Resistance to benzalkonium chloride, peracetic acid and nisin during formation of mature biofilms by Listeria monocytogenes

Increase of resistance to the application of benzalkonium chloride (BAC), peracetic acid (PA) and nisin during biofilm formation at 25 °C by three strains of Listeria monocytogenes (CECT 911, CECT 4032, CECT 5873 and BAC-adapted CECT 5873) in different scenarios was compared. For this purpose, resistance after 4 and 11-days of biofilm formation was quantified in terms of lethal dose 90% values (LD₉₀), determined according with a dose-response logistic mathematical model. Microscopic analyses after 4 and 11-days of L. monocytogenes biofilm formation were also carried out. Results demonstrated a relation between the microscopic structure and the resistance to the assayed biocides in matured biofilms. The worst cases being biofilms formed by the strain 4032 (in both stainless steel and polypropylene), which showed a complex “cloud-type” structure that correlates with the highest resistance of this strain against the three biocides during biofilm maturation. However, that increase in resistance and complexity appeared not to be dependent on initial bacterial adherence, thus indicating mature biofilms rather than planctonic cells or early-stage biofilms must be considered when disinfection protocols have to be optimized. PA seemed to be the most effective of the three disinfectants used for biofilms. We hypothesized both its high oxidizing capacity and low molecular size could suppose an advantage for its penetration inside the biofilm. We also demonstrated that organic material counteract with the biocides, thus indicating the importance of improving cleaning protocols. Finally, by comparing strains 5873 and 5873 adapted to BAC, several adaptative cross-responses between BAC and nisin or peracetic acid were identified.     

Mixed species biofilms of Listeria monocytogenes and Lactobacillus plantarum show enhanced resistance to benzalkonium chloride and peracetic acid

We investigated the formation of single and mixed species biofilms of Listeria monocytogenes strains EGD-e and LR-991, with Lactobacillus plantarum WCFS1 as secondary species, and their resistance to the disinfectants benzalkonium chloride and peracetic acid. Modulation of growth, biofilm formation, and biofilm composition was achieved by addition of manganese sulfate and/or glucose to the BHI medium. Composition analyses of the mixed species biofilms using plate counts and fluorescence microscopy with dual fluorophores showed that mixed species biofilms were formed in BHI (total count, 8-9 log₁₀ cfu/well) and that they contained 1-2 log₁₀ cfu/well more L. monocytogenes than L. plantarum cells. Addition of manganese sulfate resulted in equal numbers of both species (total count, 8 log₁₀ cfu/well) in the mixed species biofilm, while manganese sulfate in combination with glucose, resulted in 1-2 log₁₀ more L. plantarum than L. monocytogenes cells (total count, 9 log₁₀ cfu/well). Corresponding single species biofilms of L. monocytogenes and L. plantarum contained up to 9 log₁₀ cfu/well. Subsequent disinfection treatments showed mixed species biofilms to be more resistant to treatments with the selected disinfectants. In BHI with additional manganese sulfate, both L. monocytogenes strains and L. plantarum grown in the mixed species biofilm showed less than 2 log₁₀ cfu/well inactivation after exposure for 15 min to 100 μg/ml benzalkonium chloride, while single species biofilms of both L. monocytogenes strains showed 4.5 log₁₀ cfu/well inactivation and single species biofilms of L. plantarum showed 3.3 log₁₀ cfu/well inactivation. Our results indicate that L. monocytogenes and L. plantarum mixed species biofilms can be more resistant to disinfection treatments than single species biofilms.     

Irradiation and modified atmosphere packaging for the control of Listeria monocytogenes on turkey meat.

When radiation-sterilized ground turkey meat was inoculated with Listeria monocytogenes, packaged under mixtures of nitrogen and carbon dioxide, and irradiated with gamma-radiation doses of 0 to 3.0 kGy, there was a statistically significant (P < 0.05), but probably not a biologically significant, lower (0.39 log) predicted bacterial survival in the presence of 100% carbon dioxide than in the presence of 100% nitrogen. Possibly because all atmospheres contained oxygen and because a response surface design was used, gamma-radiation resistance was not significantly (P < 0.05) different in air than in modified atmosphere packaging (MAP) mixtures containing 5% O2 or containing 20, 40, 60, and 80% CO2 and balance N2. The antilisterial effects of MAP mixtures containing 17.2, 40.5, and 64% CO2 and balance N2 were compared to those associated with air and vacuum packaging on turkey inoculated with approximately 5 x 10(3) CFU/g. Samples were irradiated to doses of 0, 0.5, 1.0, 1.5, 2.0, and 2.5 kGy and were stored at 7 degrees C for up to 28 days. Irradiation treatments were significantly more lethal in the presence of air packaging than in either vacuum packaging or MAP, and in those samples that received >1.0 kGy, there was a concentration-dependent CO2 inhibition of L. monocytogenes multiplication and/or recovery.     

Effect of modified atmosphere packaging on the growth of spoilage microorganisms and Listeria monocytogenes on fresh cheese

Queso Fresco has a limited shelf life and has been shown to support the rapid growth of Listeria monocytogenes during refrigerated storage. In addition to improving quality and extending shelf life, modified atmosphere packaging (MAP) has been used to control the growth of pathogenic microorganisms in foods. The objectives of this study were to determine the effects of MAP conditions on the survival and growth of spoilage microorganisms and L. monocytogenes during storage of Queso Fresco manufactured without starter cultures. For L. monocytogenes experiments, cheeses were surface inoculated at ～4 log₁₀ cfu/g before packaging. Inoculated and uninoculated (shelf life experiments) cheeses were placed in 75-µm high-barrier pouches, packaged under 1 of 7 conditions including air, vacuum, or combinations of N₂ and CO₂ [100% N₂ (MAP1), 30% CO₂:70% N₂ (MAP2), 50% CO₂:50% N₂ (MAP3), or 70% CO₂:30% N₂ (MAP4), 100% CO₂ (MAP5)], and stored at 7°C. Samples were removed weekly through 35 d of storage. Listeria monocytogenes counts were determined for inoculated samples. Uninoculated samples were assayed for mesophilic and psychrotolerant counts, lactic acid bacteria, coliforms, and yeast and mold. In general, cheeses packaged under conditions consisting of higher contents of CO₂ had lower pH levels during storage compared with those stored in conditions with lower levels or no CO₂ at all. Similarly, the antimicrobial efficacy of MAP in controlling spoilage microorganisms increased with increasing CO₂ content, whereas conditions consisting of 100% N₂, vacuum, or air were less effective. Mean L. monocytogenes counts remained near inoculation levels for all treatments at d 1 but increased ～2 log₁₀ cfu/g on cheeses packaged in air, vacuum, and 100% N₂ (MAP1) conditions at d 7 and an additional ～1.5 log₁₀ cfu/g at d 14 where they remained through 35 d. In contrast, treatments consisting of 70% CO₂ (MAP4) and 100% CO₂ (MAP5) limited increases in mean L. monocytogenes counts to <1 log₁₀ cfu/g through 14 d and ～1.5 log₁₀ cfu/g by d 21. Mean L. monocytogenes counts increased to levels significantly higher than inoculation (d 0) on cheeses stored in MAP2 and MAP3 on d 21, on d 28 for MAP4, and on d 35 for cheeses stored under MAP5 conditions. Overall, significant treatment × time interactions were observed between air, vacuum, and MAP1 when each was compared with MAP2, MAP3, MAP4, and MAP5. These data demonstrate that packaging fresh cheese under modified atmospheres containing CO₂ may be a promising approach to extend shelf life while limiting L. monocytogenes growth during cold storage.     

Control of Listeria monocytogenes on frankfurters and cooked pork chops by irradiation combined with modified atmosphere packaging

This study was conducted to investigate the efficacy of controlling Listeria monocytogenes on frankfurters and cooked pork chops with irradiation and modified atmosphere packaging (MAP) containing a high concentration of CO(2). Frankfurters and cooked pork chops were inoculated with a five-strain cocktail of L. monocytogenes and packaged in vacuum or high-CO(2) MAP. Irradiation was applied to each product at 0, 0.5, 1.0, or 1.5 kGy. No significant packaging effect was found for the radiation sensitivity of L. monocytogenes. Radiation D(10)-values for L. monocytogenes were 0.66 ± 0.03 and 0.70 ± 0.05 kGy on frankfurters and 0.60 ± 0.02 and 0.57 ± 0.02 kGy on cooked pork chops in vacuum and high-CO(2) MAP, respectively. High-CO(2) MAP was more effective than vacuum packaging for controlling the growth of survivors during refrigerated storage. These results indicate that irradiation and high-CO(2) MAP can be used to improve control of L. monocytogenes in ready-to-eat meats.     

Active packaging of cheese with allyl isothiocyanate, an alternative to modified atmosphere packaging

The natural antimicrobial compound allyl isothiocyanate (AITC), found in mustard oil, is effective against cheese-related fungi both on laboratory media and cheese. Penicillium commune, Penicillium roqueforti, and Aspergillus flavus were more sensitive to AITC when it was added just after the spores had completed 100% germination and branching had started on Czapek yeast extract agar than were spores in the dormant phase. The use of 1 AITC label (Wasaouro interior labels, LD30D, 20 by 20 mm) in combination with atmospheric air in the packaging extended the shelf life of Danish Danbo cheese from 4 1/2 to 13 weeks. Two AITC labels extended the shelf life from 4 1/2 to 28 weeks. Both 1 and 2 labels in combination with modified atmosphere packaging extended the shelf life of the cheese from 18 to 28 weeks. This study showed that AITC was absorbed in the cheese, but it was not possible to detect any volatile breakdown products from AITC in the cheese. Cheese stored for up to 12 weeks with an AITC label had an unacceptable mustard flavor. The mustard flavor decreased to an acceptable level between weeks 12 and 28. Cheese stored in atmospheric air had a fresher taste without a CO2 off-flavor than did cheese stored in modified atmosphere packaging. AITC may be a good alternative to modified atmosphere packaging for cheese. The extended shelf life of cheese in the package is very desirable: the cheese can be transported longer distances, and the packaging can be used for the final maturing of the cheese. Furthermore, AITC can address problems such as pinholes and leaking seals in cheese packaging.     

The response of unripe Conference pears to modified atmosphere retail packaging

Unripe green Conference pears with initial firmness values of 46–55 N were sealed in modified atmosphere (MA) low density polyethylene (LDPE, 30μ) pillow packs and in perforated control packs on three dates during 1985–1986. Changes in pack atmosphere composition, skin chlorophyll content, flesh firmness and sensory quality were monitored during 14–20 days simulated shelf-life at 20°C. In MA packs, equilibrated atmospheres containing 5–9% CO₂ and < 5% O₂ developed within c. 3 days. Chlorophyll degradation was completely inhibited by MA and resumed when packs were perforated, but MA only partially retarded the rate of flesh softening. Variations in the rate of ripening changes in duplicate MA packs could not be explained simply by differences in pack atmospheres. Furthermore, pears retarded by MA packaging failed to develop the normal sweet, aromatic flavour and succulent, juicy texture of 'eating-ripe' fruit, even when the packs were perforated after 4 days.
The differences in the responses of pears and apples to MA packaging, and the limitations of using this technique for the commercial marketing of English pears are discussed.     

Modelling the respiration rate of fresh-cut Annurca apples to develop modified atmosphere packaging

In this work, the effect of temperature, oxygen, red coloration process and post-harvest storage time on the respiration rate of fresh-cut Annurca apples was studied to properly develop modified atmosphere packaging. Our results showed that the red coloration process and the post-harvest storage time did not affect the respiration rate or the respiratory quotient of fresh-cut Annurca apples in the range of temperature studied (5–20 °C). A Michaelis–Menten-type equation, with the model constants described by means of an Arrhenius-type relationship, was used for predicting respiration rate on varying the temperature and O₂ concentration in the head space. The maximal respiration rate (mL kg h⁻¹) (RR_max) and the O₂% corresponding to     values estimated at the reference temperature (12.5 °C), i.e. the average of the experimental temperature ranges, were, respectively, 6.77 ± 0.1 mL kg⁻¹ h⁻¹ and 0.68 ± 0.07% v/v, and the activation energy of the aerobic respiration rate of fresh-cut Annurca apples was estimated at 51 ± 1 kJ mol⁻¹. The model works well to develop a modified atmosphere for fresh-cut Annurca apples.     

Combined effect of aqueous chlorine dioxide and modified atmosphere packaging on inhibiting Salmonella Typhimurium and Listeria monocytogenes in mungbean sprouts

This study was conducted to investigate the effect of chlorine dioxide (ClO2) combined with modified atmosphere packaging (MAP) on inhibiting total mesophilic microorganisms, Salmonella Typhimurium, and Listeria monocytogenes in mungbean sprouts during refrigerated storage. Mungbean sprouts were packaged using 4 different methods (air, vacuum, CO2 gas, and N2 gas) following treatment with water or 100 ppm ClO2 for 5 min and stored at 5 +/- 2 degrees C. The population of total mesophilic microorganisms in mungbean sprouts was about 8.4-log(10) CFU/g and this level was not significantly reduced by treatment with water or ClO2 (P > 0.05). However, when samples were packaged under vacuum, N2 gas, or CO2 gas following treatment with ClO2, the populations of total mesophilic microorganisms were significantly reduced during storage (P < 0.05). Levels of S. Typhimurium and L. monocytogenes in mungbean sprouts following inoculation were 4.6- and 5.6-log(10) CFU/g and treatment with water followed by different packaging conditions (air, vacuum, N2 gas, and CO2 gas) had no significant effect on population reduction (P > 0.05). However, treatment with ClO2 significantly reduced populations of S. Typhimurium and L. monocytogenes by 3.0- and 1.5-log CFU/g, respectively (P < 0.05), and these reduced cell levels were maintained or decreased in samples packaged under vacuum or in N2 or CO2 gas during storage. These results suggest that the combination of ClO2 treatment and MAP such as CO2 gas packaging may be useful for inhibiting microbial contamination and maintaining quality in mungbean sprouts during storage.     

Novel approach to oxygen control in modified atmosphere packaging of bakery products

PDA plates inoculated with mould spores were packaged in air and in a mixture of CO₂/N₂ (60 : 40) with headspace oxygen adjusted to range from <0·05% to 10% (v/v) and stored at 25°C. Those packaged in air showed mould growth after 1–1·5 d, while those in the gas mixture showed growth between 4–6 d after headspace oxygen reached a minimum concentration of 0·4%. Uninoculated plates packaged in air showed no change in headspace oxygen concentration over 30 d at 25°C, while those in the gas mixture with initial headspace oxygen of <0·05% showed a steady increase of oxygen, through packaging film permeability, at an average rate of 0·06%/d. If uninoculated plates were packaged in air together with a sachet of oxygen absorbant called Ageless, the headspace oxygen was quickly reduced to <0·05% within 9 h and remained unchanged thereafter.Crusty rolls with a_w of 0·95 and pH of 5·62 developed mould after 5–6 d when packaged in air, 9–11 d in 100% N₂ and 16–18 d in CO₂/N₂ (60 : 40). However, when Ageless was incorporated into all the packages the product remained mould free for >60 d. This study shows that oxygen absorbants offer the bakery industry a viable alternative to gas flushing.     

Utilisation of modified atmosphere packaging to extend the shelf life of Khalas fresh dates

Completely matured tamr stage dates are widely available and consumed throughout the year. However, for some date varieties, the earlier khalal stage crunchy fruit is considered a premium product. This work examines the use of modified atmosphere packaging to extend the shelf life of khalal dates under refrigerated conditions. Modified atmosphere packaging with 10% or 20% CO₂ in air was successful in reducing the rate of the ripening processes while control‐packaged dates showed signs of ripening development such as significant weight loss and darkening in date colour (identified visually and as low lactic acid bacteria measurements at 18 and 27 days after packaging), similar to that observed in tamr stage fruit.     

Effects of nisin and acid on the inactivation and recovery of Listeria monocytogenes biofilms treated by high hydrostatic pressure

Food Science and Biotechnology - This study was designed to evaluate the inactivation, detachment, and recovery properties of Listeria monocytogenes biofilms treated by the combined high...     

气调包装对番石榴贮藏品质的影响

研究12℃时4%O2+5%CO2+91%N2的气调包装对番石榴贮藏品质的影响。与相同温度下未气调包装相比,气调包装能够延缓果实的软化,推迟果肉内过氧化物酶活性和还原糖含量的达峰时间,降低了VC和可滴定酸的分解速率,抑制了丙二醛的生成速率。结果表明,气调包装的番石榴保质期比未气调样品延长了一倍,其在番石榴贮藏保鲜中有一定的推广应用价值。     

Development and characterization of an antimicrobial packaging film coating containing nisin for inhibition of Listeria monocytogenes

The purpose of this study was to develop and characterize a packaging film coating containing nisin. A spot-on-lawn assay was used to determine the effect of acid type (ascorbic, acetic, hydrochloric, lactic) and nisin level (equal increments from 10,000 IU to 9 IU) to be used in the formulation of the film coating. Zones of inhibition were measured after incubation on tryptic soy agar (37 degrees C, 48 h). Low-density polyethylene films coated with differing levels of nisin were characterized by field emission scanning electron microscopy, tensile strength, elongation, and water vapor transmission rate. The MIC of nisin in solution was 157 mg/ml. All acids were equally inhibitory (P > 0.05), but acetic acid produced the largest zone of inhibition (21 mm). Field emission scanning electron microscopy confirmed that the cloudy appearance of the films was due to sodium chloride found in the commercially prepared nisin. Tensile strength increased as nisin concentration increased, which also corresponded to increasing film thickness. The nisin coatings (10,000 and 2,500 IU/ml) did not have a significant effect (P > 0.05) on the water vapor transmission rate of the low-density polyethylene film.     

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

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

Extending the quality of fresh strawberries by equilibrium modified atmosphere packaging

The impact of equilibrium-modified atmosphere packaging (EMAP) technology on extension quality [pH, acidity, brix, color (L, a, Chroma, hue)] and texture profile analyses of fresh strawberries was studied and compared during storage. Cast polypropylene (CPP), linear low density polyethylene (LLDPE) and polyethylene-terephthalate (PET)/Ethylene vinyl alcohol (EVOH)/Polyetlene-low-acetyl fractions (LAF) were used as heat-sealed lid and polyvinyl chloride (PVC/PE) tray with the purpose of obtaining equilibrium atmospheres. Among the various films used, pH of fresh strawberry was 3.275 at initial days, and increased to 3.39- for LLDPE; 3.42 for CPP and 3.44 PET/EVOH-LAF at the end of 10 days’ storage. Acidity values were 0.609 mg ml⁻¹ and decreased to 0.56 mg ml⁻¹ for LLDPE; 0.47 mg ml⁻¹ for CPP and 0.49 mg ml⁻¹ for PET/EVOH-LAF at end of storage. The strawberry brix had evolved from the initial 7.125 and reduced to about from 5.6 to 6.07 at the end of the storage. At the end of the storage, the strawberry L values had not significantly changed from the initial from 29.10 (L) to 28.9–26.46. Initial values of the firmness were 1,089 gf and reduced with ranged from 769 gf to 527 gf at end of the 10-day storage period. All the parameters in texture profile analyses showed a decline, except adhesiveness and springiness and used potential indicators of fresh strawberries. The overall results expressed that strawberry quality can be maintained effectively at least for 10 days using various polymeric lid films. PET/EVOH-LAF and CPP were much more effective then LLDPE due to barrier properties during storage periods. Quality of strawberry packaged with suitable high-barrier lid films have been prolonged significantly.     

Extending the shelf life of kohlrabi stems by modified atmosphere packaging

ABSTRACT:  Kohlrabi stems (without leaves) were stored under modified atmosphere packaging (MAP) for 60 d at 0 °C. An additional retail sale period of 3 d at 12 °C after each cold storage evaluation (30 and 60 d) was applied. Under high relative humidity (RH) and 0 °C, the stems showed low metabolic activity, as no changes in sugars and organic acids were found. From day 21 at 0 °C, air-stored stems showed a yellowing of stalks and later they fell down. This disorder severely affected the appearance of stems. A gas composition of 4.5 to 5.5 kPa O₂ plus 11 to 12 kPa CO₂ was reached using antimist oriented polypropylene plastic bags of 20-μm thicknesses. The stems in MAP conditions kept a high sensorial quality. It was enough for commercial purpose of 2 mo. The storage of kohlrabi stems in plastic bags, either MA or in perforated (control) packages, provided an additional protection reducing physical damage. The MAP conditions delayed the weight loss and development of bacterial soft and black rot, extending the shelf life of kohlrabi stems to 60 d at 0 °C plus 3 d at 12 °C. Stems are not chilling injury sensitive.     

Quality and shelf-life of washed fresh-cut asparagus in modified atmosphere packaging

Fresh-cut asparagus is one of the most popular fresh vegetables for healthy consumption. However, the level of microbial load in the raw vegetable can cause food poisoning and shorten its shelf-life of asparagus. The objectives of this work were to determine the effect of chlorinated and ozonated water in the washing process to reduce the microbial load on fresh green asparagus and the effect of modified atmosphere packaging (MAP) on asparagus quality. Washing at 10 °C for 15 min with chlorinated water (100 mg/L free Cl₂) reduced aerobic plate count which had higher efficiency on microbial reduction than the use of ozonated water (0.1 mg/L O₃). No significant differences on the amount of Escherichia coli contamination among washing methods were found. Asparagus in modified atmosphere packaging retarded the deterioration process. Changes in hue angle followed a first-order kinetic reaction. Temperature dependence of the kinetic rate constant during storage time of asparagus obeyed the Arrhenius relationship with an activation energy (E_a) 29.33 ± 4.60 kJ/mol. The shelf-life prediction equation was related well with real practice. The combination of appropriate washing process and MAP increased the food safety, maintained the quality and prolonged the shelf-life of asparagus.