P.T.V. Oliveira Instituto de Tecnologia de Alimentos, Av. Brasil 2880, 13070-178, P.O. Box 139, Campinas, Sao Paulo, Brazil *Author for correspondence: Tel.: +55-19-3743-1880, Fax: +55-19-3743-1882, E-mail: renatab@ital.sp.gov.br
Received 12 February 2004; accepted 17 August 2004

Keywords: Bacteriocin, characterization, lactic acid bacteria, Lactococcus lactis subsp. cremoris, meat, mesophilic bacteria, preservation

Summary Screening for the bacteriocin production of strains of lactic acid bacteria from various meat and meat products resulted in the detection of a bacteriocin-producing Lactococcus lactis subsp. cremoris CTC 204, isolated from chicken. The bacteriocin inhibited not only closely related lactic acid bacteria (Lactobacillus helveticus), but also pathogenic microorganisms (Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, and Clostridium perfringens). It was inactivated by a-chymotrypsin, cin, papain, and pronase E, but not by lipase or pepsin. This compound was heat stable even at autoclaving temperature (121 C for 10 min) and was produced during refrigerated storage. It was also active over a wide pH range (210), but the highest activity was observed in the lower pH range. The results indicated that dipping raw beef in the bacteriocin produced by strain CTC 204 could contribute to the extension of the shelf life of refrigerated bovine meat.

Introduction Bacteriocins are widely produced by lactic acid bacteria. These compounds have received much attention mainly because of their potential use as natural food preservatives (Cleveland et al. 2001). Furthermore, they show an interesting potential application as food additives in the control of food spoilage and pathogenic foodborn microorganisms (Yang & Ray 1994; Muriana 1996). Bacteriocins have traditionally been dened as proteinaceous compounds produced by bacteria, which inhibit (bacteriostatic) or kill (bactericidal) closely related species (Tagg et al. 1976). However, this denition has been extended to include similar compounds that act on a broader range of species and contain other functional moieties, such as carbohydrates or lipids (Eckner 1992). Bacteriocin-producing strains can be used as part of, or adjuncts to, starter cultures for fermented foods, in order to improve safety and quality (Caplice & Fitzgerald 1999; Lucke 2000). Bacteriocin-like substances produced by lactic acid bacteria from dairy products, fruits and vegetables are well known (Eckner 1992). However, bacteriocins produced by lactic acid bacteria associated with meat, such as Pediococcus, Leuconostoc, Carnobacterium, Lactobacillus, and Lactococcus genera, are likely to have a much greater potential as preservatives in their

application to meat (McMullen & Stiles 1996). According to Schillinger & Lucke (1989), lactic acid bacteria originally isolated from meat and meat products are probably the best candidates for improving the microbiological safety of these foods, because they are well adapted to the conditions in meats and should therefore be more competitive than lactic acid bacteria from other sources. Some bacteriocins have been tested for the biopreservation of meat (Hugas 1998). Nisin, produced by Lactococcus lactis subsp. lactis, has not been very successful because of its low solubility, uneven distribution and lack of stability. Moreover the required eective dose is uneconomical and exceeds the acceptable daily intake for a consumption of 100 g meat/day and an average weight of 60 kg. Pediocin, produced by Pediococcus acidilactici, is more suitable for use in meat and meat products than nisin. However, this microorganism is not an indigenous meat strain and is not able to grow and thus produce bacteriocin at retrigeration temperatures. An extensive screening of nearly 800 bacteriocinproducing lactic acid strains isolated from a variety of meat and meat products led to the selection of several strains which demonstrated antibacterial activity (R. Bromberg et al. 2004). According to the results of the well diusion assay 128 strains inhibited the growth of the indicator strains, Staphylococcus aureus CTC 033

352 and/or Listeria innocua Lin 11. The activity of the inhibitory agent was tested under conditions that eliminated the possible eect of organic acids, hydrogen peroxide and bacteriophages. Of these colonies, one designated as CTC 204 secreted inhibitory substance into the culture medium. In the present investigation, some characteristics of a bacteriocin produced by strain CTC 204, such as its antibacterial activity, stability at different pH values and temperature conditions, growth characteristics and bacteriocin production, were studied. Also, the effect of this compound on the mesophilic population present in minced raw beef was evaluated.

R. Bromberg et al. for the other microorganisms Trypticase Soya Broth (TSB, Oxoid) was used. Both media were supplemented with 15.0% glycerol. Working cultures were prepared as slants on MRS Agar (Oxoid) for the producer bacterium or TSA Agar with 0.6% yeast extract supplement (Oxoid) for the others, and stored at 4 C. Cultures for experiments were streak-plated once a week, inoculated into media from a single colony and incubated for 24 h. Before use, the microorganisms were transferred twice into MRS Broth for lactic acid bacteria or TSB Broth for the other bacteria, and incubated according to the growing conditions presented in Table 1. Unless otherwise stated, Bacillus cereus CTC 001 was used as the indicator in bacteriocin activity assays. Identication of the isolate producing the antimicrobial substance The selected strain CTC 204 was further characterized and identied on the basis of the Gram stain, catalase reaction, reduction of nitrate to nitrite, salt tolerance, ability to grow at different temperatures and pH values (assayed according to Harrigan & McCance 1976) and glucose metabolism (Elortondo et al. 1999). Carbohydrate fermentation and other tests were determined using the BBL Crystal Identication Systems, GramPositive ID Kit (Becton & Dickinson Microbiology Systems, Maryland, USA). Bacteriocin preparation

Materials and methods Microorganisms and their maintenance The bacteriocin-producing bacterium, strain CTC 204, used in this study was obtained from the microbiological culture collection at the Centro de Tecnologia de Carnes, Instituto de Tecnologia de Alimentos, Campinas, Sao Paulo, Brazil. This strain was isolated from raw chicken giblets. The indicator bacterium, the spoilage and foodborn microorganisms used in the experiments are listed in Table 1. The stock cultures of lactic acid bacteria were maintained at )80 C in de Man Rogosa Sharpe Broth (MRS, Oxoid Ltd., Basingstoke, UK) and

Table 1. Microorganisms, strains and growth conditions. Microorganisms Bacillus cereus ATCCa 14579 B. cereus CTCc 001 Clostridium perfringens CTC 042 CI. sporogenes CTC 006 Enterococcus faecalis ATCC 19433 Escherichia coli ATCC 25422 Lactobacillus helveticus (Wiesbyd) Lb. plantarum TECNOLATf 434 Leuconostoc mesenteroides ATCC 10830 Listeria innocua Lin 11 (INRAg) L. monocytogenes CTC 021 Micrococcus sp. ATCC 4698 Pseudomonas sp. CTC 032 Salmonella typhimurium ATCC 14028 Staphylococcus aureus CTC 033 Streptococcus sp. ATCC 25175 Sulphite-reducing clostridium CTC 005 Weissella viridescens CCTf 0849
a

Growth conditions TSBb 24 h/30 C TSB 24 h/30 C TSB 24 h/37 C TSB 2448 h/37 C TSB 24 h/30 C TSB 24 h/37 C MRSe 2448 h/45 C MRS 24 h/30 C MRS 24 h/30 C TSB 24 h/37 C TSB 24 h/37 C TSB 24 h/30 C TSB 24 h/37 C TSB 24 h/37 C TSB 24 h/37 C TSB 24 h/30 C TSB 24 h/37 C MRS 24 h/30 C

The bacteriocin-producing strain was grown in MRS Broth for 24 h at 30 C. Cell-free supernatants were collected by centrifugation (7500 g, 10 min, 4 C) of overnight MRS broth cultures. The supernatant uid was neutralised to pH 6.5 with 10 M NaOH and sterilized by heating at 95 C for 5 min. This crude bacteriocin preparation was kept at 5 C until use. Inhibitory activity spectrum The inhibitory spectrum of strain CTC 204 was tested against the bacteria shown in Table 1. The test organism was assayed by the serial twofold dilution assay of Mayr-Harting et al. (1972). The titre was dened as the reciprocal of the highest dilution showing an inhibition of the indicator strain multiplied by 100 to express the results as activity units per millilitre (AU/ml). Sensitivity of the bacteriocin-like substance to enzymes Cell-free supernatant at pH 6.5 was treated with the following enzymes (0.2 mg/ml): cin (3.4.22.3., Sigma Chemical Co., Dorset, England) in 20 mM sodium phosphate, pH 7.0; trypsin (EC 3.4.21.4., Sigma) in 40 mM TrisHCI, pH 8.2; a-chymotrypsin (EC 3.4.21.1., Sigma) in 20 mM TrisHCl, pH 8.0; pronase E (EC 3.4.24.4., Sigma) in 20 mM TrisHCI, pH 7.8;

ATCC American Type Culture Collection, Rockville, MD, USA. TSB Trypticase Soya Broth. c CTC Centro de Tecnologia de Carnes, Instituto de Tecnologia de Alimentos, Campinas, SP, Brazil. d Wiesby GmbH & Co. KG, Germany. e MRS de Man Rogosa Sharpe Broth. f TECNOLAT Centro de Tecnologia de Latic nios, Instituto de Tecnologia de Alimentos, Campinas, SP, Brazil. g INRA Institute National de Recherches Agronomiques, Jouyen-Josas, France. h CCT Fundacao Tropical Andre Tosello, Campinas, SP, Brazil.
b

Bacteriocin: properties and use in meat pepsin (EC 3.4.23.1., Merck Darmstad, Germany) in 0.002 M HCI; lipase (3.1.1.3., Merck) in 0.1 M potassium phosphate, pH 6.0; papain (3.4.22.2., Sigma) in 0.05 M sodium phosphate, pH 7.0. All these solutions were lter-sterilized through Millex GV 0.22 lm lters (Millipore S.A., St. Quentin-en-Yvelines, France) and then added to sterile cell-free supernatants (v/v, 1/1) at pH 6.5. The controls consisted of enzyme solutions without bacteriocin and cell-free supernatant alone in 0.1 M sodium phosphate buer. The samples and controls were incubated at 37 C for 2 h and heated in boiling water for 5 min to denature the enzymes. Cell-free supernatant uid from the bacteriocin-producing strain was obtained as described before. The remaining bacteriocin-like substance activity was determined by the serial twofold dilution assay (Mayr-Harting et al. 1972). The titre was dened as mentioned before. Resistance of the antibacterial substance under different temperature conditions and pH values To determine the thermal stability at different pH values, the cell-free. supernatant, adjusted to different pH values (2.0, 4.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 12.0) was heated at 65 C for 30 min, 100 C for 10 min, and 121 C for 10 min, or cooled at 4 C for 24 h. The treated and untreated samples were assayed for bacteriocin-like substance activity by the critical dilution assay (Mayr-Harting et al. 1972). Bacteriocin production at different temperatures In order to study the effect of incubation temperature on growth and bacteriocin production, stationary phase cells of strain CTC 204 were inoculated at 1% (v/v, 105 106 c.f.u./ml) into buered MRS Broth and incubated at its optimum temperature (37 C), abusive temperature for meat storage (25 C), refrigeration temperature (4 C) and freezing temperature ()20 C). At appropriate intervals the following determinations were performed: biomass by absorbance at OD600 nm in an u.vvisible spectrophotometer (Varian, Cary 1 E, USA), pH (Toledo Mettler, MP125, Switzerland), and the antibacterial activity of the bacteriocin-like substance by the twofold dilution assay (Mayr-Harting et al. 1972). Meat preparation and bacteriocin addition Twenty-ve-gram portions of raw minced beef were dipped into the bacteriocin preparation and maintained there for 5 min following by draining for 2 min. The samples were packed into sterile plastic bags and stored at 4 C. Samples without added bacteriocin, but dipped into sterile distilled water, were used as controls. The tests were repeated three times at selected times with three different beef samples. For the microbiological determinations, each sample was homogenized with 225 ml of 0.1% peptone water, using a stomacher

353 (Seward Laboratory, Model 400, London, England). Further decimal dilutions were prepared using 0.1% peptone water as the diluent. The dilutions were plated in duplicate on Agar Plate Count (APC, Merck 1.05463) for total mesophilic counts. Inoculated plates were incubated aerobically at 35 C for 48 h. Results were expressed as log c.fu./g. The pH values of the samples were monitored with a pH meter at the moment of sampling. The antibacterial activity of the bacteriocinlike substance was determined by the twofold dilution assay, using L. innocua Lin 11 as indicator. Specic count values were calculated by linear regression (95% condence interval).

Results and discussion Identication of the bacteriocin-producing strains Strain CTC 204 was identied as a Gram-positive, nonmotile, catalase-negative coccus, producing no gas from glucose. The strain reduced nitrate to nitrite, grew at 10 C, at pH 4.4 and in media containing 6.5% NaCl but not at 45 C, pH 9.6 or in 18% NaCl. Based on these characteristics, the analysis of the carbohydrate fermentation pattern and other reactions using the BBL Crystal Identication System (Table 2), strain CTC 204 was tentatively identied as Lactococcus lactis subsp. cremoris (0.9917%). Strains of Lc. lactis ssp., organisms traditionally associated with dairy and vegetable products, have been isolated from fermented sausage (Rodriguez et al. 1995), raw pork (Garver & Muriana 1993), vacuum-packed seafood (Mauguin & Novel 1994) and cooked poultry meat (Barakat et al. 2000). Lc. lactis ssp. has been employed in meat starter cultures for a variety of fermented meat products (Cleveland et al. 2001; Scannell et al. 2001). Antimicrobial activity According to the inhibitory spectrum of activity of the bacteriocin-like substance produced by Lc. lactis subsp. cremoris CTC 204, it presented activity against one closely related bacterium (Lb. helveticus), pathogenic strains of S. aureus CTC 033, L. monocytogenes CTC 021, Cl. perfringens CTC 042, B. cereus CTC 001 and other contaminants such as L. innocua Lin 11 (not shown). Like other bacteriocins produced by lactic acid bacteria (Piard et al. 1990), it was not eective (0 AU/ ml) against the Gram-negative bacteria tested (E. coli ATCC 25422, Pseudomonas sp. CTC 032, and Salm. typhimurium ATCC 1402). The mentioned bacteriocin activity was based on the choice of an arbitrary endpoint, the last dilution showing complete inhibition of the indicator strains. Bacteriocin produced by strain CTC 204 was two times more potent over Lb. helveticus (Wiesby) (800 AU/mt) than against L. innocua Lin 11, L. monocytogenes CTC 021, and S. aureus

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Table 2. Growth, morphological and biochemical prole of strain CTC 204. Tests employed Morphology Catalase reaction Glucose metabolism Nitrate reaction Growth at 10 C Growth at 45 C Growth in 6.5% NaCl Growth in 18% NaCI Growth at pH 4.4 Growth at pH 9.6 4MU-b-D -g1ucoside L -Valine L -Phenylalanine 4MU-a-D -glucoside L -Pyroglutamic acidAMC L -Tryptophan L -Arginine 4 MU-N-acetyl-b-D -glucosaminide 4 MU-phosphate 4 MU-b-D -glucuronide L -Isoleucine p-Nitrophenyl-b-D -glucoside p-Nitrophenyl-b-D -cellobioside p-Nitrophenyl-phosphate p-Nitrophenyl a-D -maltoside o-Nitrophenyl-b-D -galactoside (ONPG) and p-nitrophenyl-a-D -galactoside Proline and Leucine-p-nitroanilide Urea Esculin Trehalose Lactose Methyl-a- and b-glucoside Sucrose Mannitol Maltotriose Arabinose Glycerol Fructose Arginine Strain characteristics Gram positive, coccus, non-motile ) Homofermentative + + ) + ) + ) + ) + ) + ) ) ) + + ) + ) ) ) + ) ) ) ) ) ) ) ) ) ) ) + )

R. Bromberg et al. Sensitivity to proteolytic and lipolytic enzymes Bacteriocins possess a protein moiety, which is responsible for the inhibition of the target organisms. The loss of the antimicrobial activity after treatment with enzymes indicated the sensitivity of the active compounds secreted by the lactic acid strains. After treatment with a-chymotrypsin, cin, papain and pronase E, the activity of the antibacterial compound produced by Lc. lactis subsp. cremoris CTC 204 decreased from 800 to 0 AU/ml. Trypsin partially inactivated (75%) this compound, since the activity was reduced from 800 to 200 AU/ml. It was completely resistant to degradation by pepsin and lipase (800 AU/ml). Nisin is an extensively characterized bacteriocin produced by some strains of Lc. lactis subsp. lactis, isolated from dairy products and applied internationally to many food products as a preservative (Hurst 1983). In a comparison of nisin with the bacteriocin of strain CTC 204, it is important to note that this compound was also inactivated by a-chymotrypsin (Jarvis & Mahoney 1969). Pepsin aects neither the activity of nisin (Hurst 1983) nor that of the bacteriocin produced by strain CTC 204. Strains of Lc. lactis ssp. isolated from vegetables were resistant to pepsin and trypsin, but were inactivated by a-chymotrypsin (Uhlman et al. 1992). The bacteriocins produced by Lc. lactis ssp. BFE 1500 isolated from cheese (Olasupo et al. 1999) and Lc. lactis subsp. lactis A164 isolated from fermented vegetables (Choi et al. 2000) were also sensitive to achymotrypsin and resistant to pepsin. Sensitivity to cin and pronase E was reported by Moreno et al. (2000) for the bacteriocin produced by Lc. lactis subsp. lactis ATCC 11454. Lc. lactis subsp. diacetylactis S50 produces a bacteriocin-designated sensitive to pepsin, trypsin, and pronase E (Kojic et al. 1991). Lacticin 481, a bacteriocin produced by Lc. lactis 481, was completely inactivated following digestion with cin, partly inactivated by a-chymotrypsin and pronase, but unaected by trypsin (Piard et al. 1990). Sensitivity to trypsin is a characteristic of diplococcins but not of nisin (Davey & Richardson 1981). According to these authors, the proteolytic enzymes trypsin, pronase, and a-chymotrypsin completely inactivated diplococcin 346, produced by Lc. lactis subsp. cremoris. It is interesting to note that the pancreatic enzymes (trypsin and a-chymotrypsin) inactivated the bacteriocin-like substance produced by strain CTC 204. This is an important aspect with respect to food safety, since the digestive enzymes can destroy the bacteriocins (Caplice & Fitzgerald 1999). The destruction of the antimicrobial activity by proteases suggested that this compound could be a peptide or bacteriocin. Effect of temperature and pH on the antibacterial compound Hurdle technology combines different preservation methods to inhibit microbial growth. Bacteriocins often

CTC 033, that presented activity of 400 AU/ml over each one. The bacteriocin was less potent over B. cereus CTC 001 and Cl. perfringens CTC 042 (200 AU/ ml). The other strains tested (B. cereus ATCC 14578, Cl. sporogenes CTC 006, Ent. faecalis ATCC 19433, Lb. plantarum TECNOLAT 434, Leuc. mesenteroides ATCC 10830, Micrococcus sp. ATCC 4698, Streptococcus sp. ATCC 25175, sulphite-reducing clostridia CTC 005, and W. viridescens CCT 0849) were resistant (0 AU/ml) to the bacteriocin-like substance produced by this culture. Bactericidal activity against Listeria has also been reported for other bacteriocins produced by Lc. lactis ssp. (Muriana 1996; Moreno et al. 2000). Many bacteriocins produced by lactic acid bacteria act against bacteria closely related to the producer organisms and also inhibit Listeria. According to Lucke (2000), only few are eective against Bacillus, Clostridium, and Staphylococcus, as veried for strain CTC 204.

Bacteriocin: properties and use in meat have synergies with other treatments and can be used as a hurdle to improve food safety (Cleveland et al. 2001). Bacteriocins present dierent sensitivities to changes in temperature and pH. The eects of dierent pH values (from 2.0 to 12.0) and refrigeration (4 C/24 h), pasteurization (65 C/30 min, 100 C/10 min), and sterilization (121 C/10 min) temperatures on the activity of Lc. lactis subsp. cremoris CTC 204 were studied. The effects of different pH values and treatment at 4 C for 24 h on the activity of the bacteriocin under test showed that the highest level of activity presented by this strain was 3200 AU/ml at pH 2.0 and 4.0. It lost 50% of its activity from pH 6.0 to 9.0 and 75% at pH 10.0. It was completely destroyed at pH 12.0 (0 AU/ml). In comparison to refrigeration temperatures, heat treatment at 65 C for 30 min caused a slight loss of activity of the antimicrobial compound produced by strain CTC 204. The maximum activity value produced after heat treatment was at least 50% lower than that produced at 4 C. At pH 2.0 it produced 1600 AU/ml; from pH 4.0 to 9.0 the production was 800 AU/ml; at pH 10.0 it was 400 AU/ml and at pH 12.0 no activity was detected. After heat treatment at 100 C for 10 min the highest level of activity presented by the bacteriocin was 1600 AU/ml at pH 2.0. Up to pH 6.0 it presented the same pattern of production as that produced after heat treatment at 65 C for 30 min: 800 AU/ml. After that, from pH 7.0 to 10.0 it lost 50% of its activity (400 AU/ ml) and was subsequently inactivated at pH 12.0. The effect of pH and sterilization temperature (121 C for 10 min) on the activity of the antimicrobial compound showed that even at high temperatures it was still active in the pH range from 2.0 to 10.0. It maintained maximum bacteriocin production (1600 AU/ml) from pH 2.0 to 6.0 and lost 50% of its activity (800 AU/ml) from pH 7.0 to 10.0, being completely destroyed at pH 12.0. The activity level of this bacteriocin was maintained during all heat treatments under acid conditions. Many bacteriocins are active at acidic, neutral and alkaline pH values, which may reect the adaptation of these substances to the environmental conditions in which bacteriocin-producer bacteria develop (Kojic et al. 1991; Olasupo et al. 1999). Nevertheless, nisin and lactostrepcins (produced by lactococci) are exceptions to this rule, since their antimicrobial activities are dependent on this parameter. Solubility and stability of the former decrease from optimal at pH 2.0 to considerably reduced at 6.0 and irreversibly inactivated at pH 7.0 (Hurst 1981); on the other hand, lactostrepcins are stable and active within the pH range from 4.2 to 5.0 and reversibly inactivated at pH 7.0 and 8.0 (Kozak et al. 1978). However, many bacteriocin-producing lactic acid bacteria withstand exposure to a wide range of pH values (3.09.0) (Uhlman et al. 1992; Cintas et al. 2001). Tolerance to more extreme pH values (between 1.02.0 and 10.011.0) has been reported for acidocin B (produced by Lb. acidophilus M46) (Ten Brink et al. 1994), bavaricin A (produced by Lb. bavaricus MI401)

355 (Larsen et al. 1993), and the bacteriocin produced by Lc. lactis subsp. diacetylactis (Kojic et al. 1991). It is interesting that the applicability of these compounds in foods, especially in meat products, depends on some of the characteristics found in the bacteriocin produced by this strain. The bacteriocin produced by strain CTC 204 was active in a wide range of pH values and temperatures of refrigeration, pasteurization and sterilization. Bacteriocin production and bacterial growth at different temperatures and pH values The potential for stability at different pH values and temperature conditions was evaluated for cell growth and bacteriocin production by strain CTC 204 in buffered MRS Broth (Table 3). Cell growth and bacteriocin production properties were examined for 162 h in ask cultures at 20, 4, 25 and 37 C under noncontrolled pH conditions. The freezing temperature did not allow for growth and subsequent bacteriocin production by the strain tested. At this temperature, frozen food could be preserved by the direct addition of the bacteriocin produced by this strain. At the optimum temperature for the growth of lactic acid bacteria (37 C), cell growth was 58.9% greater after 16 h. After 48 h of incubation at this temperature, it presented the highest biomass production. After this, until 162 h cell density decreased by 12.6%. At the abusive temperature for food storage (25 C), the bacterial density increased 58.7% after 16 h of incubation. The maximum growth at this temperature was achieved after 24 h of incubation and it was 17.1% lower than at 37 C. At 4 C, cell density presented a slower growth rate during the rst 24 h. From that point on, accentuated growth was observed, reaching a peak at 162 h of incubation. At 0 C, the initial density was reduced by 9.5% after the rst 24 h, gradually decreasing until 162 h. Cell growth increased with temperature, reaching a maximum at 37 C. At 4, 25, and 37 C the maximum bacteriocin production by strain CTC 204 was 800 AU/ml. At 25 and 37 C, maximum bacteriocin production was detected after 16 h when the bacterial growth was approximately 2.4 times higher than the initial value. At )20 C the maximum bacteriocin production occurred after 20 h of incubation when the cell growth was 1.2 times higher than at the beginning. Bacteriocin production decreased 50% after 24 h of incubation at 4 and 37 C and after 48 h of incubation at 25 C. During the period of incubation the pH decreased about 2 units at 25 and 37 C, while at 4 C it decreased 1.3 units. Due to an absence of growth at )20 C, no variation in pH value was observed. According to Hugas et al. (1998), the maximum sakacin K production by Lb. sake CTC 494 was detected after 7 days at 4 C, while at 25 C it was detected after 9 h. The rate of cell growth increased according to the temperature, reaching a maximum at 30 C, but bacteriocin production was higher at the lower temperatures.

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Table 3. Growth, bacteriocin-like substance production and pH resistance of Lc. lactis subsp. ceremoris CTC 204 incubated in dierent temperatures. Time (h) ODa 0 16 18 20 22 24 48 114 162 0.281 0.684 0.772 0.768 0.742 0.726 0.808 0.738 0.706 37 C BAb 0 800 800 800 800 400 400 200 0 pH 6.43 4.60 4.58 4.54 4.52 4.49 4.8 4.47 4.46 OD 0.264 0.640 0.636 0.624 0.616 0.670 0.644 0.562 0.546 25 C BA 0 800 800 800 800 800 400 400 0 pH 6.43 5.08 5.00 4.94 4.86 4.80 4.68 4.52 4.46 OD 0.292 0.343 0.354 0.362 0.398 0.412 0.588 0.636 0.666 4 C BA 0 0 400 800 800 400 400 400 0 pH 6.46 6.30 6.25 6.20 6.14 6.10 5.82 5.31 5.16 OD 0.326 0.295 0.276 0.243 0.240 0 C BA 0 0 0 0 0 pH 6.34 6.34 6.32 6.36 6.35

not detected. Absorbance at 600 nm. b Bacteriocin activity (AU/ml).

a

However, Yang & Ray (1994) reported that the amount of bacteriocin produced by a lactic acid bacterium isolated from vacuum-packaged meat products was 23 times higher at the abusive temperature (25 C) than at refrigeration temperature (4 C). Effect of the bacteriocin on the mesophilic population present in meat Raw meat stored aerobically under chilled conditions spoils predominantly due to Gram-negative bacteria, especially Pseudomonas (Lucke 2000). Moreover, bacte rial pathogens of greater signicance to the consumer of raw meat (Salmonella sp., Campylobacter sp., Yersinia enterocolitica, Escherichia coli 0157:H7) are also Gramnegative. Under these conditions, lactic acid bacteria compete poorly and the use of the bacteriocin extract would be an alternative to control undesirable microorganisms. Figure 1 shows the inhibitory eect on aerobic mesophilic bacteria during the storage of minced beef submitted to treatment with the bacteriocin produced by strain CTC 204, conrming the results of three experiments. In these tests, ca. 500 AU/ml of bacteriocin produced by strain CTC 204 were added to the minced beef samples. The initial mesophilic counts in the meat samples varied from 2.2 to 7.9 log10 c.f.u./g (data not shown). It means that each beef sample constitutes a closed system in which the natural microbiota (types and concentrations of the microorganisms presented) cannot be reproducible, so it is important to consider

each sample analysed as a dierent one. Based on this fact, a linear regression curve to express the way the mesophilic bacterium population was aected by the bacteriocin produced by strain CTC 204 was obtained. The correlation coecient (R2) showed a value of 0.95 and the eectivity of the treatment was 7% (1)0.934) considering P (0.899 < 0.934 < 0.970) 95% as condence interval. The bacteriocin activity was not inuenced by the pH value once it decreased approximately 0.75 units in both control and test samples during the experiment. According to Muriana (1996), the eectiveness of bacteriocins may also depend on the amount of bacteriocin inactivated by interaction with food components. Because of their relatively large size (48 kDa), bacteriocins may also be considered a nite population of macromolecular inhibitors and, therefore, the relative amount of bacteriocin required to inactivate target cells may depend on the population of cells that may be present. There is little information on the effect of lactic acid culture treatments and their bacteriocins on the mesophilic aerobic plate counts of meat during refrigerated storage. Cell suspensions of Lc. lactis subsp. lactis biovar. diacetylactis have been used to inhibit the growth of gram-negative bacterial populations in refrigerated ground beef (Daly et al. 1972). Meat stored in air is rapidly spoiled by bacteria, which are responsible for discoloration and production of o-odours, resulting in consumer rejection (Labadie 1999). In our study, the colour of both the treated and control samples was

Figure 1. Eect of the bacteriocin produced by Lc. lactis subsp. cremoris CTC 204 on the mesophilic counts of minced beef during refrigerated storage.

Bacteriocin: properties and use in meat veried during the storage period. The appearance of a green colour was detected in the samples dipped in water at the end of the storage time. The green uorescent pigment (pyoverdin) is produced by many uorescent species of Pseudomonas (Labadie 1999). Generally, the storage life depends largely on the bacterial counts of the meat at the beginning of the storage period, notably the proportion of Pseudomonas spp. within the ora (Dainty & Mackey 1992). The results of this study revealed that the bacteriocin produced by Lc. lactis subsp. cremoris CTC 204 was inhibitory towards the mesophilic aerobic bacteria associated with red meat. A low concentration of bacteriocin reduced the number of bacteria present by 1 cycle log. This dierence was found in some heavily contaminated beef samples, being considered satisfactory. Considering its bactericidal activity, proteinaceous nature and heat resistance, the antimicrobial compound produced by Lc. lactis subsp. cremoris CTC 204 can be classied as a bacteriocin. It presented characteristics of resistance to proteolytic enzymes and temperatures similar to those of nisin. It was thermostable and could thus be used in pasteurized food products. It was also stable over a wide range of pH values and could be employed in acid and non-acid foods. As it was stable at low temperatures, the microorganism could act as a potential barrier to inhibit the growth of psychrotrophic or mesophilic spoilage and foodborn pathogens, such as Lactobacillus ssp., L. monocytogenes, S. aureus, B. cereus, and Cl. perfringens, frequently found in foods stored under refrigeration. Further work to evaluate the potential of this compound in meat after purication is in progress.

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Acknowledgements The authors thank FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo) for their nancial support (Process 99/12314-0) and Homero F. Gumerato for the statistic analysis.

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