Synergistic effect of nisin and garlic shoot juice against Listeria monocytogenes in milk

The aim of this research was to determine the synergistic effect of nisin and garlic shoot juice (GSJ) against Listeria monocytogenes ATCC 19118 found in whole (3.5%), low (1%) and skim (no fat content) milk. Garlic shoot juice (GSJ) at concentrations of 2.5%, 5% and 10% revealed strong and similar patterns of antilisterial effect against L. monocytogenes ATCC 19118 in all categories of milk. Nisin only at concentrations of 62.5, 125, 250 and 500 IU/ml displayed a strong antilisterial effect as compared to the control group. Also, the synergistic combinations of GSJ (2.5%, 5%) and nisin (62.5, 125, 250 and 500 IU/ml) had a remarkable antilisterial activity in all categories of whole, low and skim milk after 14 days. Results of this study indicated the synergistic effect of GSJ and nisin as a potential antilisterial agent for the food industry.     

Bactericidal activity of lauric arginate in milk and Queso Fresco cheese against Listeria monocytogenes cold growth

Lauric arginate (LAE) at concentrations of 200 ppm and 800 ppm was evaluated for its effectiveness in reducing cold growth of Listeria monocytogenes in whole milk, skim milk, and Queso Fresco cheese (QFC) at 4°C for 15 to 28 d. Use of 200 ppm of LAE reduced 4 log cfu/mL of L. monocytogenes to a nondetectable level within 30 min at 4°C in tryptic soy broth. In contrast, when 4 log cfu/mL of L. monocytogenes was inoculated in whole milk or skim milk, the reduction of L. monocytogenes was approximately 1 log cfu/mL after 24 h with 200 ppm of LAE. When 800 ppm of LAE was added to whole or skim milk, the initial 4 log cfu/mL of L. monocytogenes was nondetectable following 24 h, and no growth of L. monocytogenes was observed for 15 d at 4°C. With surface treatment of 200 or 800 ppm of LAE on vacuum-packaged QFC, the reductions of L. monocytogenes within 24 h at 4°C were 1.2 and 3.0 log cfu/g, respectively. In addition, the overall growth of L. monocytogenes in QFC was decreased by 0.3 to 2.6 and by 2.3 to 5.0 log cfu/g with 200 and 800 ppm of LAE, respectively, compared with untreated controls over 28 d at 4°C. Sensory tests revealed that consumers could not determine a difference between QFC samples that were treated with 0 and 200 ppm of LAE, the FDA-approved level of LAE use in foods. In addition, no differences existed between treatments with respect to flavor, texture, and overall acceptability of the QFC. Lauric arginate shows promise for potential use in QFC because it exerts initial bactericidal activity against L. monocytogenes at 4°C without affecting sensory quality.     

Potential production and preservation of dahi by Lactococcus lactis W8, a nisin-producing strain

Lactococcus lactis W8 produced nisin concomitantly while fermenting milk to “dahi”, a traditional Indian fermented milk. The activity of nisin was detected at 3 h of fermentation, which increased in parallel to growth of the organism and reached its maximum at 6 h. The activity remained essentially stable thereafter. At 7 h of fermentation of milk with the strain L. lactis W8 the pH of the medium dropped to 4.2, when the milk became converted to dahi. The produced dahi displayed antibacterial property against spoilage and pathogenic bacteria including Listeria monocytogenes. When L. monocytogenes was mixed with dahi at 5.2 log CFU/ml and stored at 4 °C, the number of L. monocytogenes gradually decreased and became undetectable at 10 h. L. lactis W8 appeared to be a suitable starter culture for production of dahi from milk and preservation of the dahi.     

Fat content increases the lethality of ultra-high-pressure homogenization on Listeria monocytogenes in milk

Listeria monocytogenes CCUG 15526 was inoculated at a concentration of approximately 7.0 log₁₀ cfu/mL in milk samples with 0.3, 3.6, 10, and 15% fat contents. Milk samples with 0.3 and 3.6% fat content were also inoculated with a lower load of approximately 3.0 log₁₀ cfu/mL. Inoculated milk samples were subjected to a single cycle of ultra-high-pressure homogenization (UHPH) treatment at 200, 300, and 400 MPa. Microbiological analyses were performed 2 h after the UHPH treatments and after 5, 8, and 15 d of storage at 4°C. Maximum lethality values were observed in samples treated at 400 MPa with 15 and 10% fat (7.95 and 7.46 log₁₀ cfu/mL), respectively. However, in skimmed and 3.6% fat milk samples, complete inactivation was not achieved and, during the subsequent 15 d of storage at 4°C, L. monocytogenes was able to recover and replicate until achieving initial counts. In milk samples with 10 and 15% fat, L. monocytogenes recovered to the level of initial counts only in the milk samples treated at 200 MPa but not in the milk samples treated at 300 and 400 MPa. When the load of L. monocytogenes was approximately 3.0 log₁₀ cfu/mL in milk samples with 0.3 and 3.6% fat, complete inactivation was not achieved and L. monocytogenes was able to recover and grow during the subsequent cold storage. Fat content increased the maximum temperature reached during UHPH treatment; this could have contributed to the lethal effect achieved, but the amount of fat of the milk had a stronger effect than the temperature on obtaining a higher death rate of L. monocytogenes.     

Microstructure and physico-chemical evaluation of nano-emulsion-based antimicrobial peptides embedded in bioactive packaging films

Customized application of antimicrobial peptide (AMP) ‘nisin’ directly into food (neither in active packaging nor encapsulated form) is expensive and associated with loss of activity due to deactivation in complex food systems. The purpose of the present study was to fusion the two concepts for improved bioavailability i.e. AMP nanoencapsulation and biopolymer immobilizing to formulate the next generation biodegradable films embedded with either active agent, nano-encapsulated active agent or both of them. Nanoliposomes were prepared using soy-lecithin by microfluidizer at 2000 bar with 5 cycles to generate an average size of 151 ± 4 nm with 50 ± 3% encapsulation efficiency. For active films, nisin had demonstrated no negative impact on transparency, thickness and water sorption behavior obtained by GAB model (25 °C, 0–0.95 a_w). For nano-active films, the results clearly illustrated that different physico-chemical properties including barrier (oxygen and water vapor permeability), color and transparency (200–900 nm) remained comparable to native hydroxypropyl methylcellulose (HPMC) films and were significantly improved than using lecithin directly without nano-scale restructuring. The microstructure studies (topography and morphology) by scanning and transmission electron microscopes (SEM/TEM) revealed different (pore, lamellar, fusion) modes of nisin release from nanoliposomes embedded in HPMC matrix. As microbiological worth, nisin nano-emulsion (encapsulated and free nisin) films were effective against potential foodborne pathogen Listeria monocytogenes. This innovative concept of biodegradable nano-active films may thus be a preventive system toward improved food safety.     

Release and antilisterial properties of nisin from zein capsules spray-dried at different temperatures

Nisin is an effective antimicrobial against a broad spectrum of Gram-positive bacteria. It has been proposed that reduced efficacy of nisin in foods can be improved by technologies such as encapsulation to protect it from interferences by food matrix components. The potential of spray drying, a practical technology, was studied in this work for encapsulation of nisin in zein microcapsules at four inlet temperatures between 75 and 120 °C. At 95 °C and above, no apparent loss of nisin activity was observed after spray drying. At pH 6.0, burst release of nisin was impacted by spray drying temperature more than equilibrium release, possibly due to influences on capsule structures. At pH 2.0, complete release of nisin in 30 min was observed, contrasting to limited release over 8 d at pH 8.0. Capsules produced at an inlet temperature of 105 °C showed the most sustained release of nisin at pH 6.0. For these capsules, sustained release of nisin to >80% was observed at pH 6.0 and 8.0 when NaCl was used at 0.5 mol/L. Finally, at 400 IU/mL, the encapsulated nisin demonstrated slightly improved antilisterial properties than free nisin in reduced fat (2g/100g) milk but much work is still needed to enhance the antimicrobial effectiveness.     

Effect of fat content on survival of Listeria monocytogenes during simulated digestion of inoculated beef frankfurters stored at 7 °C

Potential effects of the fat content of frankfurters on the gastrointestinal survival of Listeria monocytogenes were investigated. At various stages of storage (7 °C, up to 55 days), inoculated frankfurters of low (4.5%) and high (32.5%) fat content were exposed to a dynamic gastrointestinal model (37 °C) and L. monocytogenes counts were determined at intervals during exposure in each gastrointestinal compartment (gastric, GC; intestinal, IC). Bacterial survival curves in each compartment were fitted with the Baranyi and Roberts mathematical model. L. monocytogenes populations on low- and high-fat frankfurters exceeded 8.0 log CFU/g at 39 and 55 days of storage, respectively. Major declines in populations occurred after 60 min on low-fat frankfurters in the GC, with reductions of 2.6 to >7.2 log CFU/g at 120 min on days 1 and 39 of storage, respectively. L. monocytogenes reductions in high-fat frankfurters ranged from 1.6 (day-1) to 5.2 (day-55) log CFU/g. Gastric inactivation rates were 0.080–0.194 and 0.030–0.097 log CFU/g/min for low- and high-fat samples, respectively. Since gastric emptying began while the gastric pH was >5, initial counts (enumerated 30 min after ingestion) reaching the IC depended on initial contamination levels on each product, which increased during storage. Subsequent reductions during the intestinal challenge were 0.1–1.4 log CFU/g. Findings indicated protective effects of fat against gastric destruction of L. monocytogenes. However, since the effects of fat were observed mainly at later stages of gastric exposure, they did not influence numbers of viable cells reaching the IC.     

Antimicrobial activity of acid-hydrolyzed Citrus unshiu peel extract in milk

Citrus fruit (Citrus unshiu) peels were extracted with hot water and then acid-hydrolyzed using hydrochloric acid. Antimicrobial activities of acid-hydrolyzed Citrus unshiu peel extract were evaluated against pathogenic bacteria, including Bacillus cereus, Staphylococcus aureus, and Listeria monocytogenes. Antilisterial effect was also determined by adding extracts at 1, 2, and 4% to whole, low-fat, and skim milk. The cell numbers of B. cereus, Staph. aureus, and L. monocytogenes cultures treated with acid-hydrolyzed extract for 12 h at 35°C were reduced from about 8 log cfu/mL to <1 log cfu/mL. Bacillus cereus was more sensitive to acid-hydrolyzed Citrus unshiu peel extract than were the other bacteria. The addition of 4% acid-hydrolyzed Citrus unshiu extracts to all types of milk inhibited the growth of L. monocytogenes within 1 d of storage at 4°C. The results indicated that Citrus unshiu peel extracts, after acid hydrolysis, effectively inhibited the growth of pathogenic bacteria. These findings indicate that acid hydrolysis of Citrus unshiu peel facilitates its use as a natural antimicrobial agent for food products.     

Anti-Listeria monocytogenes activity of enterocins microencapsulated by ionic gelation

The encapsulation of enterocins synthesized by Enterococcus faecium CRL1385 through ionic gelation with calcium ions was analyzed. Different enterocins samples were lyophilised and encapsulated using low-methoxyl pectin as the coating material. Lipids present in milk butter were also added to control the release of antimicrobial peptides from the capsules. The morphology of fresh and freeze-dried capsules was examined using light microscopy and scanning electron microscopy, respectively. Antimicrobial activity of encapsulated bacteriocins was assessed against Listeria monocytogenes 01/155 using the agar diffusion technique and direct contact in microplates. The capsules with higher lipid content showed a more spherical and uniform shape. Pathogen inhibition was observed for capsules prepared with different bacteriocin solutions both on solid (halo diameter = 8.5–13.5 mm) and in an aqueous medium (ca. 2 log orders decline in L. monocytogenes viability). The outcomes suggest that bacteriocin encapsulation through ionic gelation can be a potential alternative for the application of these antimicrobial peptides as biopreservatives in food.     

Growth characteristics of Listeria monocytogenes as affected by a native microflora in cooked ham under refrigerated and temperature abuse conditions

This study examined the growth characteristics of Listeria monocytogenes as affected by a native microflora in cooked ham at refrigerated and abuse temperatures. A five-strain mixture of L. monocytogenes and a native microflora, consisting of Brochothrix spp., isolated from cooked meat were inoculated alone (monocultured) or co-inoculated (co-cultured) onto cooked ham slices. The growth characteristics, lag phase duration (LPD, h), growth rate (GR, log₁₀ cfu/h), and maximum population density (MPD, log₁₀ cfu/g), of L. monocytogenes and the native microflora in vacuum-packed ham slices stored at 4, 6, 8, 10, and 12 °C for up to 5 weeks were determined. At 4-12 °C, the LPDs of co-cultured L. monocytogenes were not significantly different from those of monocultured L. monocytogenes in ham, indicating the LPDs of L. monocytogenes at 4-12 °C were not influenced by the presence of the native microflora. At 4-8 °C, the GRs of co-cultured L. monocytogenes (0.0114-0.0130 log₁₀ cfu/h) were statistically but marginally lower than those of monocultured L. monocytogenes (0.0132-0.0145 log₁₀ cfu/h), indicating the GRs of L. monocytogenes at 4-8 °C were reduced by the presence of the native microflora. The GRs of L. monocytogenes were reduced by 8-7% with the presence of the native microflora at 4-8 °C, whereas there was less influence of the native microflora on the GRs of L. monocytogenes at 10 and 12 °C. The MPDs of L. monocytogenes at 4-8 °C were also reduced by the presence of the native microflora. Data from this study provide additional information regarding the growth suppression of L. monocytogenes by the native microflora for assessing the survival and growth of L. monocytogenes in ready-to-eat meat products.     

Growth kinetics of Listeria monocytogenes and spoilage microorganisms in fresh-cut cantaloupe

The main objective of this study was to investigate the growth kinetics of Listeria monocytogenes and background microorganisms in fresh-cut cantaloupe. Fresh-cut cantaloupe samples, inoculated with three main serotypes (1/2a, 1/2b, and 4b) of L. monocytogenes, were incubated at different temperatures, ranging from 4 to 43 °C, to develop kinetic growth models. During storage studies, the population of both background microorganisms and L. monocytogenes began to increase almost immediately, with little or no lag phase for most growth curves. All growth curves, except for two growth curves of L. monocytogenes 1/2a at 4 °C, developed to full curves (containing exponential and stationary phases), and can be described by a 3-parameter logistic model. There was no significant difference (P = 0.28) in the growth behaviors and the specific growth rates of three different serotypes of L. monocytogenes inoculated to fresh-cut cantaloupe. The effect of temperature on the growth of L. monocytogenes and spoilage microorganisms was evaluated using three secondary models. For L. monocytogenes, the minimum and maximum growth temperatures were estimated by both the Ratkowsky square-root and Cardinal parameter models, and the optimum temperature and the optimum specific growth rate by the Cardinal parameter model. An Arrhenius-type model provided more accurate estimation of the specific growth rate of L. monocytogenes at temperatures <4 °C. The kinetic models developed in this study can be used by regulatory agencies and food processors for conducting risk assessment of L. monocytogenes in fresh-cut cantaloupe, and for estimating the shelf-life of fresh-cut products.     

Antimicrobial Activity of Vanillin and Mixtures with Cinnamon and Clove Essential Oils in Controlling <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 in Milk

The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of vanillin against Listeria monocytogenes Scott A and Escherichia coli O157:H7 was determined in tripticase soy broth (TSB), pH 7 and 6, incubated at 35 °C/24 h and in semi-skim milk incubated at 35 °C/24 h and 7 °C/14 days. The influence of the fat content of milk on the antimicrobial activity of vanillin was tested in whole and skim milk incubated at 7 °C/14 days. Mixtures of clove and cinnamon with vanillin were also evaluated in semi skim milk incubated at 7 °C. The MICs for L. monocytogenes were 3,000 ppm in TSB (pH 7) and 2,800 ppm in TSB (pH 6). The MICs for E. coli O157:H7 were 2,800 ppm in TSB (pH 7) and 2,400 ppm in TSB (pH 6). The MBCs in TSB were 8,000 ppm for L. monocytogenes and 6,000 ppm for E. coli O157:H7. The pH values assayed did not influence significantly the MIC or MBC in TSB. The MICs in semi-skim milk for L. monocytogenes and E. coli O157:H7 were 4,000 and 3,000 ppm at 35 °C/24 h, and 2,500 and 1,000 ppm at 7 °C/7 days, respectively. The MBCs were 20,000 ppm for L. monocytogenes and 11,000 ppm for E. coli O157:H7. High incubation temperatures did not affect the MBC but increased the MIC of the vanillin in milk. This effect could be attributed to the increased membrane fluidity and to the membrane perturbing activity of vanillin at low temperatures. The fat in milk reduced significantly the antimicrobial activity of vanillin, probably due to effect protective of the fat molecules. Mixtures of clove and cinnamon leaves inhibited the growth of L. monocytogenes in a similar way that vanillin alone but had a synergistic effect on the E. coli O157:H7. Mixtures of cinnamon bark and vanillin had always a synergistic effect and some of the combination assayed showed bactericidal activity on the population of L. monocytogenes and E. coli O 157:H7.     

Lactose oxidase: An enzymatic approach to inhibit Listeria monocytogenes in milk

Listeria monocytogenes is a ubiquitous pathogen that can cause morbidity and mortality in immunocompromised individuals. Growth of L. monocytogenes is possible at refrigeration temperatures due to its psychrotrophic nature. The use of antimicrobials in dairy products is a potential way to control L. monocytogenes growth in processes with no thermal kill step, thereby enhancing the safety of such products. Microbial-based enzymes offer a clean-label approach for control of L. monocytogenes outgrowth. Lactose oxidase (LO) is a microbial-derived enzyme with antimicrobial properties. It oxidizes lactose into lactobionic acid and reduces oxygen, generating H₂O₂. This study investigated the effects of LO in UHT skim milk using different L. monocytogenes contamination scenarios. These LO treatments were then applied to raw milk with various modifications; higher levels of LO as well as supplementation with thiocyanate were added to activate the lactoperoxidase system, a natural antimicrobial system present in milk. In UHT skim milk, concentrations of 0.0060, 0.012, and 0.12 g/L LO each reduced L. monocytogenes counts to below the limit of detection between 14 and 21 d of refrigerated storage, dependent on the concentration of LO. In the 48-h trials in UHT skim milk, LO treatments were effective in a concentration-dependent fashion. The highest concentration of LO in the 21-d trials, 0.12 g/L, did not show great inhibition over 48 h, so concentrations were increased for these experiments. In the lower inoculum, after 48 h, a 12 g/L LO treatment reached levels of 1.7 log cfu/mL, a reduction of 1.3 log cfu/mL from the initial inoculum, whereas the control grew out to approximately 4 log cfu/mL, an increase of 1 log cfu/mL from the inoculum on d 0. When a higher challenge inoculum of 5 log cfu/mL was used, the 0.12 g/L and 1.2 g/L treatments reduced the levels by 0.2 to 0.3 log cfu/mL below the initial inoculum and the 12 g/L treatment by >1 log cfu/mL below the initial inoculum by hour 48 of storage at refrigeration temperatures. After the efficacy of LO was determined in UHT skim milk, LO treatments were applied to raw milk. Concentrations of LO were increased, and the addition of thiocyanate was investigated to supplement the effect of the lactoperoxidase system against L. monocytogenes. When raw milk was inoculated with 2 log cfu/mL, 1.2 g/L LO alone and combined with sodium thiocyanate reduced ~0.8 log cfu/mL from the initial inoculum on d 7 of storage, whereas the control grew out to >1 log cfu/mL from the initial inoculum. Furthermore, in the higher inoculum, 1.2 g/L LO combined with sodium thiocyanate reduced L. monocytogenes counts from the initial inoculum by >1 log cfu/mL, whereas the control grew out 2 log cfu/mL from the initial inoculum. Results from this study suggest that LO is inhibitory against L. monocytogenes in UHT skim milk and in raw milk. Therefore, LO may be an effective treatment to prevent L. monocytogenes outgrowth, increase the safety of raw milk, and be used as an effective agent to prevent L. monocytogenes proliferation in fresh cheese and other dairy products. This enzymatic approach is a novel application to control the foodborne pathogen L. monocytogenes in dairy products.     

Inactivation of Listeria monocytogenes in Skim Milk and Liquid Egg White by Antimicrobial Bottle Coating with Polylactic Acid and Nisin

ABSTRACT: This study was to develop an antimicrobial bottle coating method to reduce the risk of outbreaks of human listeriosis caused by contaminated liquid foods. Liquid egg white and skim milk were inoculated with Listeria monocytogenes Scott A and stored in glass jars that were coated with a mixture of polylactic acid (PLA) polymer and nisin. The efficacy of PLA per nisin coating in inactivating L. monocytogenes was investigated at 10 and 4 °C. The pathogen grew well in skim milk without PLA/nisin coating treatments, reaching 8 log CFU/mL after 10 d and then remained constant up to 42 d at 10 °C. The growth of Listeria at 4 °C was slower than that at 10 °C, taking 21 d to obtain 8 log CFU/mL. At both storage temperatures, the PLA coating with 250 mg nisin completely inactivated the cells of L. monocytogenes after 3 d and throughout the 42-d storage period. In liquid egg white, Listeria cells in control and PLA coating without nisin samples declined 1 log CFU/mL during the first 6 d at 10 °C and during 28 d at 4 °C, and then increased to 8 or 5.5 log CFU/mL. The treatment of PLA coating with 250 mg nisin rapidly reduced the cell numbers of Listeria in liquid egg white to undetectable levels after 1 d, then remained undetectable throughout the 48 d storage period at 10 °C and the 70 d storage period at 4 °C. These data suggested that the PLA/nisin coating treatments effectively inactivated the cells of L. monocytogenes in liquid egg white and skim milk samples at both 10 and 4 °C. This study demonstrated the commercial potential of applying the antimicrobial bottle coating method to milk, liquid eggs, and possibly other fluid products.     

Antimicrobial activity of nisin, reuterin, and the lactoperoxidase system on Listeria monocytogenes and Staphylococcus aureus in cuajada, a semisolid dairy product manufactured in Spain

The inhibitory activity of nisin (N), reuterin (R), and the lactoperoxidase system (LPS), added individually or in combination, against Listeria monocytogenes and Staphylococcus aureus was investigated in “cuajada” (curdled milk), a semisolid dairy product manufactured in Spain. Cuajada was manufactured from UHT skim milk separately inoculated with L. monocytogenes and Staph. aureus, each at approximately 4 log cfu/mL, and held under conditions of temperature abuse (10°C). On d 3, a synergistic bactericidal activity was observed for the combinations of biopreservatives assayed, with L. monocytogenes counts of only 0.30 log cfu/mL in cuajada made with N + R + LPS vs. 8.31 log cfu/mL in control cuajada. After 12 d, L. monocytogenes could not be detected in cuajada made with added N + LPS or N + R + LPS. Staphylococcus aureus was more resistant than L. monocytogenes to biopreservatives added individually. On d 3, the synergistic effect of the 3 biopreservatives against Staph. aureus resulted in counts of 3.03 log cfu/mL in cuajada made with N + R + LPS vs. 6.40 in control cuajada. After 12 d, Staph. aureus counts were 2.61 log cfu/mL in cuajada made with N + R + LPS, whereas they ranged from 6.11 to 7.70 log cfu/mL in control cuajada and in cuajada made with other combinations of biopreservatives. The most pronounced decrease in pathogen counts was achieved by the triple combination N + R + LPS, which acted synergistically on the inactivation of L. monocytogenes and Staph. aureus in cuajada over 12 d at 10°C. The treatment combining these 3 natural biopreservatives at low concentrations, within the hurdle concept of food preservation, might be a useful tool to control the growth of pathogenic microorganisms in nonacidified dairy products.     

Phospholipid nanovesicles containing a bacteriocin-like substance for control of Listeria monocytogenes

Bacillus licheniformis strain P40 produces a bacteriocin-like substance (BLS) that has potential to be used as a natural biopreservative for control of pathogenic and food microorganisms. The objective of this study was the encapsulation of BLS in phosphatidylcholine vesicles, evaluating its antimicrobial activity against Listeria monocytogenes. The size of the nanovesicles with the BLS was around 570 nm and of the nanovesicles without BLS was of approximately 484.8 nm, as determined by light scattering with a He–Ne laser (λ = 632.8 nm) as light source. The encapsulated BLS showed inhibitory activity against L. monocytogenes as observed by agar diffusion assay. Complete inhibition of L. monocytogenes growth was observed with the addition of 100 and 50 AU mL^− 1 of encapsulated and free BLS, respectively. A reduction in the number of viable cells to zero was observed after 10 min incubation with 400 AU mL^− 1 of either encapsulated or free BLS. The encapsulated BLS was stable for up to 30 days at 4 °C. These results indicate that nanovesicles containing BLS may have potential for use as food preservative.Industrial relevanceThe increased concern on minimally processed food and natural additives has been stimulated many studies on the utilization of antimicrobial peptides as biopreservatives. The incorporation of bacteriocins into nanovesicles may represents an interesting alternative for controlled release and increased stability of bacteriocins.     

Efficacy of bacteriocin-containing cell-free culture supernatants from lactic acid bacteria to control Listeria monocytogenes in food

Consumer demands have led to an increased interest in the use of natural antimicrobials for food protection. With the objective of developing novel products for enhancing the microbial safety of food, we have tested cell-free culture supernatants (CFS's) of eight antagonistic bacterial strains for their efficacy to inhibit Listeria monocytogenes in different food matrices. The antagonistic strains represented different members of the order Lactobacillales as well as one isolate of Staphylococcus sciuri and all showed strong inhibition of L. monocytogenes on agar plates. Cell-free supernatants were obtained after growing the bacteria in a yeast extract-glucose broth. In six of the CFS's, different class IIa bacteriocins, namely leucocin A, leucocin B, mundticin L, pediocin PA-1, sakacin A, and sakacin X, were identified as the major anti-listerial compounds. For the other two strains, the active substances could not be ascertained conclusively. The minimal effective concentration (MEC) of the individual CFS's to achieve a 2.3 log₁₀ reduction of L. monocytogenes was determined in culture broth, whole milk, and ground beef at 4 °C. While all bacteriocin-containing CFS's were effective in broth at concentrations from 52 to 205 AU/ml, significant higher concentrations were needed when applied in food. Best results were obtained using CFS's containing pediocin PA-1, that displayed only three- and ten-times higher MEC's in milk (307 AU/ml) and ground meat (1024 AU/g) compared to broth, respectively. A twenty-fold increase in the MEC (2048 AU/ml) was observed for a mundticin L-containing fermentate, and a CFS containing leucocin A and B was inactivated more than fifty-fold (> 1280 AU/ml) in both food matrices. Remarkably, the sakacin A and sakacin X containing CFS's displayed very selective inactivation rates, in which sakacin A was only effective in meat (512 AU/g), while sakacin X was only effective in milk (2048 AU/ml). In all cases, inhibition of L. monocytogenes was only transient and surviving or resistant bacteria started growing after prolonged storage. These results highlight the importance of careful testing the effectiveness of bacteriocins in the food systems for which they are intended to be applied against the selected target and non-target bacteria. Furthermore, the outgrowth of surviving or resistant bacterial populations points out that the tested bacteriocins are not suited to assure full inhibition of L. monocytogenes in a food product, if not applied in combination with additional preservative measures.     

Effect of exopolysaccharides (EPSs) produced by Lactobacillus delbrueckii subsp. bulgaricus strains to bacteriophage and nisin sensitivity of the bacteria

Lactobacillus strains used in this study were isolated from village-type yogurt and raw milk. The isolates were identified as Lactobacillus delbrueckii subsp. bulgaricus by 16 s rDNA sequence analysis and API 50 CHL identification systems. The exopolysaccharide (EPS) production of the strains growth in skim milk were investigated. In addition sensitivity and insensitivity of these strains against domestic bacteriophages and nisin were examined. It was deduced that those strains which had relatively high EPS-producing capacity were insensitive against phages and nisin. Linear relationships were determined between EPS production of the bacteria and bacteriophage and nisin insensitivity of the bacteria.There was a negative correlation between EPS production quantity and phage and nisin sensitivity of the bacteria. Of all the strains, L. delbrueckii subsp bulgaricus B3 produced the highest EPS quantity, and it was insensitive against phages and nisin. Based on these results, it is suggested that L. delbrueckii subsp bulgaricus B3 can be used with the starter culture in dairy industry for stable and high-quality yogurt production.     

Antimicrobial activity of nanoliposomes co-encapsulating nisin and garlic extract against Gram-positive and Gram-negative bacteria in milk

Nisin and garlic extract (GE) were co-encapsulated into phosphatidylcholine nanoliposomes. The mean diameter and zeta potential of the nanoparticles were 179 nm and − 27.7 mV, respectively, with an entrapment efficiency of about 82% and 90% for nisin and GE, respectively. The efficiency of free and encapsulated nisin-GE to control the development of Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli and Staphylococcus aureus was assessed over time in whole milk at 37 °C. At such abuse temperature conditions, both free and liposomal nisin-GE resulted a difference of 1–4 log CFU/ml against the strains tested, when compared with free nisin and GE separately. A difference of 5–6 log CFU/ml in viable counts of Gram-positive strains and 3–4 log CFU/ml for Gram-negative bacteria was observed for treatments with nisin-GE when compared to the control. The effect of nisin-GE on L. monocytogenes was evaluated under refrigeration (7 ± 1 °C) for up to 25 days. Viable counts for treatments with free and encapsulated nisin-GE were 4–5 log CFU/ml lower than the values reached by the control. Liposome encapsulation of natural antimicrobials with synergistic effect may be important to overcome stability issues and undesirable interaction with food components. The results of this study indicated that nanoliposome-encapsulated nisin-GE has potential as an antimicrobial formulation for food use.Industrial relevanceThere is increased interest for minimally processed foods and natural additives, which agrees with the use of natural antimicrobials as food preservatives. Co-encapsulation of antimicrobials may extend the inhibitory spectrum and effectiveness in controlling food pathogens. The use of nanoliposomes for delivery of natural antimicrobials in dairy products represents an interesting alternative for controlled release of biopreservatives and improvement of food quality and shelf life.