An effective lacticin biopreservative in fresh pork sausage

Lacticin 3147 is a novel heat-stable bacteriocin, produced by Lactococcus lactis DPC 3147, that exhibits a broad-range inhibition spectrum similar to nisin. In this study, the effect of lacticin 3147 and nisin on the shelf life of fresh pork sausage and their ability to control pathogens (Clostridium perfringens DSM 756, Salmonella Kentucky AT1) and nonpathogenic Listeria innocua DPC 1770 was investigated. The following preservative regimens were evaluated, both in broth and sausage systems: (i) 450 ppm of sodium metabisulphite; (ii) 500 IU g(-1) or ml(-1) of nisin, (iii) 2500 arbitary units (AU) g(-1) or ml(-1) of lacticin 3147; (iv) 2% sodium lactate and 500 IU of nisin; (v) 2% sodium citrate and 500 IU g(-1) or ml(-1) of nisin; (vi) 2% sodium lactate and 2500 AU g(-1) or ml(-1) of lacticin 3147, (vii) 2% sodium citrate and 2500 AU g(-1) or ml(-1) of lacticin 3147, (viii) 2% sodium lactate, and (ix) 2% sodium citrate. There was no significant difference in the activity of nisin and lacticin 3147 against any of the target strains used, both bacteriocins performing significantly better than sodium metabisulfite against gram-positive strains in broth systems. Trends indicate that the combination of organic acids with either bacteriocin enhanced its activity against Salmonella Kentucky and L. innocua and was particularly effective in the inhibition of C. perfringens in fresh pork sausage. In addition, lacticin 3147 combined with either sodium citrate or sodium lactate maintained significantly lower (P < 0.05) total aerobic plate counts for the duration of the trials and may function as an alternative to sodium metabisulfite in the preservation of fresh pork sausage.     

Development of a lacticin 3147-enriched whey powder with inhibitory activity against foodborne pathogens.

The broad-spectrum bacteriocin lacticin 3147, produced by Lactococcus lactis DPC3147, is inhibitory to a wide range of gram-positive food spoilage and pathogenic organisms. A 10% solution of demineralized whey powder was fermented with DPC3147 at a constant pH of 6.5. The fermentate was spray dried, and the resulting powder exhibited inhibitory activity. The ability of the lacticin 3147-enriched powder to inhibit Listeria monocytogenes Scott A and Staphylococcus aureus 10 was assessed in buffer at both acidic (pH 5) and neutral (pH 7) pH. In addition, the ability of the powder to inhibit L. monocytogenes Scott A in an infant milk formulation was assessed. Resuspension of approximately 10(8) midexponential phase L. monocytogenes Scott A cells in a 10% solution of the lacticin 3147-enriched powder resulted in a 1,000-fold reduction in viable cells at pH 5 and pH 7 after 3 h at 30 degrees C. In the case of S. aureus 10, resuspension of 2.5 x 10(7) midexponential phase cells in a 15% solution of the lacticin 3147-enriched powder at pH 5 resulted in only a 10-fold reduction in viable cell counts, compared with a 1,000-fold reduction at pH 7, following incubation for 3 h at 30 degrees C. The use of the lacticin 3147 powder in an infant milk formulation resulted in greater than a 99% kill of L. monocytogenes within 3 h at 30 degrees C. These results suggest that this bioactive lacticin 3147 food ingredient may find applications in many different foods, including those with pH close to neutrality.     

Preservation and fermentation: past,present and future

Preservation of food and beverages resulting from fermentation has been an effective form of extending the shelf-life of foods for millennia. Traditionally, foods were preserved through naturally occurring fermentations, however, modern large scale production generally now exploits the use of defined strain starter systems to ensure consistency and quality in the final product. This review will mainly focus on the use of lactic acid bacteria (LAB) for food improvement, given their extensive application in a wide range of fermented foods. These microorganisms can produce a wide variety of antagonistic primary and secondary metabolites including organic acids, diacetyl, CO2 and even antibiotics such as reuterocyclin produced by Lactobacillus reuteri. In addition, members of the group can also produce a wide range of bacteriocins, some of which have activity against food pathogens such as Listeria monocytogenes and Clostridium botulinum. Indeed, the bacteriocin nisin has been used as an effective biopreservative in some dairy products for decades, while a number of more recently discovered bacteriocins, such as lacticin 3147, demonstrate increasing potential in a number of food applications. Both of these lactococcal bacteriocins belong to the lantibiotic family of posttranslationally modified bacteriocins that contain lanthionine, beta-methyllanthionine and dehydrated amino acids. The exploitation of such naturally produced antagonists holds tremendous potential for extension of shelf-life and improvement of safety of a variety of foods.     

Cell membrane damage induced by lacticin 3147 enhances aldehyde formation in Lactococcus lactis IFPL730

Amino acid catabolism is mainly initiated in Lactococcus lactis by a transamination reaction that leads to the formation of alpha-keto acids. In addition, a novel alpha-keto acid decarboxylase enzyme, rare in lactic acid bacteria, responsible for the conversion of alpha-keto acids into aldehydes has been reported in L. lactis IFPL730. The effect of lacticin 3147-induced cell damage on both amino acid transamination and alpha-keto acid decarboxylation by L. lactis IFPL730 leading to the formation of aldehydes from amino acids was investigated. Cell membrane permeabilization induced by lacticin 3147 facilitated the diffusion of amino acids into the cells and thus, enhanced amino acid transamination and formation of alpha-keto acids. However, alpha-keto acid decarboxylation was not affected by cell membrane permeabilization since decarboxylation of alpha-keto acids in both control and lacticin 3147-treated cells were similar, suggesting that these substrates could freely diffuse inside the cells. Nevertheless, the formation of 2-methylbutyraldehyde from isoleucine was enhanced in lacticin 3147-treated cells. The increase in alpha-keto acids formation rate by L. lactis IFPL730 due to lacticin 3147-induced cell damage, led to a concomitant increase in the subsequent decarboxylation reaction that complete the metabolic pathway to aldehyde production from amino acids. The present study points out to the use of the food grade lacticin 3147 along with L. lactis IFPL730 as a valuable tool in the development of cheese flavour.     

Acid-adaptation does not increase the resistance of Listeria monocytogenes to irradiation in a seafood salad

Stress adaptation of microbial cells enables the cells to survive better when they are subsequently exposed to other types of stresses. In the food industry, pathogens are commonly stressed during food processing and this is a concern where pathogens such as Listeria monocytogenes are involved. Research was conducted to determine if acid adaptation of L. monocytogenes provides resistance to ionizing irradiation. Three different strains of L. monocytogenes were acid-adapted using three different acids (acetic, citric, lactic) in Tryptic Soy Broth, at a pH 5.5 for 1 h, 4 h, or continuous acid exposure. The acid-adapted L. monocytogenes were then exposed to a low level of gamma irradiation (0.59-0.72 kGy) along with a non-acid adapted L. monocytogenes control. In a test tube study, the 1-h acetic acid-adapted L. monocytogenes strains showed the greatest difference from the control, a reduced kill of 1.1 log CFU/g but this difference was not significant by ANOVA (p=0.054). The reduction achieved after 4 h and continuous acid exposure also did not significantly differ from the control. To determine whether acid adaptation affected radiation resistance within a food product, a refrigerated storage shelf-life study was completed. Acetic acid was used to acid adapt a three-strain cocktail of L. monocytogenes for a period of 1 h. The organisms were then inoculated into a seafood salad (pH 5.15) and subsequently exposed to low dose gamma irradiation (0.7 to 4.5 kGy). L. monocytogenes was reduced or eliminated by irradiation regardless of acid adaptation; no increased resistance was observed.     

Development of bioactive food packaging materials using immobilised bacteriocins lacticin 3147 and nisaplin

Immobilisation of the bacteriocins nisin and lacticin 3147 to packaging materials was investigated. Stability of both cellulose-based bioactive inserts and anti-microbial polyethylene/polyamide pouches was examined over time. Anti-microbial activity against the indicator strain Lactococcus lactis subsp. lactis HP, in addition to Listeria innocua DPC 1770 and Staphylococcus aureus MMPR3 was observed for all bacteriocin-adsorbed materials. Activity retention of the inserts showed an initial decrease in the first week of storage but remained stable for the remaining 3 months of the trial. However, adsorption of lacticin 3147 to plastic film was unsuccessful, nisin bound well and the resulting film maintained its activity for 3-month period, both at room temperature and under refrigeration. When applied to food systems, the anti-microbial packaging reduced the population of lactic acid bacteria in sliced cheese and ham stored in modified atmosphere packaging (MAP) at refrigeration temperatures, thus extending the shelf life. Nisin-adsorbed bioactive inserts reduced levels of Listeria innocua by ≥2 log units in both products, and Staphylococcus aureus by 1.5 log units in cheese, and 2.8 log units in ham. Similar reductions were observed in cheese vacuum-packaged in nisin-adsorbed pouches.     

Growth and bacteriocin production by lactic acid bacteria in vegetable broth and their effectiveness at reducing Listeria monocytogenes in vitro and in fresh-cut lettuce

The fresh-cut fruit and vegetable industry is searching for alternatives to replace chemical treatments with biopreservative approaches that ensure the safety of the product and fulfil consumer preferences for minimally processed foods. In this study, the use of bacteriocins produced by lactic acid bacteria has been tested as a substitute for chemical disinfection of fresh-cut iceberg lettuce. First, the ability of several non-plant origin bacteriocinogenic strains (nisin Z(+), plantaricin C(+), lacticin 481(+), coagulin(+) or pediocin PA-1(+)) to grow in a lettuce extract at 4 degrees C, 10 degrees C and 32 degrees C was tested. All strains were able to grow, but bacteriocin production was predominantly detected at 32 degrees C. Addition of bacteriocinogenic supernatants (nisin(+), coagulin(+) and a nisin-coagulin(+) cocktail) to tryptic-soy agar plates inoculated with Listeria monocytogenes reduced Listeria counts by approximately 1-1.5 log units compared with the control plates without bacteriocin, after 48 h of storage at 4 degrees C. The effect of washing with bacteriocin-containing solutions on survival and proliferation of Listeria monocytogenes was also evaluated in fresh-cut lettuce packaged in macro-perforated polypropylene bags and stored for 7 days at 4 degrees C. Washing fresh-cut lettuce with these solutions decreased the viability of Listeria monocytogenes by 1.2-1.6 log units immediately after treatment, but, during storage at 4 degrees C, bacteriocin treatments only exerted minimal control over the growth of the pathogen. Natural microbiota were little affected by bacteriocins during storage.     

Synergy between nisin and select lactates against Listeria monocytogenes is due to the metal cations

Listeria monocytogenes, a major foodborne pathogen, has been responsible for many outbreaks and recalls. Organic acids and antimicrobial peptides (bacteriocins) such as nisin are produced by lactic acid bacteria and are commercially used to control pathogens in some foods. This study examined the effects of lactic acid (LA) and its salts in combination with a commercial nisin preparation on the growth of L. monocytogenes Scott A and its nisin-resistant mutant. Because of an increase in its activity at a lower pH, nisin was more active against L. monocytogenes when used in combination with LA. Most of the salts of LA, including potassium lactate, at up to 5% partially inhibited the growth of L. monocytogenes and had no synergy with nisin. Zinc and aluminum lactate, as well as zinc and aluminum chloride (0.1%), worked synergistically with 100 IU of nisin per ml to control the growth of L. monocytogenes Scott A. No synergy was observed when zinc or aluminum lactate was used with nisin against nisin-resistant L. monocytogenes. The nisin-resistant strain was more sensitive to Zn lactate than was wild-type L. monocytogenes Scott A; however, the cellular ATP levels of the nisin-resistant strain were not significantly affected. Changes in the intracellular ATP levels of the wild-type strain support our hypothesis that pretreatment with zinc lactate sensitizes cells to nisin. The similar effects of thesalts of hydrochloric and lactic acids support the hypothesis that metal cations are responsible for synergy with nisin.     

Effect of milk inoculation with bacteriocin-producing lactic acid bacteria on a Lactobacillus helveticus adjunct cheese culture

The effect of eight strains of lactic acid bacteria (two strains of Enterococcus, one strain of Lactobacillus, and five strains of Lactococcus, which produce enterocin AS-48, enterocin 607, nisin A, nisin Z, plantaricin 684, lacticin 481, or nisin Z plus lacticin 481) on acid production and proteolytic activity of Lactobacillus helveticus LH 92 (a highly peptidolytic strain used as an adjunct in cheese making) was evaluated in mixed cultures in milk. Acid production by mixed cultures depended on the sensitivity of L. helveticus LH 92 to the different bacteriocins and on the acidification rates of bacteriocin-producing strains. Proteolysis values of mixed cultures were, in all cases, lower than those of L. helveticus LH 92 single culture (control). Cell-free aminopeptidase activity values after 9 h of incubation did not increase in the presence of enterocin producers or the nisin A producer, whereas in the presence of the nisin Z producer, cell-free aminopeptidase activity was, at most, 3.7-fold greater than the control value. In mixed cultures with the plantaricin producer, a progressive lysis of L. helveticus LH 92 took place, with cell-free aminopeptidase activity values after 9 h being, at most, 10.5-fold greater than the control value. The highest cell-free aminopeptidase activity values after 9 h were recorded in the presence of lacticin 481 producers, with the values being, at most, 25.1-fold greater than the control value. L. helveticus LH 92 was extremely sensitive to small variations in the concentration of the inoculum of the nisin Z plus lacticin 481 producer, with there being a narrow optimum for the release of intracellular aminopeptidases. Plantaricin and lacticin 481 producers seemed the most promising strains to be combined with L. helveticus LH 92 as lactic cultures for cheese manufacture,because of the accelerated release of intracellular aminopeptidases.     

Survival of the acid-adapted Bacillus cereus in acidic environments

In this study, the acid tolerance of Bacillus cereus 1-4-1 after adaptation at pH 5.5 for 1, 2 and 4 h was first determined. The survival of acid-adapted and non-adapted cells of B. cereus in phosphate buffer solution (PBS pH 4.0) containing various organic acids such as acetic, propionic, citric, lactic or tartaric acid as well as in a commercial acidic beverage of mixed fruits and vegetables (pH 3.7) was then examined. Results revealed that acid adaptation time influenced the increased tolerance of B. cereus in PBS (pH 4.0). The 2 h-adapted cells exhibited the highest acid tolerance in PBS. The presence of chloramphenicol during the acid adaptation reduced the extent of increased acid tolerance. Acid adaptation was also found to enhance the tolerance of the test organism in the presence of the various organic acids tested. While the extent of increased acid tolerance varied with the organic acid examined. Acid-adapted B. cereus cells exhibited the largest extent of increased tolerance, showing an increased survival of ca. 1000 folds, in the propionic acid-containing PBS. Additionally, a higher survival percentage was noted with the acid-adapted than the non-adapted cells of B. cereus in the acidic beverage stored at 4 or 25 degrees C.     

Acid adaptation does not promote survival or growth of Listeria monocytogenes on fresh beef following acid and nonacid decontamination treatments

The objective of this study was to evaluate the survival and growth of acid-adapted and nonadapted Listeria monocytogenes inoculated onto fresh beef subsequently treated with acid or nonacid solutions. Beef slices (2.5 by 5 by 1 cm) from top rounds were inoculated with acid-adapted or nonadapted L. monocytogenes (4.6 to 5.0 log CFU/cm2) and either left untreated (control) or dipped for 30 s in water at 55 degrees C, water at 75 degrees C, 2% lactic acid at 55 degrees C, or 2% acetic acid at 55 degrees C. The beef slices were vacuum packaged and stored at 4 or 10 degrees C and were analyzed after 0, 7, 14, 21, and 28 days of storage. Dipping in 75 degrees C water, lactic acid, and acetic acid resulted in immediate pathogen reductions of 1.4 to 2.0, 1.8 to 2.6, and 1.4 to 2.4 log CFU/cm2, respectively. After storage at 10 degrees C for 28 days, populations of L. monocytogenes on meat treated with 55 degrees C water increased by ca. 1.6 to 1.8 log CFU/cm2. The pathogen remained at low population levels (1.6 to 2.8 log CFU/cm2) on acid-treated meat, whereas populations on meat treated with 75 degrees C water increased rapidly, reaching levels of 3.6 to 4.6 log CFU/cm2 by day 14. During storage at 4 degrees C, there was no growth of the pathogen for at least 21 days in samples treated with 55 and 75 degrees C water, and periods of no growth were longer for acid-treated samples. There were no differences between acid-adapted and nonadapted organisms across treatments with respect to survival or growth. In conclusion, the dipping of meat inoculated with L. monocytogenes into acid solutions reduced and then inhibited the growth of the pathogen during storage at 4 and 10 degrees C, while dipping in hot water allowed growth despite initial reductions in pathogen contamination. The results of this study indicate a residual activity of acid-based decontamination treatments compared with water-based treatments for refrigerated (4 degrees C) or temperature-abused (10 degrees C) lean beef tissue in vacuum packages, and these results also indicate that this activity may not be counteracted by prior acid adaptation of L. monocytogenes.     

Effect of liposome-encapsulated nisin and bacteriocin-like substance P34 on Listeria monocytogenes growth in Minas frescal cheese

The efficacy of liposome-encapsulated nisin and bacteriocin-like substance (BLS) P34 to control growth of Listeria monocytogenes in Minas frescal cheese was investigated. Nisin and BLS P34 were encapsulated in partially purified soybean phosphatidylcholine (PC-1) and PC-1-cholesterol (7:3) liposomes. PC-1 nanovesicles were previously characterized. PC-1-cholesterol encapsulated nisin and BLS P34 presented, respectively, 218 nm and 158 nm diameters, zeta potential of -64 mV and -53 mV, and entrapment efficiency of 88.9% and 100%. All treatments reduced the population of L. monocytogenes compared to the control during 21 days of storage of Minas frescal cheese at 7°C. However, nisin and BLS P34 encapsulated in PC-1-cholesterol liposomes were less efficient in controlling L. monocytogenes growth in comparison with free and PC-1 liposome-encapsulated bacteriocins. The highest inhibitory effect was observed for nisin and BLS P34 encapsulated in PC-1 liposomes after 10 days of storage of the product. The encapsulation of bacteriocins in liposomes of partially purified soybean phosphatidylcholine may be a promising technology for the control of foodborne pathogens in cheeses.     

Modeling survival of Listeria monocytogenes in the traditional Greek soft cheese Katiki

In the present work, survival of Listeria monocytogenes in the traditional Greek soft, spreadable cheese Katiki was studied throughout the shelf life of the product. Samples of finished cheese were inoculated with a cocktail of five L. monocytogenes strains (ca. 6 log CFU g(-1)) and stored at 5, 10, 15, and 20 degrees C. Acid-stress adaptation or cross-protection to the same stress was also investigated by inoculation of acid-adapted cells in the product. The results showed that pathogen survival was biphasic. Various mathematical equations (Geeraerd, Cerf, Albert-Mafart, Whiting, Zwietering, and Baranyi models) were fitted to the experimental data. A thorough statistical analysis was performed to choose the best model. The Geeraerd model was finally selected, and the results revealed no acid tolerance acquisition (no significant differences, P > 0.05, in the survival rates of the non-acid-adapted and acid-adapted cells). Secondary modeling (second-order polynomial with a(0) = 0.8453, a(1) = -0.0743, and a(2) = 0.0059) of the survival rate (of sensitive population), and other parameters that were similar at all temperatures (fraction of initial population in the major population = 99.98%, survival rate of resistant population = 0.10 day(-1), and initial population = 6.29 log CFU g(-1)), showed that survival of the pathogen was temperature dependent with bacterial cells surviving for a longer period of time at lower temperatures. Finally, the developed predictive model was successfully validated at two independent temperatures (12 and 17 degrees C). This study underlines the usefulness of predictive modeling as a tool for realistic estimation and control of L. monocytogenes risk in food products. Such data are also useful when conducting risk assessment studies.     

Resistance of Escherichia coli and Salmonella against nisin and curvacin A.

We have determined the effects of the following factors on the resistance of Gram-negative bacteria against nisin and curvacin A: (i) chemotype of the lipopolysaccharide (LPS), (ii) addition of agents permeabilizing the outer membrane, (iii) the fatty acid supply of the growth medium, and (iv) the adaptation to acid and salt stress. Bacteriocin activity was determined against growing and resting cells as well as protoplasts. All smooth strains of Escherichia coli and Salmonella enterica serovar Typhimurium were highly resistant towards the bacteriocins, whereas mutants that possess the core of the LPS, but not the O antigen, as well as deep rough LPS mutants were sensitive. Antibiotics with outer membrane permeabilizing activity, polymyxin B and polymyxin B nonapeptide, increased the sensitivity of smooth E. coli towards nisin, but not that of deep rough mutants. Incorporation of 1 g l(-1) of either oleic acid or linoleic acid to the growth media greatly increased the susceptibility of E. coli LTH1600 and LTH4346 towards bacteriocins. Both strains of E. coli were sensitive to nisin and curvacin A at a pH of less than 5.5 and more than 3% (w/v) NaCl. Adaptation to sublethal pH or higher NaCl concentrations (pH 4.54 and 5.35 or 4.5% (w/v) NaCl) provided only limited protection against the bacteriocidal activity of nisin and curvacin A. Adaptation to 4.5% (w/v) NaCl did not result in cross protection to bacteriocin activity at pH 4.4, but rendered the cells more sensitive towards bacteriocins.     

Characterization of bacteriocins from two Lactococcus lactis subsp. lactis isolates

In this study, bacteriocins from two Lactococcus lactis subsp. lactis isolates from raw milk samples in Turkey designated OC1 and OC2, respectively, were characterized and identified. The activity spectra of the bacteriocins were determined by using different indicator bacteria including Listeria, Bacillus and Staphylococcus spp. Bacteriocins were tested for their sensitivity to different enzymes, heat treatments and pH values. Loss of bacteriocin activities after alpha-amylase treatment suggested that they form aggregates with carbohydrates. Molecular masses of the purified bacteriocins were determined by SDS-PAGE. PCR amplification was carried out with specific primers for the detection of their structural genes. As a result of these studies, the two bacteriocins were characterized as nisin and lacticin 481, respectively. Examination of plasmid contents of the isolates and the results of plasmid curing and conjugation experiments showed that in L. lactis subsp. lactis OC1 strain the 39.7-kb plasmid is responsible for nisin production, lactose fermentation and proteolytic activity, whereas the 16.0-kb plasmid is responsible for lacticin 481 production and lactose fermentation in L. lactis subsp. lactis OC2 strain.     

Elevated enzyme release from lactococcal starter cultures on exposure to the lantibiotic lacticin 481, produced by Lactococcus lactis DPC5552

A Lactococcus lactis subsp. lactis strain (DPC5552), which causes the lysis of other lactococcal cultures, was isolated during a screening of raw milk samples for bacteriocin producers. Purification of the bacteriocin produced revealed that production of the lantibiotic, lacticin 481, was associated with the bacteriolytic capability of the strain. However, unlike bacteriocin-induced lysis observed with bacteriocins such as lacticin 3147 and lactococcins A, B, and M (where the target strain is killed), the DPC5552 supernatant gave rise to a situation whereby the target strain continued to grow (albeit at a lower rate) with simultaneous release of the intracellular enzymes lactate dehydrogenase (LDH) and post-proline dipeptidyl aminopeptidase (Pep X). In parallel experiments, 32 AU/ml of the inhibitory activity from L. lactis DPC5552 resulted in a 10- and 6-fold-higher LDH release after 5 h than that with 32 AU/ml of either lacticin 3147 or lactococcin A, B, and M. Laboratory-scale Cheddar cheese-making trials also demonstrated that lacticin 481-producing cultures induced the release of elevated levels of LDH from the starter L. lactis HP, without severely compromising its acid-producing capabilities. These results indicate that lacticin 481-producing strains may provide improved adjuncts for delivering lactococcal intracellular enzymes into the cheese matrix and, thus, improve cheese quality and flavor.     

Effective control of Listeria monocytogenes by combination of nisin formulated and slowly released into a broth system

In order to identify conditions for efficient food preservation by nisin, the sensitivity of Listeria monocytogenes to this preservative was studied under the following three model conditions: (1) the instantaneous addition of nisin into broth medium to simulate the formation of nisin in foods, (2) the slow delivery of nisin solution into broth medium using a pump to simulate the slow release of nisin from packaging materials to foods, (3) a combination of the two delivery methods. Based on the following results, we conclude that the antimicrobial effectiveness of nisin strongly depends on its mode of delivery. The instantaneous and slow methods for adding nisin inhibited L. monocytogenes, but over time of exposure, L. monocytogenes developed tolerance to nisin. Our data indicate that cells treated with instantaneously added nisin developed resistance to higher concentrations of nisin (200 IU/ml), compared to cells treated with slowly added nisin at the same total amount of the antimicrobial. Further studies indicated that nisin-tolerant cells recovered from treatments in which 200 IU/ml nisin was added instantaneously were likely to be mutants, which became resistant to the bacteriocin. In contrast, when 200 IU/ml of the antimicrobial was added slowly to the cells, only a temporary tolerance was developed; these cells became nisin-sensitive after passage through nisin-free medium. Due to the development of nisin-resistant cells, excessive amounts of nisin in the model system did not further inhibit L. monocytogenes. These results signify that excess nisin in foods does not necessarily improve the efficiency of controlling L. monocytogenes. Our data suggest that the combination of packaging material containing nisin used in conjunction with nisin-containing foods will provide the most effective means of preventing L. monocytogenes growth.     

Influence of acid adaptation on the tolerance of Escherichia coli O157:H7 to some subsequent stresses

Three stains of Escherichia coli O157:H7, including ATCC 43889, ATCC 43895, and 933, were first subjected to acid adaptation at a pH of 5.0 for 4 h. Thermal tolerance at 52 degrees C and survival of the acid-adapted as well as the nonadapted cells of E. coli O157:H7 in the presence of 10% sodium chloride, 0.85% bile salt, or 15.0% ethanol were investigated. Results showed that the effect of acid adaptation on the survival of E. coli O157:H7 varied with the strains and types of subsequent stress. Acid adaptation caused an increase in the thermal tolerance of E. coli O157:H7 ATCC 43889 and ATCC 43895, but no significant difference in the thermal tolerance was noted between acid-adapted and nonadapted cells of E. coli O157:H7 933. Although the magnitude of increase varied with strains of test organisms, acid adaptation generally led to an increase in the tolerance of E. coli O157:H7 to sodium chloride. On the other hand, the susceptibility of acid-adapted cells of the three strains of E. coli O157:H7 tested did not show a significant difference from that of their nonadapted counterparts when stressed with bile salt. The acid-adapted cells of E. coli O157:H7 ATCC 43889 and ATCC 43895 were less tolerant than the nonadapted cells to ethanol, whereas the tolerance of adapted and nonadapted cells of E. coli O157:H7 933 showed no significant differences.     

Effect of acid tolerance response (ATR) on attachment of Listeria monocytogenes Scott A to stainless steel under extended exposure to acid or/and salt stress and resistance of sessile cells to subsequent strong acid challenge

The aim of this study was to investigate the potential effect of adaptive stationary phase acid tolerance response (ATR) of Listeria monocytogenes Scott A cells on their attachment to stainless steel (SS) under low pH or/and high salt conditions and on the subsequent resistance of sessile cells to strong acid challenge. Nonadapted or acid-adapted stationary-phase L. monocytogenes cells were used to inoculate (ca. 10? CFU/ml) Brain Heart (BH) broth (pH 7.4, 0.5% w/v NaCl) in test tubes containing vertically placed SS coupons (used as abiotic substrates for bacterial attachment). Incubation was carried out at 16 °C for up to 15 days, without any nutrient refreshment. L. monocytogenes cells, prepared as described above, were also exposed to low pH (4.5; adjusted with HCl) or/and high salt (5.5% w/v NaCl) stresses, during attachment. On the 5th, 10th and 15th day of incubation, cells attached to SS coupons were detached (through bead vortexing) and enumerated (by agar plating). Results revealed that ATR significantly (p<0.05) affected bacterial attachment, when the latter took place under moderate acidic conditions (pH 4.5, 0.5 or 5.5% w/v NaCl), with the acid-adapted cells adhering slightly more than the nonadapted ones. Regardless of acidity/salinity conditions during attachment, ATR also enhanced the resistance of sessile cells to subsequent lethal acid challenge (exposure to pH 2 for 6 min; pH adjusted with either hydrochloric or lactic acid). The trend observed with viable count data agreed well with conductance measurements, used to indirectly quantify remaining attached bacteria (following the strong acid challenge) via their metabolic activity. To sum, this study demonstrates that acid adaptation of L. monocytogenes cells during their planktonic growth enhances their subsequent attachment to SS under extended exposure (at 16 °C for up to 15 days) to mild acidic conditions (pH 4.5), while it also improves the resistance of sessile cells to extreme acid treatment (pH 2). Therefore, the ATR of bacterial cells should be carefully considered when applying acidic decontamination strategies to eradicate L. monocytogenes attached to food processing equipment.     

Comparative Study on Antibacterial Effect of Pediocin ACCEL and Nisin Against Fluorescencestained Listeria monocytogenes BCRC 14845

ABSTRACT: Effects of pediocin ACCEL (Class IIa, pediocin-like bacteriocins) and nisin on carboxyfluorescein diacetate (cFDA) -stained Listeria monocytogenes BCRC 14845 was carried out. Decrease of fluorescence intensity in stained cells and increase in efflux were observed with the increase in bacteriocin concentrations up to 12.5 μg/mL. No further decreases in fluorescence intensity and cell numbers were obtained when the concentration of bacteriocins was over 12.5μg/mL. The leakage of efflux was further confirmed by the release of intracel-lular ultraviolet-absorbing substances. Comparing the leaking time of fluorescence, the intensity of cFDA in nisin-treated cells within 1 min incubation was almost comparable to that in pediocin-treated samples after 40 min incubation. Considering the leakage of cFDA, UV-absorbing substances, changes in cell numbers during incubation of cFDA-stained cells, and restoration ability, nisin seemed to reveal mainly bacteriostatic effect and partially bactericidal effect on L. monocytogenes and to cause easier-to-repair membrane damage. However, pediocin ACCEL appeared to have both bacteriostatic and bactericidal effects on L. monocytogenes and to cause more difficult-to-repair membrane damage.