Evaluation of anti-Listeria meat borne Lactobacillus for biofilm formation on selected abiotic surfaces

The ability of meat borne anti-Listeria Lactobacillus to form biofilms under different in vitro conditions and on abiotic surfaces was investigated. Biofilm formation by the adhesion to polystyrene microtiter plates was determined, this being higher for Lactobacillus curvatus CRL1532 and CRL705 and Lactobacillus sakei CRL1862. The physicochemical properties of the cell surface were relatively hydrophilic and acidic in character; L. sakei CRL1862 exhibiting the strongest autoaggregation. The adhesion of lactobacilli to stainless steel (SS) and polytetrafluoroethylene (PTFE) supports at 10 °C was found to be maximal for L. sakei CRL1862 on SS after 6 days. When biofilm architecture was characterized by epifluorescence and SEM, L. sakei CRL1862 homogeneously covered the SS surface while cell clusters were observed on PTFE; the extracellular polymeric substance matrix adapted to the topography and hydrophilic/hydrophobic characteristics of each material. The feasibility of L. sakei CRL1862 to form biofilm on materials used in meat processing highlights its potential as a control strategy for Listeria monocytogenes biofilms.     

The control of Listeria innocua and Lactobacillus sakei in broth and meat slurry with the bacteriocinogenic strain Lactobacillus casei CRL705

The effect of the bacteriocin-producing strain, Lactobacillus casei CRL705, in the control of Listeria innocua 7 and Lactobacillus sakei CRL1424 in MRS medium and meat slurry during the storage under vacuum at chill temperatures was evaluated. L. sakei CRL 1424 isolated from vacuum-packaged contaminated raw meat was identified as the predominant indigenous lactic acid bacterial flora. Co-inoculation of MRS broth at 8°C with L. casei CRL705 caused growth inhibition of L. sakei CRL1424 and L. innocua 7 after 10 and 4 days of storage, respectively. At 4°C the complete inhibition of both strains occurred within 14 and 8 days for L. sakei CRL1424 and L. innocua 7, respectively. Bacteriocin activity in broth was observed to be maximal at 8°C reaching 2130 AU ml⁻¹ after10 days and 400 AU m1⁻¹ after 8 days of storage, while at 4°C maximal activities, 690 and 30 AU ml⁻¹ were obtained at 14 and 10 days, respectively. Addition of bacteriocinogenic strain to the meat slurry did not allow the growth of L. sakei and L. innocua, showing a bacteriostatic effect during 21 days of storage at 4°C. In addition, L. casei CRL705, as a protective culture did not change significantly the pH of the meat slurry in the assayed storage conditions. The results presented here confirm that the bacteriocinogenic strain L. casei CRL705 can be used as a useful tool to improve the microbial stability and safety in the commercial meat preservation.     

Development and characterization of an active polyethylene film containing Lactobacillus curvatus CRL705 bacteriocins

The development and characterization of a bacteriocin-containing polyethylene-based film is described, incorporating lactocin 705 and lactocin AL705, produced by Lactobacillus curvatus CRL705, and nisin. Three different procedures to obtain lactocin 705 and AL705 solution were evaluated, with the partially purified aqueous bacteriocin solution showing the highest inhibitory activity against indicator strains (Lactobacillus plantarum CRL691 and Listeria innocua 7). Pouch contact, soaking and a contact method were compared for incorporating bacteriocins onto PE-based films. Contact between the PE film and bacteriocin solution was the most effective, resulting in a more uniform distribution of bacteriocins on the film surface and using less active solution. The minimal inhibitory concentration of bacteriocin solution was 267 AU cm^?3 (lactocin 705) and 2133 AU cm^?3 (lactocin AL705), while the minimal contact time was 1 h. When relative inhibition area for antilisterial activity of the active films was compared, those treated with L. curvatus CRL705 bacteriocins displayed higher inhibitory activity than nisin-treated films. Functional properties of active PE-films containing lactocin 705 and AL705 showed no differences compared with non-active control films. Bacteriocin-active PE-based films are shown to be highly effective in inhibiting growth of Listeria. The potential use of commercially available packaging films as bacteriocins carriers may benefit active-packaging systems.     

Development of an active wheat gluten film with Lactobacillus curvatus CRL705 bacteriocins and a study of its antimicrobial performance during ageing

Antimicrobial wheat gluten film was obtained at pilot scale by Lactobacillus curvatus CRL705 bacteriocins inclusion in the film-forming solution. Bacteriocins’ minimum inhibitory concentration for the film activation was 2133 AU cm^?3 (lactocin AL705) and 267 AU cm^?3 (lactocin 705). Mechanical and barrier properties as well as film ageing kinetics were not significantly affected by the addition of bacteriocins. The antimicrobial film performance during ageing was assessed. Film activity against Listeria innocua 7 and Lactobacillus plantarum CRL691 was observed over 50 days of ageing. Even when the release of bacteriocins from the film upon water contact was observed for both bacteriocins at the beginning of the ageing period, and anti-Listeria activity was delivered to the simulant up to the 15th day of ageing, film residual activity for both bacteriocins was observed over 50 days. The results confirm the potential of a gluten film doped with L. curvatus CRL705 bacteriocins as a carrier of bacteriocins to avoid Listeria and lactic acid bacterial growth, thus enhancing quality and safety in foods.     

A Weibull model to describe antimicrobial kinetics of oregano and lemongrass essential oils against Salmonella Enteritidis in ground beef during refrigerated storage

The antimicrobial effect of oregano (Origanum vulgare L.) and lemongrass (Cymbopogon citratus (DC.) Stapf.) essential oils (EOs) against Salmonella enterica serotype Enteritidis in in vitro experiments, and inoculated in ground bovine meat during refrigerated storage (4 ± 2 °C) for 6 days was evaluated. The Weibull model was tested to fit survival/inactivation bacterial curves (estimating of p and δ parameters). The minimum inhibitory concentration (MIC) value for both EOs on S. Enteritidis was 3.90 μl/ml. The EO concentrations applied in the ground beef were 3.90, 7.80 and 15.60 μl/g, based on MIC levels and possible activity reduction by food constituents. Both evaluated EOs in all tested levels, showed antimicrobial effects, with microbial populations reducing (p ≤ 0.05) along time storage. Evaluating fit-quality parameters (RSS and RSE) Weibull models are able to describe the inactivation curves of EOs against S. Enteritidis. The application of EOs in processed meats can be used to control pathogens during refrigerated shelf-life.     

Characterization of a multilayer film activated with Lactobacillus curvatus CRL705 bacteriocins

BACKGROUND: Bacteriocins produced by lactic acid bacteria offer enormous promise for food safety preservation. In this study an active multilayer film obtained by the incorporation of lactocin 705 and lactocin AL705, two bacteriocins produced by Lactobacillus curvatus CRL705 with antimicrobial activity against Lactobacillus plantarum CRL691 and Listeria innocua 7, respectively, was characterized for its potential application in active packaging technology. Film activity performance at different storage conditions, bacteriocins transfer into water and sunflower oil, and film surface properties were evaluated. RESULTS: Film activity against L. innocua 7 was maintained during 2, 4 and 6 weeks at 30, 10 and 5 °C respectively. At 30 and 10 °C, activity loss against L. plantarum CRL691 was observed on the second week of storage and after the fourth week at 5 °C. Results showed no significant difference for active multilayer film contact angle and seal properties compared to the control (without bacteriocins). A decrease in lactocin 705 inhibitory activity after sunflower oil contact was observed, while lactocin AL705 remained unaffected. After water contact, film activity was retained for both bacteriocins. CONCLUSIONS: As demonstrated by antimicrobial activity and physico‐mechanical properties retention, lactocin 705 and AL705 active multilayer film present potential for application in active packaging technology. Copyright © 2011 Society of Chemical Industry     

Fate of post-processing inoculated Listeria monocytogenes on vacuum-packaged pepperoni stored at 4, 12 or 25 °C

If present, Listeria monocytogenes may not be eliminated during processing of pepperoni or may be introduced during peeling, slicing, or packaging. We evaluated the fate of the pathogen on sliced inoculated pepperoni during vacuum-packaged storage, and potential differences in survival among three types of inocula, including nonacid-adapted, acid-adapted and pepperoni extract-habituated cultures. Commercial pepperoni (two replicates, three samples per treatment) was sliced and inoculated (3 to 4 log CFU/cm²), before vacuum-packaging and storage for up to 180 days at 4, 12 or 25 °C. Samples were periodically analyzed for pathogen counts (PALCAM agar) and total bacterial counts (tryptic soy agar with 0.6% yeast extract). The pH of the product was relatively stable (4.50–4.81) throughout storage. Overall, levels of the pathogen (all inocula) and total counts decreased continuously during storage at all temperatures. The pathogen died slower at 4 °C than at 12 and 25 °C, while at 12 and 25 °C the death rates were similar. Death rates depended on type of inoculum and generally decreased in the order: acid-adapted, extract-habituated and nonacid-adapted inoculum. At day 60, pathogen levels were below the detection limit and remained undetectable throughout the rest of the 180-day storage period, regardless of inoculum type and storage temperature. Therefore, storage of sliced vacuum-packaged pepperoni, especially at ambient temperature, prior to consumption may reduce the potential risk of listeriosis.     

Effects of carbon monoxide-modified atmosphere packaging and irradiation on E. coli K12 survival and raw beef quality

The objective of this study was to evaluate the combined effects of irradiation and carbon monoxide in modified atmosphere packaging (CO-MAP) on total plate counts, Escherichia coli K12, color, and odor of fresh beef during refrigerated storage. Beef was packaged aerobically or in CO-MAP, and irradiated at 0, 0.5, 1.0, 1.5 or 2.0 kGy then held at 4 °C for 28 days. Raw beef odor decreased and acid/sour, rancid and grassy odors increased starting on day 14. Initially, no difference existed for visual green color scores due to gas atmosphere. After 14 days of storage, aerobically packaged beef was greener and less red than CO-MAP packaged beef. The a∗ value of CO-MAP packaged beef was higher than that of aerobically packaged beef. Red color of CO-MAP packaged samples decreased slightly in some irradiated samples after 14 days of storage. On day 0 and thereafter, no coliforms were detect after irradiation at 1.5 or 2.0 kGy regardless of packaging format. These findings suggest that CO-MAP could be used to preserve beef color irradiated at doses sufficient to reduce microbial loads to safe levels during 28 days of storage.     

A bacteriocin from Carnobacterium piscicola for the control of Listeria monocytogenes in vacuum-packaged meat

The purpose of this study was to determine the inhibitory capacity of a bacteriocin-like substance produced by Carnobacterium piscicola L103 against Listeria monocytogenes , when inoculated into vacuum-packaged meat. The bacteriocin was partially purified by ammonium sulfate precipitation and gel filtration on Sephadex G-25, followed by lyophilization. Beef steaks were inoculated with the bacteriocin and a sensitive strain of L. monocytogenes . The vacuum-packaged meat was stored at 4°C and microbial analyses were carried out every 7 days, over 21 days. Results showed that in the control steaks, without bacteriocin, counts of L. monocytogenes stayed at the initial inoculation level during the complete storage period. The naturally growing lactic acid bacteria (LAB) reached log 7·4 cfu cm⁻²after 14 days of storage and no inhibitory effect on the growth of the LAB was observed in the presence of the bacteriocin. The pH of the meat dropped from 5·7 to 5·0 in the inoculated as well as in the control samples, while colour and odour remained acceptable up to 14 days of storage. On the meat inoculated with bacteriocin, L. monocytogenes was completely inhibited after 14 days of storage at 4°C. It was concluded that the use of the inhibitory substance from C. piscicola L103 could be an important contribution to meat safety, when inoculated into vacuum-packaged meat.     

Effects of curing additives on the control ofListeria monocytogenesby lactocin 705 in meat slurry

Meat slurry inoculated withListeria monocytogenes(4.00 cfu g⁻¹) was mixed with different levels of curing additives and their influence on the inhibitory effect of lactocin 705 (17,000 AU ml⁻¹) was evaluated at 20°C. Inhibition ofL. monocytogeneswas 1.90 and 1.00 log less in meat slurry with 5 and 7% NaCl than in meat slurry without added sodium chloride. When nitrite and bacteriocin were added together, less nitrite (200 μg g⁻¹) was required to obtain the sameListeriapopulation (3.00 log cfu g⁻¹) as when 800 μg g⁻¹NaNO₂was used. However, when compared with lactocin 705 alone, lessListeriainhibition was observed showing also a protective effect of NaNO₂. When ascorbic acid and alginate meat binder were assayed in the presence of the bacteriocin, the inhibition ofL. monocytogeneswas less effective, but when sodium lactate (2%) was added to the meat slurry, almost no protective effect was observed. These results indicated that the use of lactocin 705 to controlL. monocytogeneswas less effective in the presence of curing ingredients.     

Effect of freeze-thaw cycles on physicochemical properties and color stability of beef semimembranosus muscle

The effects of freeze-thaw cycle on the physicochemical properties and color stability of beef semimembranosus muscle during cold storage were investigated. Meat was tested for effects on color (CIE L^?, a^?, b^?), myoglobin content (%), metmyoglobin reducing ability (MRA), thiobarbituric acid reactive substances (TBARS), heme pigment content (ppm), SDS-PAGE profile, and tissue microstructure. Fresh meat showed significantly (p < 0.05) higher values of CIE a^?, CIE b^?, and oxymyoglobin content (%) compared to freeze-thawed meat during cold storage. However, metmyoglobin content (%) was higher in freeze-thawed meat than in fresh meat during 7 days of cold storage. There were no significant differences (p > 0.05) in MRA among the treatments during cold storage. The TBARS value (0.387 mg/kg) of fresh meat was significantly (p < 0.05) higher than those of freeze-thawed meats after 5 days of cold storage. These results suggest that the freeze-thaw process could accelerate the deterioration of meat color.     

Proteomic and peptidomic insights on myofibrillar protein hydrolysis in a sausage model during fermentation with autochthonous starter cultures

The hydrolysis of myofibrillar proteins during fermentation of sausage models by an autochthonous starter culture was investigated. In order to provide a whole map of the generated products, proteomic and peptidomic were used and complemented with the amino acid profile. Beaker sausages (BS) were used as models which were inoculated or not with Lactobacillus curvatus CRL705 and Staphylococcus vitulinus GV318 as starter cultures. The hydrolysis of actin, myosin light chain 1/3 (MLC 1/3), myosin regulatory light chain-2 (MRLC-2) and myosin heavy chain (MHC) was evidenced by two-dimensional gel electrophoresis (2-DE). In addition, a total of 33 peptides arisen from troponin T, MRLC-2 and particularly from actin were identified by LC–MS/MS. These results showed that the starter culture significantly enhanced the proteolysis of the proteins named above, even when the endogenous enzymes induced a clear breakdown. L. curvatus CRL705 highly enriched both peptide pattern and amino acid concentrations. When the autochthonous starter culture was inoculated, although proteolysis was remarkably reinforced, a reduction in peptide and amino acid composition was observed. Regarding actin primary structure, three regions of this protein were highly susceptible to degradation by the starter culture. Additionally, the essential role of exopeptidases – from meat and bacteria – in diversity of actin peptides during fermentation was shown. This study improved the knowledge of the proteolysis of myofibrillar proteins and the involved enzymes, as well as, completed the previously reported degradation of sarcoplasmic proteins by the same autochthonous starter culture. The singular peptides and amino acids pattern generated might contribute to the uniqueness of produced fermented sausages while they may be used as quality markers.     

<Emphasis Type="Italic">Listeria</Emphasis> Inactivation by the Combination of High Hydrostatic Pressure and Lactocin AL705 on Cured-Cooked Pork Loin Slices

The effect of the bacteriocin lactocin AL705 in combination with high hydrostatic pressure (HHP) on the inactivation of Listeria innocua 7, a nonpathogenic indicator for Listeria monocytogenes, deliberately inoculated (ca. 6.4 log CFU/g) onto the surface of ready-to-eat (RTE) sliced cured-cooked pork loin, was evaluated. Nontreated pork slices (control) and treatments subjected to lactocin AL705 (105 AU/ml) and/or HHP (400 or 600 MPa) were prepared. L. innocua 7 was monitored at days 1, 20, and 40 of storage at 4 °C. The results showed a complete inhibition of L. innocua 7 after the combined treatment with lactocin AL705 and 600 MPa and no regrowing of cells up to 40-day storage. The treatment at 600 MPa alone was not enough to avoid regrowth of L. innocua. Ultrastructural cell damage was observed at the cytoplasm and cell membrane/wall levels with all treatments; however, complete cell lysis was observed only with the combined treatment. HHP in combination with lactocin AL705 provided a wider margin of safety as post-processing listericidal treatment of RTE cured-cooked meat products.     

Changes in microbiological, biochemical and physico-chemical properties of Nham inoculated with different inoculum levels of Lactobacillus curvatus

The effect of inoculum level of Lactobacillus curvatus on Nham fermentation was studied. Nham inoculated with L. curvatus at 10⁴ (LC104) and 10⁶ cfu/g (LC106) exhibited a higher rate of fermentation than naturally fermented Nham (control) as indicated by greater rate of pH drop, lactic acid production, and changes in protein compositions. Based on the pH, the fermentation was completed within 72, 48 and 36 h for control, LC104, and LC106, respectively. Higher extent of proteolysis and lipolysis were observed during fermentation of Nham inoculated with starter bacteria (P<0.05). Due to higher acid production rate and extent, texture development of inoculated Nham was more rapid. Inoculated Nham exhibited higher TPA hardness and adhesiveness but lower fracturability than naturally fermented Nham (P<0.05). In terms of acceptability, control and LC104 had higher ratings on the overall liking than LC106 (P<0.05). However, LC104 was better accepted than control in terms of flavour, sourness, saltiness, and texture. Unusual smell was detected only in Nham inoculated at 10⁶ cfu/g. Based on physico-chemical properties and consumer acceptability, L. curvatus is a potential starter for Nham fermentation. However, inoculation of L. curvatus at high level may cause off-flavour in the product.     

Production of salami from beef, horse, mutton, Blesbok (Damaliscus dorcas phillipsi) and Springbok (Antidorcas marsupialis) with bacteriocinogenic strains of Lactobacillus plantarum and Lactobacillus curvatus

Lactobacillus plantarum 423, producer of bacteriocin 423, Lactobacillus curvatus DF38, producer of curvacin DF38, and a bacteriocin-negative mutant of L. plantarum 423 (423m) were evaluated as starter cultures in the production of salami from beef, horse, mutton, Blesbok (Damaliscus dorcas phillipsi) and Springbok (Antidorcas marsupialis). Growth of L. plantarum 423 and L. curvatus DF38 was best supported in Blesbok salami, as revealed by the highest growth rate during sweating, cold smoking and maturation, and final cell numbers after 70 days (1 × 10⁸ and 5 × 10⁷ cfu/g, respectively). Growth of Listeria innocua was the best suppressed in Blesbok salami fermented with L. plantarum 423 and L. curvatus DF38. Growth of L. innocua in horse salami was best suppressed when fermented with L. curvatus DF38. The final pH of salami fermented with L. plantarum 423 and L. plantarum 423m was slightly lower (4.4) compared to the pH of salami produced with L. curvatus DF38 (pH 4.7). No significant differences (P > 0.05) were recorded by a trained sensory taste panel amongst the three starter cultures regarding colour and venison like aroma. Horse, Blesbok and Springbok salami were rated significantly higher (P ? 0.05) in salami flavour than mutton salami, which was rated the lowest for this attribute. Blesbok salami was rated the highest for sour meat aroma, while beef salami was rated the lowest. Springbok salami was rated the highest in terms of oily mouth feel. Beef salami had the most compact structure and horse salami the softest structure of all meat types fermented. In general, salami produced with L. plantarum 423 yielded the best sour meat aroma, colour, texture, venison like flavour, sour meat flavour and oily mouthfeel and is considered superior to the L. plantarum mutant (strain 423m) and L. curvatus DF38.     

Enhancing the thermal destruction of Escherichia coli O157:H7 in ground beef patties by trans-cinnamaldehyde

The effect of trans-cinnamaldehyde (TC) on the inactivation of Escherichia coli O157:H7 in undercooked ground beef patties was investigated. A five-strain mixture of E. coli O157:H7 was inoculated into ground beef (7.0 log CFU/g), followed by addition of TC (0, 0.15, and 0.3%). The meat was formed into patties and stored at 4 °C for 5 days or at −18 °C for 7 days. The patties were cooked to an internal temperature of 60 or 65 °C, and E. coli O157:H7 was enumerated. The numbers of E. coli O157:H7 did not decline during storage of patties. However, cooking of patties containing TC significantly reduced (P < 0.05) E. coli O157:H7 counts, by >5.0 log CFU/g, relative to the reduction in controls cooked to the same temperatures. The D-values at 60 and 65 °C of E. coli O157:H7 in TC-treated patties (1.85 and 0.08 min, respectively) were significantly lower (P < 0.05) than the corresponding D-values for the organism in control patties (2.70 and 0.29 min, respectively). TC-treated patties were more color stable and showed significantly lower lipid oxidation (P < 0.05) than control samples. TC enhanced the heat sensitivity of E. coli O157:H7 and could potentially be used as an antimicrobial for ensuring pathogen inactivation in undercooked patties. However detailed sensory studies will be necessary to determine the acceptability to consumers of TC in ground beef patties.     

Modeling the growth of lactic acid bacteria in sliced ham processed by high hydrostatic pressure

The main responsible for the spoilage of cooked cured meat products stored under refrigerated and anaerobic conditions are lactic acid bacteria. The application of high hydrostatic pressure (HHP) reduces the lactic acid bacterial growth extending the product shelf-life and preserving natural taste, texture, color and vitamin content. This work studied the influence of pressure level and holding time on the lactic acid bacterial growth in vacuum-packaged sliced ham. Modified Gompertz and Logistic models were used to fit experimental data obtained from post-treatment microbial counts carried out along the product storage. Samples of sliced vacuum-packaged ham treated by HHP and control samples (non-treated) were stored at 8 °C until the microorganism population reached 10⁷ CFU/g. An experimental planning 2² with triplicate at the central point was designed to determine the influence of pressure level (200, 300, and 400 MPa) and holding time (5, 10, and 15 min) on the product shelf-life. The results have shown that the pressure intensity and the holding time significantly influenced microbial population over the product storage. Shelf-life of ham treated at 400 MPa for 15 min was extended from 19 (control samples) to 85 days.     

Incorporation of bacteriocin in plastic retains activity and inhibits surface growth of bacteria on meat

The bacteriocin, nisin, was incorporated into a polyethylene based plastic film and retained activity against the indicator bacteria Lactobacillus helveticus and Brochothrix thermosphacta . Beef carcass surface tissue sections (BCT) topically inoculated with the psychrotrophic spoilage bacterium B. thermosphacta were vacuum-packaged both with and without wrapping with the nisin impregnated plastic and held at 4° C. An initial reduction of 2 log₁₀cycles of B. thermosphacta was observed with nisin-impregnated wrapped BCT within the first 2 days of storage. After 20 days of refrigerated storage, B. thermosphacta populations from nisin impregnated plastic wrapped samples were significantly less than (P<0·05) control vacuum-packaged samples; log₁₀5·8 vs 7·2 cfu cm⁻²respectively. Temperature abuse was simulated by shifting inoculated packs from 4° C (after 2 days) to 12° C. Again, by 20 days, the B. thermosphacta populations of treated samples wrapped with nisin impregnated plastic were significantly less than (P<0·05) control vacuum-packaged samples; log₁₀3·6 vs 6·3 cfu cm⁻²respectively. This work highlights the potential for incorporating antimicrobial peptides with a wider and different range of inhibitory activity directly into plastics of different properties for use in controlling food spoilage as well as preservation to enhance product microbial safety.     

Combined antimicrobial effect of oregano essential oil and caprylic acid in minced beef

Oregano essential oil (OEO) and caprylic acid (CA) are highly aromatic natural antimicrobials with limited individual application in food. We proved their combined additive effect when used in meat. Application of 0.5% CA and 0.2% OEO (v/w) with 0.1% of citric acid in vacuum packed minced beef inoculated with Listeria monocytogenes at a concentration of 5 log cells/g reduced counts of lactic acid bacteria by 1.5 log CFU/g and counts of psychrotrophic bacteria and L. monocytogenes by more than 2.5 log CFU/g at the end of storage at 3 °C for 10 days. In sensory evaluation the samples with OEO showed during the whole experiment statistically better scores than control, whereas the samples treated with CA showed worse colour attributes.     

Reductions ofListeria innocuaandBrochothrix thermosphactaon beef following nisin spray treatments and vacuum packaging

Sections of UV sterilized lean and adipose tissues from the surfaces of post-rigor (24 h post-mortem) beef carcasses were inoculated withBrochothrix thermosphactaorListeria innocuato obtain approximately 4.50 log₈cfu cm⁻²and subjected to spray treatments with sterile water or nisin (5000 AU ml⁻¹). Untreated and spray treated samples were vacuum-packaged, and incubated at 4°C for up to 4 weeks. Bacterial populations from untreated vacuum-packaged tissues and spray treated, vacuum-packaged tissues were enumerated on non-selective and selective media at 0, 7, 14, 21 or 28 days. Nisin spray treatments of lean and adipose vacuum-packaged tissues reduced the numbers ofL. innocuaup to 2.83 log₈cfu cm⁻². Additionally, nisin sprays and vacuum packaging effectively suppressedL. innocuaduring the 4-week incubation such that the remaining bacteria did not grow to the same level as untreated or water-treated, vacuum-packaged tissues. Nisin spray treatments and vacuum packaging of lean and adipose tissues reducedB. thermosphactato undetectable levels. Data from this study demonstrate that nisin spray treatments followed by vacuum packaging under refrigerated conditions could increase the shelf life by suppressing or inhibiting the growth of undesirable bacteria present on fresh beef.