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.     

Inhibition of Listeria monocytogenes by exposure to a combination of nisin and cold-pressed terpeneless Valencia oil

Listeria monocytogenes is an important foodborne pathogen and its control in foods is a significant challenge. This study evaluated the effectiveness of nisin and cold-pressed terpeneless Valencia oil (CPTVO) on limiting L. monocytogenes growth. Disk diffusion assays were performed to determine the effects of CPTVO and nisin individually and in combination. Together, these antimicrobials produced a zone of inhibition that was significantly larger (P < 0.05) than zones correlating to CPTVO or nisin individually. Furthermore, L. monocytogenesΔsigB had an increased sensitivity to the combination treatment. Growth experiments performed in brain heart infusion (BHI) broth revealed the effects of nisin and CPTVO, individually and in combination on L. monocytogenes growth rate. When L. monocytogenes was grown in BHI containing 0.025% CPTVO and 26 IU/mL nisin, no growth inhibition was observed relative to the control. However, exposure to CPTVO at 0 h followed by the introduction of nisin at 15 h resulted in a statistically significant (P < 0.05) reduction in growth. This approach to inhibiting L. monocytogenes has potential as an all-natural, generally-recognized-as-safe multiple hurdle intervention that may be applicable for ready-to-eat products in which L. monocytogenes is likely to cause foodborne illness.     

The antimicrobial effect of thyme essential oil, nisin, and their combination against Listeria monocytogenes in minced beef during refrigerated storage

The antimicrobial effect of thyme essential oil (EO) at 0.3%, 0.6%, or 0.9%, nisin at 500 or 1000IU/g, and their combination against Listeria monocytogenes was examined in both tryptic soy broth (TSB) and minced beef meat. Thyme EO at 0.3% possessed a weak antibacterial activity against the pathogen in TSB, whereas at 0.9% showed unacceptable organoleptic properties in minced meat. Thus, only the level of 0.6% of EO was further examined against the pathogen in minced meat. Treatment of minced beef meat with nisin at 500 or 1000IU/g showed antibacterial activity against L. monocytogenes, which was dependent on the concentration level of nisin and the strains used. Treatment of minced beef meat with EO at 0.6% showed stronger inhibitory activity against L. monocytogenes than treatment with nisin at 500 or 1000IU/g. All treatments showed stronger inhibitory activity against the pathogens at 10 degrees C than at 4 degrees C. The combined addition of EO at 0.6% and nisin at 500 or 1000IU/g showed a synergistic activity against the pathogen. Most efficient among treatments was the combination of EO at 0.6% with nisin at 1000IU/g, which decreased the population of L. monocytogenes below the official limit of the European Union recently set at 2logcfu/g, during storage at 4 degrees C.     

Inactivation of Listeria innocua in nisin-treated salmon (Oncorhynchus keta) and sturgeon (Acipenser transmontanus) caviar heated by radio frequency

Recent regulatory concerns about the presence of the pathogen Listeria monocytogenes in ready-to-eat aquatic foods such as caviar has prompted the development of postpackaging pasteurization processes. However, caviar is heat labile, and conventional pasteurization processes affect the texture, color, and flavor of these foods negatively. In this study, chum salmon (Oncorhynchus keta, 2.5% total salt) caviar or ikura and sturgeon (Acipenser transmontanus, 3.5% total salt) caviar were inoculated with three strains of Listeria innocua in stationary phase at a level of more than 10(7) CFU/g. L innocua strains were used because they exhibit an equivalent response to L monocytogenes for many physicochemical processing treatments, including heat treatment. The products were treated by immersion in 500 IU/ml nisin solution and heat processed (an 8-D process without nisin or a 4-D process with 500 IU/ml nisin) in a newly developed radio frequency (RF; 27 MHz) heating method at 60, 63, and 65 degrees C. RF heating along with nisin acted synergistically to inactivate L. innocua cells and total mesophilic microorganisms. In the RF-nisin treatment at 65 degrees C, no surviving L. innocua microbes were recovered in sturgeon caviar or ikura. The come-up times in the RF-heated product were significantly lower compared with the water bath-heated caviar at all treatment temperatures. The visual quality of the caviar products treated by RF with or without nisin was comparable to the untreated control.     

Inactivation of Listeria innocua in skim milk by pulsed electric fields and nisin

Pulsed electric fields (PEF) is an emerging nonthermal processing technology used to inactivate microorganisms in liquid foods such as milk. PEF results in loss of cell membrane functionality that leads to inactivation of the microorganism. There are many processes that aid in the stability and safety of foods. The combination of different preservation factors, such as nisin and PEF, to control microorganisms should be explored. The objective of this research was to study the inactivation of Listeria innocua suspended in skim milk by PEF as well as the sensitization of PEF treated L. innocua to nisin. The selected electric field intensity was 30, 40 and 50 kV/cm and the number of pulses applied was 10.6, 21.3 and 32. The sensitization exhibited by PEF treated L. innocua to nisin was assessed for 10 or 100 IU nisin/ml. A progressive decrease in the population of L. innocua was observed for the selected field intensities, with the greatest reduction being 2 1/2 log cycles (U). The exposure of L. innocua to nisin after PEF had an additive effect on the inactivation of the microorganism as that exhibited by the PEF alone. As the electric field, number of pulses and nisin concentration increased, synergism was observed in the inactivation of L. innocua as a result of exposure to nisin after PEF. The reduction of L. innocua accomplished by exposure to 10 IU nisin/ml after 32 pulsed electric fields was 2, 2.7, and 3.4 U for an electric field intensity of 30, 40, and 50 kV/cm, respectively. Population of L. innocua subjected to 100 IU nisin/ml after PEF was 2.8-3.8 U for the selected electric field intensities and 32 pulses. The designed model for the inactivation of L. innocua as a result of the PEF followed by exposure to nisin proved to be accurate in the prediction of the inactivation of L. innocua in skim milk containing 1.2 or 37 IU nisin/ml. Inactivation of L. innocua in skim milk containing 37 IU nisin/ml resulted in a decrease in population of 3.7 U.     

Destruction of Listeria monocytogenes in sturgeon (Acipenser transmontanus) caviar by a combination of nisin with chemical antimicrobials or moderate heat

The objective of this study was to investigate the effect of nisin in combination with heat or antimicrobial chemical treatments (such as lactic acid, chlorous acid, and sodium hypochlorite) on the inhibition of Listeria monocytogenes and total mesophiles in sturgeon (Acipenser transmontanus) caviar. The effects of nisin (250, 500, 750, and 1,000 IU/ml), lactic acid (1, 2, and 3%), chlorous acid (134 and 268 ppm), sodium hypochlorite (150 and 300 ppm), and heat at 60 degrees C for 3 min were evaluated for a five-strain mixture of L. monocytogenes and total mesophiles in sturgeon caviar containing 3.5% salt. Selected combinations of these antimicrobial treatments were also tested. Injured and viable L. monocytogenes cells were recovered using an overlay method. Treating caviar with > or =500 IU/ml nisin initially reduced L. monocytogenes by 2 to 2.5 log units. Chlorous acid (268 ppm) reduced L. monocytogenes from 7.7 log units to undetectable (<0.48 log units) after 4 days of storage at 4 degrees C. However, there were no synergistic effects observed for combinations of nisin (500 or 750 IU/ml) plus either lactic acid or chlorous acid. Lactic acid caused a slight reduction (approximately 1 log unit) in the microbial load during a 6-day period at 4 degrees C. Sodium hypochlorite was ineffective at the levels tested. Mild heating (60 degrees C for 3 min) with nisin synergistically reduced viable counts of L. monocytogenes and total mesophiles. No L. monocytogenes cells (<0.48 log units) were recovered from caviar treated with heat and nisin (750 IU/ml) after a storage period of 28 days at 4 degrees C.     

Transmission electron microscopy study of Listeria monocytogenes treated with nisin in combination with either grape seed or green tea extract

The objective of this study was to use transmission electron microscopy to investigate the morphological changes that occurred in Listeria monocytogenes cells treated with grape seed extract (GSE), green tea extract (GTE), nisin, and combinations of nisin with either GSE or GTE. The test solutions were prepared with (i) 1% GSE, 1% GTE, 6,400 IU of nisin, and the combination of these dilutions with nisin or with (ii) the pure major phenolic constituents of GSE (0.02% epicatechin plus 0.02% catechin) or GTE (0.02% epicatechin plus 0.02% caffeic acid) and their combinations with 6,400 IU of nisin in tryptic soy broth with 0.6% yeast extract (TSBYE). Test solutions were inoculated with L. monocytogenes at approximately 10(6) CFU/ml and incubated for 3 or 24 h at 37 degrees C. After 3 h of incubation, cells were harvested and evaluated under a transmission electron microscope (JEOL-100 CX) operating at 80 kV (50,000X). Microscopic examination revealed an altered cell membrane and condensed cytoplasm when L. monocytogenes cells were exposed to a combination of nisin with either GSE or GTE or to pure compounds of the major phenolic constituents in combination. After 24 h of incubation at 37 degrees C, the combinations of nisin with GSE and nisin with GTE reduced the L. monocytogenes population to undetectable levels and 3.7 log CFU/ml, respectively. These observations indicate that the combination of nisin with either GSE or GTE had a synergistic effect, and the combinations of nisin with the major phenolic constituents were most likely associated with the L. monocytogenes cell damage during inactivation in TSBYE at 37 degrees C.     

Synergistic inhibition of Listeria monocytogenes by nisin and garlic extract

Minimum Inhibitory Concentrations (MICs) were determined for nisin and an aqueous garlic extract (AGE) for six strains of Listeria monocytogenes, including a nisin-resistant mutant. When used in combination in broth a synergistic effect was observed between nisin and AGE in all strains. Increasing the pH reduced the activity of both nisin and garlic. During storage of the aqueous garlic extract at 4°C and at 20°C for 14 days, the MIC increased over the first 8 days but remained stable thereafter, indicating at least two antimicrobial components, one of which was relatively stable. Aqueous garlic extract was not appreciably bactericidal although there was a positive interaction with nisin in terms of its bactericidal effect in broth at 4°C. The effect of combining nisin and garlic in a food system, hummus, was also studied. Sub-MIC combinations of AGE and nisin were effective at preventing listerial growth and did enhance the slight bactericidal effect of nisin. The results indicate that combined use of nisin with garlic could help overcome problems of nisin resistance in Gram-positive organisms. Such interactions could also be a significant factor in traditional lactic fermented foods where garlic is an ingredient.     

Enhancement of nisin, lysozyme, and monolaurin antimicrobial activities by ethylenediaminetetraacetic acid and lactoferrin

A microtiter plate assay was employed to systematically assess the interaction between ethylenediaminetetraacetic acid (EDTA) or lactoferrin and nisin, lysozyme, or monolaurin against strains of Listeria monocytogenes, Escherichia coli, Salmonella enteritidis, and Pseudomonas fluorescens. Low levels of EDTA acted synergistically with nisin and lysozyme against L. monocytogenes but EDTA and monolaurin interacted additively against this microorganism. EDTA synergistically enhanced the activity of nisin, monolaurin, and lysozyme in tryptic soy broth (TSB) against two enterohemorrhagic E. coli strains. In addition, various combinations of nisin, lysozyme, and monolaurin with EDTA were bactericidal to some gram-negative bacteria whereas none of the antimicrobials alone were bactericidal. Lactoferrin alone (2000 microg ml(-1)) did not inhibit any of the bacterial strains, but did enhance nisin activity against both L. monocytogenes strains. Lactoferrin in combination with monolaurin inhibited growth of E. coli O157:H7 but not E. coli O104:H21. While lactoferrin combined with nisin or monolaurin did not completely inhibit growth of the gram-negative bacteria, there was some growth inhibition. All combinations of EDTA or lactoferrin with antimicrobials were less effective in 2% fat UHT milk than in TSB. S. enteritidis and P. fluorescens strains were consistently more resistant to antimicrobial combinations. Resistance may be due to differences in the outer membrane and/or LPS structure.     

Inhibition of Bacillus subtilis and Listeria innocua by nisin in combination with some naturally occurring organic compounds

The application of combined preservative factors (hurdle technology) is very effective in controlling the growth of food spoilage and foodborne pathogenic bacteria. Antimicrobial activity of nisin alone and in combination with some natural organic compounds (carvacrol, cinnamic acid, eugenol, diacetyl, and thymol) on the growth of gram-positive bacteria Bacillus subtilis and Listeria innocua was-investigated. All the organic compounds tested exhibited antimicrobial activity against the microorganisms used; however, the MICs varied between 0.8 and 15.0 mM depending on the potency of the compound or the sensitivity of the target strain. Investigation of the interaction between the organic compounds and nisin against the test organisms revealed different patterns, varying from synergistic to antagonistic. Combinations of nisin with carvacrol, eugenol, or thymol resulted in synergistic action against both test organisms. Activity of nisin and cinnamic acid together was synergistic against L. innocua, but only additive against B. subtilis. In contrast, the combination of diacetyl and nisin resulted in an antagonistic effect against both test organisms. This study highlights the potential of the combination of these compounds with nisin to inhibit pathogen growth in food.     

Inactivation of Listeria innocua in liquid whole egg by pulsed electric fields and nisin

Consumer demand for fresh-like products with little or no degradation of nutritional and organoleptic properties has led to the study of new technologies in food preservation. Pulsed electric fields (PEF) is a nonthermal preservation method used to inactivate microorganisms mainly in liquid foods. Microorganisms in the presence of PEF suffer cell membrane damage. Nisin is a natural antimicrobial known to disrupt cell membrane integrity. Thus the combination of PEF and nisin represents a hurdle for the survival of Listeria innocua in liquid whole egg (LWE). L. innocua suspended in LWE was subjected to two different treatments: PEF and PEF followed by exposure to nisin. The selected frequency and pulse duration for PEF was 3.5 Hz and 2 micros, respectively. Electric field intensities of 30, 40 and 50 kV/cm were used. The number of pulses applied to the LWE was 10.6, 21.3 and 32. The highest extent of microbial inactivation with PEF was 3.5 log cycles (U) for an electric field intensity of 50 kV/cm and 32 pulses. Treatment of LWE by PEF was conducted at low temperatures, 36 degrees C being the highest. Exposure of L. innocua to nisin following the PEF treatment exhibited an additive effect on the inactivation of the microorganism. Moreover, a synergistic effect was observed as the electric field intensity, number of pulses and nisin concentration increased. L. innocua exposed to 10 IU nisin/ml after PEF exhibited a decrease in population of 4.1 U for an electric field intensity of 50 kV/cm and 32 pulses. Exposure of L. innocua to 100 IU nisin/ml following PEF resulted in 5.5 U for an electric field intensity of 50 kV/cm and 32 pulses. The model developed for the inactivation of L. innocua by PEF and followed by exposure to nisin proved to be accurate (p = 0.05) when used to model the inactivation of the microorganism by PEF in LWE with 1.2 or 37 IU nisin/ml. The presence of 37 IU nisin/ml in LWE during the PEF treatment for an electric field intensity of 50 kV/cm and 32 pulses resulted in a decrease in the population of L. innocua of 4.4 U.     

The effect of homogenization of whole milk, skim milk and milk fat on nisin activity against Listeria innocua

Whole milk, skim milk and an emulsion of milk fat in water, inoculated with approx. 10(5) cfu/ml of Listeria innocua, were treated at 30 degrees C with 100 IU/ml of nisin, homogenization at 200 bar or both procedures. Nisin activity and survival of L. innocua after treatments were determined. Recovery of nisin activity from non-homogenized whole milk treated with 100 IU/ml of nisin was complete, whereas a loss of 18 to 28% of activity was detected in non-homogenized fat-in-water emulsion. Loss in nisin activity due to homogenization represented up to 64% in whole milk and 62% in fat-in-water emulsion. Nisin addition by itself achieved a reduction in L. innocua counts of 3.7-3.8 log units in whole milk and 3.6 log units in fat-in-water emulsion compared to numbers in untreated samples. When nisin-containing whole milk and fat-in-water emulsion were homogenized, L. innocua counts were only reduced by 2.6-2.9 log units and 2.5 log units, respectively, compared to numbers in untreated samples. Homogenization of nisin-containing skim milk resulted in a loss of nisin activity of 20% but achieved a reduction of 3.0 log units in L. innocua counts.     

Inhibition of Listeria monocytogenes using nisin with grape seed extract on turkey frankfurters stored at 4 and 10 degrees C

Recontamination of cooked ready-to-eat (RTE) chicken and beef products with Listeria monocytogenes has been a major safety concern. Natural antimicrobials in combinations can be an alternative approach for controlling L. monocytogenes. Therefore, the objectives of this study were to evaluate the inhibitory activities against L. monocytogenes of nisin (6,400 IU/ ml), grape seed extract (GSE; 1%), and the combination of nisin and GSE both in tryptic soy broth with 0.6% yeast extract (TSBYE) and on the surface of full-fat turkey frankfurters. TSBYE was incubated at 37 degrees C for 72 h and turkey frankfurters at 4 or 10'C for 28 days. Inocula were 6.7 or 5 log CFU per ml or g for TSBYE or frankfurters, respectively. After 72 h in TSBYE, nisin alone did not show any inhibitory activity against L. monocytogenes. The combination of nisin and GSE gave the greatest inhibitory activity in both TSBYE and on turkey frankfurters with reductions of L. monocytogenes populations to undetectable levels after 15 h and 21 days, respectively. This combination of two natural antimicrobials has the potential to control the growth and recontamination of L. monocytogenes on RTE meat products.     

INDIVIDUAL AND COMBINED LISTERICIDAL EFFECTS OF SODIUM LACTATE, POTASSIUM SORBATE, NISIN AND CURING SALTS AT REFRIGERATION TEMPERATURE

The individual and combined antilisterial activity of the preservatives sodium lactate (4%), potassium sorbate (0.3%) and nisin (400 IU/ml), in either the presence or absence of the curing salts nitrite (125 ppm) and polyphosphate (0.5%), was assessed in buffered BHI broth (pH 5.5) during incubation at 4C. A cocktail of the strain Listeria monocytogenes NCTC 7973 and two food derived strains was used as the inoculum in challenge studies. In the absence of the preservatives , L. monocytogenes grew in the presence of polyphosphate but not in the presence of nitrite or a combination of nitrite and polyphosphate. The antilisterial action of nitrite was predominantly bacteriostatic in nature. Lactate acting alone, or in the presence of curing salts, produced a bacteriostatic effect. Sorbate acting alone had a bacteriostatic effect. Sorbate acting in the presence of nitrite or nitrite and polyphosphate produced a marked listericidal effect, reducing the population by 6.7 logs and 5.4 logs, respectively. Nisin acting alone produced an almost immediate 90% listericidal response (1 log population decrease), but initial numbers were restored within 14 days by regrowth. Regrowth was eliminated when nisin was used in combination with lactate, nitrite or nitrite and polyphosphate. The combination of lactate and sorbate offered no advantage over the use of sorbate alone. Sorbate and nisin acting in combination produced an enhanced listericidal effect, which was also seen in the presence of curing salts, but was delayed. No listericidal advantage over sorbate-nisin combination was achieved through the use of the lactate-sorbate-nisin combination. The combined use of sorbate and nisin offers promise as a means of eliminating L. monocytogenes from low pH cured meat products. The efficacy of this preservative combination remains to be evaluated in a meat system before its practical application can be considered .     

Synergistic inhibition of Listeria monocytogenes on cold-smoked rainbow trout by nisin and sodium lactate

The inhibition of Listeria monocytogenes and mesophilic aerobic bacteria in cold-smoked rainbow trout by nisin, sodium lactate or their combination was studied. Nisin (4000-6000 IU/ml), sodium lactate (60%) or their combination (1:1) were injected into rainbow trout at an industrial scale before the smoking process, or injected into the finished smoked product. Both types of fish samples were smoked, sliced and vacuum-packed according to normal practice in the plant. Packages were opened and L. monocytogenes was inoculated (10(3)-10(4) log colony forming units (cfu)/g) onto the fish samples, which were then vacuum packed again. Samples were stored at 8 degrees C for 17 days or at 3 degrees C for 29 days. Listeria and mesophilic aerobic bacteria counts were measured once a week. The effects of treatments on sensory characteristics and storage stability were also analyzed. Both nisin and lactate inhibited the growth of L. monocytogenes in smoked fish, but the combination of the two compounds was even more effective. The combination of nisin and sodium lactate injected into smoked fish decreased the count of L. monocytogenes from 3.26 to 1.8 log cfu/g over 16 days of storage at 8 degrees C. The level of L. monocytogenes remained almost constant (4.66-4.92 log cfu/g) for 29 days at 3 degrees C in the samples injected before smoking and which contained both nisin and sodium lactate. The treatments did not affect the sensory characteristics of cold-smoked rainbow trout. Based on a triangle test, the sensory quality of all test samples remained unchanged for 23 days of storage at 3 degrees C, whereas the control fish prepared without additives or additional salt remained unchanged only for 16 days.     

Inhibition of Listeria monocytogenes in pH-adjusted pasteurized liquid whole egg

Although the transmission of L. monocytogenes to humans via pasteurized egg products has not been documented, L. monocytogenes and other Listeria species have been isolated from commercially broken raw liquid whole egg (LWE) in both the United States and Ireland. Recent Listeria thermal inactivation studies indicate that conventional minimal egg pasteurization processes would effect only a 2.1- to 2.7-order-of-magnitude inactivation of L. monocytogenes in LWE; thus, the margin of safety provided by conventional pasteurization processes is substantially smaller for L. monocytogenes than for Salmonella species (a 9-order-of-magnitude process). The objective of this study was to evaluate the inhibitory effects of nisin on the survival and growth of L. monocytogenes in refrigerated and pH-adjusted (pH 6.6 versus pH 7.5) ultrapasteurized LWE and in a liquid model system. The addition of nisin (1,000 IU/ml) to pH-adjusted ultrapasteurized LWE reduced L. monocytogenes populations by 1.6 to > 3.3 log CFU/ml and delayed (pH 7.5) or prevented (pH 6.6) the growth of the pathogen for 8 to 12 weeks at 4 and 10 degrees C. Bioactive nisin was detected in LWE at both pH values for 12 weeks at 4 degrees C. In subsequent experiments, Listeria reductions of > 3.0 log CFU/ml were achieved within 24 h in both LWE and broth plus nisin (500 IU/ml) at pH 6.6 but not at pH 7.5, and antilisterial activity was enhanced when nisin was added as a solution rather than in dry form.     

Efficacy of nisin in combination with protective cultures against Listeria monocytogenes Scott A in tofu.

Nisin can be used as a biopreservative to control growth of Listeria monocytogenes in various minimally processed foods. Tofu is an example of a non-fermented soybean product, which may allow growth of Listeria at refrigeration temperatures and in which nisin may be applied to prevent multiplication of Listeria. The efficacy of nisin against Listeria may be compromised by the emergence of spontaneous nisin-resistant mutants. Exposure of L. monocytogenes Scott A to nisin in a culture medium or in a food product results in an initial reduction of Listeria population which is followed by regrowth of survivors to nisin during further incubation. In vitro studies using Standard I Nutrient broth showed that Enterococcus faecium BFE 900-6a and Lactobacillus sakei Lb 706-1a used as protective cultures in combination with nisin were able to suppress proliferation of Listeria cells not killed by nisin at 10 degrees C. Growth and bacteriocin production of these two strains and a third protective culture, Lactococcus lactis BFE 902 was also observed in soymilk and tofu at 10 degrees C. Inoculation studies with tofu prepared with nisin and protective cultures showed that lower amounts of nisin are required for an effective inhibition of L. monocytogenes Scott A when either E. faecium BFE 900-6a or Lc. lactis BFE 902 are used in addition. The combination of nisin with these bacteriocinogenic lactic acid bacteria (LAB) resulted in a complete suppression of listerial growth in homemade tofu stored at 10 degrees C for 1 week. Lb. sakei Lb 706-1a was less effective and did not prevent a slight increase of L. monocytogenes Scott A numbers during storage.     

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.     

Antimicrobial activity of nisin incorporated in pectin and polylactic acid composite films against Listeria monocytogenes

An extruded composite food packaging film containing pectin, polylactic acids (PLAs) and nisin was developed to inhibit Listeria monocytogenes . The mechanical properties and surface structure of the film were also examined. Cells of L. monocytogenes were reduced by 2.1, 4.5 and 3.7 log units mL⁻¹ by the pectin plus PLA (pectin/PLA) film containing nisin (1000 IU mL⁻¹ of tested liquid) in Brain Heart Infusion (BHI) broth, liquid egg white and orange juice, respectively, after 48 h at 24 °C. Pectin played an important roll in embedding nisin into the film. The pectin/PLA film had a similar stiffness but lower tensile strength, elongation and fracture energy than the pure PLA film. These data suggested that nisin incorporated into the pectin/PLA film was an effective approach to reducing L. monocytogenes in a typical growth medium (e.g. BHI broth) as well as in foods (e.g. orange juice and liquid egg).     

Survival of Listeria monocytogenes Scott A on vacuum-packaged raw beef treated with polylactic acid, lactic acid, and nisin

Low-molecular-weight polylactic acid (LMW-PLA) and lactic acid (LA) were used to inhibit growth of Listeria monocytogenes Scott A on vacuum-packaged beef. Nisin was also used simultaneously as an additional hurdle to the growth of this pathogen. Inoculated beef cubes were immersed in a solution of 2% LMW-PLA, 2% LA, 400 IU/ml of nisin, or combinations of each acid and nisin for 5 min and drip-dried for 15 min. The cubes were then vacuum-packaged and stored at 4 degrees C for up to 42 days. Surface pH values of beef cubes treated with 2% LMW-PLA, the combination of 400 IU/ml of nisin and 2% LMW-PLA (2% NPLA), or 400 IU/ml of nisin alone were significantly reduced from 5.59 to 5.18, 5.01, and 5.19, respectively, whereas those decontaminated with 2% LA or 400 IU/ml of nisin and 2% LA (2% NLA) were significantly decreased from 5.59 to 4.92 and 4.83, respectively, at day 0 (P < or = 0.05). The 2% LMW-PLA, 2% LA, 2% NPLA, 2% NLA, and 400 IU/ml of nisin showed immediate bactericidal effects on L. monocytogenes Scott A (1.22-, 1.56-, 1.57-, 1.94-, and 1.64-log10 reduction, respectively) compared with the initial number of 5.33 log10 CFU/cm2 of the untreated control at day 0 (P < or = 0.05). These treatments, combined with vacuum-packaging and refrigeration temperature, succeeded to inhibit growth of L. monocytogenes during storage up to 42 days. At the end of 42 days, the numbers of L. monocytogenes Scott A remaining viable on these samples were 1.21, 0.36, 2.21, 0.84, and 0.89 log10 CFU/cm2, respectively.