Simulated Food Processing Research Articles

A novel Fe3O4@Pt synthetized via a self-assembly strategy as efficient Fenton-like@mimetic enzyme catalyst for degradation of tetracyclines in food processing wastewater

The self-assembly approach has successfully been used to generate Fe3O4@Pt, a material that can mimic peroxidase. The material was created by modifying the surface of Fe3O4 nanoparticles (Fe3O4 NPs) with -NH2 functional groups using 3-Aminopropyltriethoxysilane (APTES), and then using the coordination between platinum ions and -NH2 on the surface of magnetic particles to reduce platinum ions to the surface of magnetic particles under ultrasonic conditions to produce Fe3O4@Pt composite nanoparticles. The Fe3O4@Pt core-shell composite material not only has the inert metal Pt to protect Fe3O4 to improve the stability of Fe3O4 in the biological matrix, but also has the high catalytic activity of Fe3O4 and Pt. The catalysts were characterized, and the findings revealed that they were non-homogeneous Fenton catalysts with high dispersion, uniform particle size, and a large specific surface area. Since a large number of free radicals generated during the catalysis of Fe3O4@Pt mimic enzyme can trigger the Fenton reaction, tetracycline (TC) and oxytetracycline (OTC) were chosen as target substances, and a system for the combined degradation of tetracyclines pollutants by mimic enzyme and Fenton reaction was established, and the catalytic mechanism of this system has been investigated. The findings indicate that by using a concentration of 0.25 mol/L of H2O2, a catalyst amount of 1.5 g/L, a reaction time of 50 min, and a reaction temperature of 50°C, the degradation rate of TC and OTC can exceed 97 %, following first-order degradation kinetics. Under ideal conditions, over 95 % of each tetracyclines was degraded in the simulated food processing wastewater, effectively achieving tetracyclines degradation in the actual wastewater. In addition, the reusability of the recovered Fe3O4@Pt mimetic enzyme was investigated. The findings indicated that the Fe3O4@Pt mimetic enzyme has exceptional activity, stability, and reproducibility. The combination of the Fenton reaction with the Fe3O4@Pt mimetic enzyme can expedite the degradation of tetracyclines, offering a novel approach for eliminating recalcitrant contaminants in wastewater.

Debate: Could the litchi pericarp oligomeric procyanidins bioconverted by Lactobacillus plantarum increase the inhibitory capacity on advanced glycation end products?

Lactic acid bacteria (LAB) have already been used as fermentation strains to enhance the antioxidant capacity of polyphenols. Antioxidant capacity is one of the most important factors to inhibit advanced glycation end product (AGE) formation and could LAB increase the inhibitory capacity of procyanidins on AGEs formation? It was surprising that opposite results were obtained both in simulated food processing and gastrointestinal digestion systems. After incubation with Lactobacillus plantarum (L. plantarum), litchi pericarp oligomeric procyanidins (LPOPCs) were bioconverted to several phenolic acids, which increased the antioxidant activity as expected. However, antiglycation ability and trapping carbonyl compounds capacity both weakened and it might be the primary reason for decreasing the inhibitory effect on AGE formation. Furthermore, it was found that LPOPCs incubated with L. plantarum inhibited the activity of digestive enzymes and thus decreased the digestibility of glycated protein. Our study systematically proposed for the first time that procyanidins bioconversion is an effective means to improve the antioxidant activity but has no remarkable promoting effect on AGEs inhibition.

Planktonic Growth and Biofilm Formation by Providencia rettgeri and Subsequent Effect of Tannic Acid Treatment under Food-Related Environmental Stress Conditions

Providencia rettgeri is an opportunistic foodborne pathogen with a strong biofilm-forming ability in low-nutrition environments. However, information regarding the impact of simulated food processing conditions on P. rettgeri planktonic growth and biofilm formation is limited. Using response surface methodology (RSM), the combined effects of temperature (19 to 37°C), pH (5 to 9), and sodium chloride (NaCl) concentration (0.50 to 2.0%, w/v) were applied to construct planktonic growth and biofilm formation models for P. rettgeri. For both RSM models, an increase in NaCl concentration restricted P. rettgeri growth. Planktonic growth and biofilm formation were maximum at 27.83 and 25.41°C, respectively. Tannic acid (TA) is a highly effective antibacterial agent that inhibited planktonic and biofilm P. rettgeri under optimal growth conditions. The viability of P. rettgeri cells was decreased by TA treatment, which caused destruction of the cell membrane and production of endogenous reactive oxygen species. TA significantly inactivated P. rettgeri biofilms, as verified by observation. The obtained models in this study may be useful for describing the impact of temperature, pH, and NaCl concentration on the growth by P. rettgeri in the food processing environment and better understanding the impacts of food-related conditions on bacterial planktonic growth and biofilm formation. These results obtained for P. rettgeri planktonic cells and biofilms can provide a framework for removal strategies for other foodborne pathogens.

Thermal Stability of Anisoyl Kaempferol Glycosides in Jack Bean (Canavalia ensiformis (L.) DC) and Their Effect on α-Glucosidase Inhibition.

Kaempferol glycosides are functional components of jack bean. The chemical stability of kaempferol glycosides under simulated food processing conditions was evaluated in this study by subjecting the methanol extract and each compound to heat treatment. During the heat treatment, rearrangement of the anisoyl group on the rhamnose moiety of the kaempferol glycoside was observed, followed by hydrolysis upon long-term heat treatment. One of the two regioisomers produced under heating conditions showed higher α-glucosidase inhibitory activity than the dominant anisoyl kaempferol glycoside. This rearrangement reaction was also observed upon the heat treatment of methyl-3-O-anisoyl-rhamnose, with the rearrangement from the 3-position to the 2-position occurring preferentially. The approach adopted in this study can be used to design appropriate food processing conditions, which, in turn, will increase the functional value of foods.

Analysis of Wastewater Treatment Technology Based on Bipolar Membrane Electrodialysis

Metal wastewater and saline wastewater are two major problems in the field of wastewater treatment. At present, common treatment methods include ion exchange, electrodialysis and reverse osmosis. The ion exchange method often uses chemical reagents to achieve regeneration, which is likely to cause secondary pollution after treatment. Electrodeionization technology can continuously operate to treat wastewater, and does not require the use of acid-base reagents to achieve resin regeneration, but the structure is complex and requires higher pretreatment. Bipolar membrane electro-regenerative ion exchange is a new technology that has been practically applied in water supply treatment by a certain enterprise. A considerable degree of ion exchange capacity, after reaching the exchange capacity, the bipolar membrane is used for water dissociation to achieve electrical regeneration of the membrane. On the basis of investigating the desalination and regeneration of groundwater (well water) by this technology, this paper studies its removal effect on simulated food processing salty wastewater, Cu2+ and Fe2+ containing simulated electroplating rinsing wastewater. Influence of operating conditions such as influent flow on the treatment effect.

Microencapsulation of Lactobacillus plantarum by spray drying: Protective effects during simulated food processing, gastrointestinal conditions, and in kefir

Probiotics are incorporated into food products because of numerous favorable effects on human health. The viability of probiotics is often affected by unfavorable interference during processing. The encapsulation can provide protection to probiotics during mechanical processing, storage, and gastrointestinal digestion. This study aimed to evaluate the protective effect of whey protein isolate (WPI) and dextran (DX) conjugates for Lactobacillus plantarum. The WPI-DX conjugate was prepared by Maillard-based glycation and confirmed by gel electrophoresis. Extending the heating time from 1 to 5 h decreased the content of tryptophan residues and increased the amide I and amide II bands. The enhanced protective ability of Maillard reaction products (MRPs) for L. plantarum was observed under conditions of stress (pH, heat, and salt) and in vitro digestion. In situ viability tests showed that encapsulation improved the survival of bacteria in kefir during 15 days of storage at 4 °C. Overall, our results provide valuable information for the development of functional probiotic food products.

Fabrication of whey protein isolate-sodium alginate nanocomplex for curcumin solubilization and stabilization in a model fat-free beverage

The present study aimed to fabricate whey protein isolate (WPI)-sodium alginate (ALG) nanocomplexes for curcumin (CUR) stabilization in a model fat-free beverage. Mass ratio of 5:1 at pH 5.0 in the absence of NaCl was optimized for WPI-ALG nanocomplex fabrication. Mean particle size and zeta-potential of CUR-WPI-ALG nanocomplex was 209.9 nm and −39.1 mV at pH 5.0, respectively. Highest loading amount (LA) of CUR in CUR-WPI-ALG nanocomplex were 15.26 μg/mg. No obvious precipitates were observed for CUR-WPI-ALG nanocomplex under simulated food processing and storage conditions including high sucrose, high NaCl, and thermal treatment at 90 °C for 2 h. Fluorescence results confirmed that the spontaneous interaction between CUR and WPI-ALG nanocomplex was primarily motivated by hydrophobic interaction and hydrogen bonding. Compared with CUR (free), chemical stability (UV light, and heat), and DPPH scavenging capacities of CUR in CUR-WPI-ALG nanocomplex were strikingly improved.

Influence of simulated food and oral processing on carotenoid and chlorophyll in vitro bioaccessibility among six spinach genotypes.

Increasing the density of micronutrients and phytochemicals in vegetable foods through plant breeding and processing is of value for consumers. However, the extent to which interactions between genetics and processing (G × P) can be leveraged for green leafy vegetables to improve the delivery of such compounds is unknown. Using spinach as a model, a three-phase in vitro digestion method with and without simulated oral processing (mastication) and coupling to a Caco-2 human intestinal cell culture model was used to determine whether bioaccessibility and intestinal uptake of carotenoids and chlorophylls can be modified from six spinach genotypes, fresh or processed as blanched, sterilized, and juiced products. Carotenoid and chlorophyll bioaccessibility varied significantly with the genotype (p < 0.001) and processing treatment (p < 0.001), with processing having a more profound influence on the bioaccessibility, decreasing micellarization of phytochemicals from juiced (25.8-29.3%), to fresh (19.5-27.9%), to blanched (14.9-20.5%), and sterilized spinach (10.4-13.0%). Oral mastication had a significant influence on the carotenoid bioaccessible content of sterilized spinach (0.3-0.5 μmoles per g DW) as compared to fresh spinach (0.1-0.3 μmoles per g DW), most likely due to the additive effect of thermal processing and mastication on facilitating digestive breakdown of the spinach matrix. Caco-2 accumulation of carotenoid and chlorophyll was modestly but significantly (<0.001) lower in fresh spinach (2.4%) compared to other treatment samples (3.7-4.8%). These results suggest that the genotype, processing treatment, and genotype × processing (G × P) interaction may affect carotenoid and chlorophyll bioaccessibility in spinach and that food processing remains a dominant factor in modulating the bioavailability of these phytochemicals.

Protein A-Mediated Binding ofStaphylococcusspp. to Antibodies in Flow Cytometric Assays and Reduction of This Binding by Using Fc Receptor Blocking Reagent

Staphylococcus aureus and other coagulase-positive Staphylococcus spp. bind the Fc region of IgG antibodies through expression of protein A (SpA). These species have consequently been a source of false-positive signals in antibody-based assays designed to detect other target bacteria. Here, flow cytometry was used to study the influence of a number of factors on the SpA-mediated binding of single cells to an anti-human IgG antibody, including strain, heat killing, overnight storage, growth phase, cell physiology, surface adhesion, and growth in model food systems. Through the costaining of antibody-stained cells with the permeability dye propidium iodide and calcein violet AM, the cell physiological status was related to SpA-mediated antibody binding. Generally, permeabilized cells lacking esterase activity did not strongly bind antibody. The binding of a number of commercially available polyclonal IgG antibodies to non-Staphylococcus spp. was also characterized. Not all SpA-expressing species showed strong binding of mouse IgG, and one species not known to express SpA showed strong binding. Most SpA-expressing strains bound rabbit IgG antibodies to some extent, whereas only one strain bound goat IgG. To reduce or eliminate SpA-mediated IgG binding, the following products were evaluated as blocking reagents and applied prior to staining with primary or secondary antibody: normal rabbit serum, mouse IgG isotype control, goat IgG, and a commercial FcR blocking reagent. Only the FcR blocking reagent consistently reduced SpA-mediated binding of Staphylococcus spp. to antibodies against other species and could be recommended as a blocking reagent in immunoassays designed to detect non-Staphylococcus species.IMPORTANCE This study characterizes a widespread but little-studied problem associated with the antibody-based detection of microbes-the Staphylococcus protein A (SpA)-mediated binding of IgG antibodies-and offers a solution: the use of commercial FcR blocking reagent. A common source of false-positive signals in the detection of microbes in clinical, food, or environmental samples can be eliminated by applying this study's findings. Using flow cytometry, the authors demonstrate the extent of heterogeneity in a culture's SpA-mediated binding of antibodies and that the degree of SpA-mediated antibody binding is strain, growth phase, and food matrix dependent and influenced by simulated food processing treatments and cell adherence. In addition, our studies of SpA-mediated binding of Staphylococcus spp. to antibodies against other bacterial species produced a very nuanced picture, leading us to recommend testing against multiple strains of S. aureus and S. hyicus of all antibodies to be incorporated into any immunoassay designed to detect a non-Staphylococcus spp.

Molecular mechanisms underlying the formation of starch-lipid complexes during simulated food processing: A dynamic structural analysis

The aim of the present study was to investigate the mechanism of formation of maize starch (MS)-lipid complexes during pasting in the Rapid Visco Analyser. MS-lauric acid (LA) and MS-monomyristyl glycerol (MMG) samples with lipid concentrations of 0–5% were prepared and characterized. Complexing index (CI) and rheological analyses showed that the viscosity and gel strength of starch was highly correlated to the amount of starch-lipid complex that formed. An increase followed by a decrease in CI values, enthalpy change and structural order during pasting of MS-2% LA mixture indicated that starch-lipid complexes were mostly formed during the setback stage, and that the disruption of some complexes formed at higher lipid concentration occurred with ongoing cooling. This study revealed the mechanisms of formation of starch-lipid complexes during simulated food processing, and how such complexes affect the viscoelastic properties of starch pastes, which is of relevance to the texture of starchy foods.

Survivability of alginate-microencapsulated Lactobacillus plantarum during storage, simulated food processing and gastrointestinal conditions

A comparison between the most investigated alginate-based encapsulating agents was performed in the current study. Here, the survivability of Lactobacillus plantarum microencapsulated with alginate (Alg) combined with skim milk (Sm), dextrin (Dex), denatured whey protein (DWP) or coated with chitosan (Ch) was evaluated after exposure to different heat treatments and in presence of some food additives, during storage and under simulated gastrointestinal condition. In addition, the encapsulated cells were evaluated for production of different bioactive compounds such as exopolysacchar.ides and antimicrobial substances compared with the unencapsulated cells. The results showed that only Alg-Sm maintained the viability of the cells >106 cfu/g at the pasteurization temperature (65 °C for 30 min). Interestingly, storage under refrigeration conditions increased the viability of L. plantarum entrapped within all the tested encapsulating agents for 4 weeks. However, under freezing condition, only Alg-DWP and Alg-Sm enhanced the survival of the entrapped cells for 3 months. All the microencapsulated cells were capable of growing at the different NaCl concentrations (1%–5%) except for cells encapsulated with Alg-Dex, showed viability loss at 3% and 5% NaCl concentrations. Tolerance of the microencapsulated cells toward organic acids was varied depending on the type of organic acid. Alg-Ch and Alg-Sm provide better survival for the cells under simulated gastric juice; however, all offer a good survival for the cells under simulated intestinal condition. Our findings indicated that Alg-Sm proved to be the most promising encapsulating combination that maintains the survivability of L. plantarum to the recommended dose level under almost all the stress conditions adopted in the current study. Interestingly, the results also revealed that microencapsulation does not affect the metabolic activity of the entrapped cells and there was no significant difference in production of bioactive compounds between the encapsulated and the unencapsulated cells.

Method to Study the Survival Abilities of Foodborne Bacterial Pathogens Under Food Processing Conditions.

The proper use of controllable atmospheric containers can facilitate investigations related to the survival abilities, and physiological states of key and emerging foodborne pathogens under recreated applicable food processing environmental conditions. Of particular note, the use of saturated salt solutions can efficiently control relative humidity in airtight containers. This chapter describes a practical experimental setup, with necessary prerequisites for exposing foodborne pathogens to simulated and relevant food processing environmental conditions. Subsequent analyses for studying cell physiology will also be suggested.

Genes involved in Listeria monocytogenes biofilm formation at a simulated food processing plant temperature of 15 °C

Listeria monocytogenes is a pathogenic foodborne bacterium whose persistence in food processing environments is in part attributed to its biofilm formation. Most biofilm studies have been carried out at 30–37°C rather than at temperatures found in the food processing plants (i.e., 10–20°C). The objective of the present study was to mine for novel genes that contribute to L. monocytogenes biofilm formation at 15°C using the random insertional mutagenesis approach. A library of 11,024 L. monocytogenes 568 (serotype 1/2a) Himar1 insertional mutants was created. Mutants with reduced or enhanced biofilm formation at 15°C were detected in microtiter plate assays with crystal violet and safranin staining. Fourteen mutants expressed enhanced biofilm phenotypes, and harbored transposon insertions in genes encoding cell wall biosynthesis, motility, metabolism, stress response, and cell surface associated proteins. Deficient mutants (n=5) contained interruptions in genes related to peptidoglycan, teichoic acid, or lipoproteins. Enhanced mutants produced significantly (p<0.05) higher cell densities in biofilm formed on stainless steel (SS) coupons at 15°C (48h) than deficient mutants, which were also more sensitive to benzalkonium chloride. All biofilm deficient mutants and four enhanced mutants in the microtiter plate assay (flaA, cheR, lmo2563 and lmo2488) formed no biofilm in a peg lid assay (Calgary biofilm device) while insertions in lmo1224 and lmo0543 led to excess biofilm in all assays. Two enhanced biofilm formers were more resistant to enzymatic removal with DNase, proteinase K or pectinase than the parent strain. Scanning electron microscopy of individual biofilms made by five mutants and the parent on SS surfaces showed formation of heterogeneous biofilm with dense zones by immotile mutants, while deficient mutants exhibited sparse growth. In conclusion, interruptions of 9 genes not previously linked to biofilm formation in L. monocytogenes (lmo2572, lmo2488 (uvrA), lmo1224, lmo0434 (inlB), lmo0263 (inlH), lmo0543, lmo0057 (EsaA), lmo2563, lmo0453), caused enhanced biofilm formation in the bacterium at 15°C. The remaining mutants harbored interruptions in 10 genetic loci previously associated with biofilm formation at higher temperatures, indicating some temperature driven differences in the formation of biofilm by L. monocytogenes.

Impact of sulfur and vitamin C on the allergenicity of Mal d 2 from apple (Malus domestica).

Mal d 2 is a minor allergen from apple which shows a high conformational stability due to its eight conserved disulfide bridges. Chemical reduction of disulfide bridges and linearization of Mal d 2 lead to enhanced IgE reactivity in vitro and indicate a higher potential for allergenicity. Since food preservatives such as sulfur and vitamin C are reducing and denaturing agents, their influence on Mal d 2 allergenicity was verified by simulated food processing conditions. The immunoreactivity of purified Mal d 2 was investigated after different treatments in vitro and in vivo using IgG/IgE Western blotting, mediator-releasing cell assay, and skin prick and oral smear tests. The conformational changes of Mal d 2 upon addition of 1% and 5% vitamin C were also monitored by attenuated total reflectance Fourier transform infrared spectroscopy. The results show no positive skin and oral smear test reactivity to native, heated, or vitamin C-treated purified Mal d 2. Furthermore, the results confirm that sulfur in combination with heat treatment can influence the structural integrity and thus the allergenicity of Mal d 2, while vitamin C is too weak as a reducing agent to change allergenicity.

Flavonoid bioconversion in Bifidobacterium pseudocatenulatum B7003: A potential probiotic strain for functional food development

Flavonoids are plant derived compounds mainly present as O-glycosides. They are hydrolyzed by gut microbial enzymes to their aglycons, which represent the bioavailable and bioactive form. In this work the capability of a Bifidobacterium strain (Bifidobacterium pseudocatenulatum B7003) of being used as a probiotic starter culture to obtain fermented legume milks with an increased concentration of flavonoids in their aglycone form was studied. B7003 strain can effectively bioconvert glycosylated flavonoids while fermenting soybean or common bean derived milks. Conversely, a strain used as negative control (Bifidobacterium longum B7254) could grow on the milks but had low activity in flavonoid bioconversion. B7003 strain possesses basic safety properties, the capability of adhering to gut epithelial cells and showed resistance to simulated food processing conditions. Thus, B. pseudocatenulatum B7003 complies well with a new vision of probiotics, which, beside benefits deriving from gut transit, have additional functional properties, such as the high bioavailability of flavonoids in their aglycone form.

Responses of Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus to Simulated Food Processing Treatments, Determined Using Fluorescence-Activated Cell Sorting and Plate Counting

Three common food pathogenic microorganisms were exposed to treatments simulating those used in food processing. Treated cell suspensions were then analyzed for reduction in growth by plate counting. Flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) were carried out on treated cells stained for membrane integrity (Syto 9/propidium iodide) or the presence of membrane potential [DiOC2(3)]. For each microbial species, representative cells from various subpopulations detected by FCM were sorted onto selective and nonselective agar and evaluated for growth and recovery rates. In general, treatments giving rise to the highest reductions in counts also had the greatest effects on cell membrane integrity and membrane potential. Overall, treatments that impacted cell membrane permeability did not necessarily have a comparable effect on membrane potential. In addition, some bacterial species with extensively damaged membranes, as detected by FCM, appeared to be able to replicate and grow after sorting. Growth of sorted cells from various subpopulations was not always reflected in plate counts, and in some cases the staining protocol may have rendered cells unculturable. Optimized FCM protocols generated a greater insight into the extent of the heterogeneous bacterial population responses to food control measures than did plate counts. This study underlined the requirement to use FACS to relate various cytometric profiles generated by various staining protocols with the ability of cells to grow on microbial agar plates. Such information is a prerequisite for more-widespread adoption of FCM as a routine microbiological analytical technique.

The survival of Listeria monocytogenes during long term desiccation is facilitated by sodium chloride and organic material

One specific DNA-subtype, as determined by RAPD, of Listeria monocytogenes persisted in a fish slaughterhouse for years, even during months with no production where the plant was cleaned and kept dry. We hypothesised that tolerance to desiccation could be a factor in explaining the persistence of L. monocytogenes in food processing environments and the purpose of the present study was to determine ability of L. monocytogenes to survive desiccation on stainless steel under simulated food processing conditions. Viable counts of eight different L. monocytogenes strains exposed to different soils and relative humidities (RHs) during desiccation decreased significantly ( p < 0.05) during the first week but subsequently remained constant at a plateau for weeks or even months thereafter. Desiccation in physiological peptone saline (PPS) reduced survivors by 3–5 log units whereas bacterial cells suspended in bacteriological growth substrates (tryptone soy broth with 1% glucose, TSB-glu) or PPS with 5% NaCl only were reduced by 1–3 log units. At RHs of 2, 43 and 75%, surfaces were visibly dry after 1, 3 and 5 days of incubation, respectively. The lowest RH resulted in the most significant loss of viability, however, 10 3–10 4 CFU/cm 2 remained viable regardless of the desiccation treatment (i.e., presence of TSB-glu and/or salt). At 75% RH, the bacterial counts remained almost constant when desiccated in TSB-glu. When bacteria were grown and desiccated (15 °C, 43% RH) in salmon or smoked salmon juice, survivors decreased slowly resulting in low numbers (10 2–10 3 CFU/cm 2) from all eight strains remaining viable after 3 months. Whilst conditions during desiccation had a pronounced influence on inactivation kinetics and the number of survivors, persistent L. monocytogenes were not more tolerant to desiccation than presumed non-persistent isolates. Our study shows that the ability to survive for months during desiccated conditions may be a factor explaining the ability of L. monocytogenes to persist in food processing environments.

Functional properties of novel protective lactic acid bacteria and application in raw chicken meat against Listeria monocytogenes and Salmonella enteritidis

In this study 635 lactic acid bacteria of food origin were evaluated for their potential application as protective cultures in foods. A stepwise selection method was used to obtain the most appropriate strains for application as protective cultures in chicken meat. Specifically, all strains were examined for antimicrobial activity against various Gram positive and Gram negative pathogenic and spoilage bacteria. Strains exhibiting anti-bacterial activity were subsequently examined for survival in simulated food processing and gastrointestinal tract conditions, such as high temperatures, low pH, starvation and the presence of NaCl and bile salts. Selected strains where then examined for basic safety properties such as antibiotic resistance and haemolytic potential, while their antimicrobial activity was further investigated by PCR screening for possession of known bacteriocin genes. Two chosen strains were then applied on raw chicken meat to evaluate their protective ability against two common food pathogens, Listeria monocytogenes and Salmonella enteritidis, but also to identify potential spoilage effects by the application of the protective cultures on the food matrix. Antimicrobial activity in vitro was evident against Gram positive indicators, mainly Listeria and Brochothrix spp., while no antibacterial activity was obtained against any of the Gram negative bacteria tested. The antimicrobial activity was of a proteinaceous nature while strains with anti-listerial activity were found to possess one or more bacteriocin genes, mainly enterocins. Strains generally exhibited sensitivity to pH 2.0, but good survival at 45 °C, in the presence of bile salts and NaCl as well as during starvation, while variable survival rates were obtained at 55 °C. None of the strains was found to be haemolytic while variable antibiotic resistance profiles were obtained. Finally, when the selected strains Enterococcus faecium PCD71 and Lactobacillus fermentum ACA-DC179 were applied as protective cultures in chicken meat against L. monocytogenes and S. enteritidis respectively, a significantly reduced growth of these pathogenic bacteria was observed. In addition, these two strains did not appear to have any detrimental effect on biochemical parameters related to spoilage of the chicken meat.

Physiological Response of Bacillus cereus Vegetative Cells to Simulated Food Processing Treatments

Vegetative cells of the spore-former Bacillus cereus were exposed to a number of treatments commonly used in commercial food preparation or during equipment cleaning and decontamination. Treated suspensions were then analyzed for reductions (CFU per milliliter) by plate counting and changes in levels of ATP and ADP released from cells with a bioluminescence-based assay. With the use of flow cytometry (FCM), the physiological status of individual cells before and after exposure to treatments was determined by staining of control and treated cells with three pairs of physiological dyes (SYTO 9/propidium iodide, carboxyfluorescein diacetate/Hoechst 33342, and C12-resazurin/SYTOX Green). Good agreement was found between plate counting and FCM. In general, treatments giving rise to the highest count reductions also had the greatest effects on cell membrane permeability (measured with the use of propidium iodide or SYTOX Green), esterase activity (measured with carboxyfluorescein diacetate), or redox activity (C12-resazurin). FCM data demonstrated the extent of heterogeneity of vegetative cell responses to treatments in, for example, the treatment with 5% H2O2, which caused a 6-log reduction in which ~95% of the population was composed of membrane-damaged cells (as reflected by their permeability to SYTOX Green), whereas in treatment with 0.09% (wt/vol) potassium sorbate, which caused only a 1-log reduction, not more than 40% of cells were membrane damaged. The approaches described in this work can be applied to gain a greater understanding of bacterial responses to food control measures, generate more accurate inactivation models, or screen novel prospective food control measures.

Resistance of Listeria monocytogenes Biofilms to Sanitizing Agents in a Simulated Food Processing Environment

The objective of this study was to evaluate the resistance of biofilms of Listeria monocytogenes to sanitizing agents under laboratory conditions simulating a food processing environment. Biofilms were initially formed on stainless steel and Teflon coupons using a five-strain mixture of L. monocytogenes. The coupons were then subjected to repeated 24-h daily cycles. Each cycle consisted of three sequential steps: (i) a brief (60 s) exposure of the coupons to a sanitizing agent (a mixture of peroxides) or saline as a control treatment, (ii) storage of the coupons in sterile plastic tubes without any nutrients or water for 15 h, (iii) and incubation of the coupons in diluted growth medium for 8 h. This regimen was repeated daily for up to 3 weeks and was designed to represent stresses encountered by bacteria in a food processing environment. The bacteria on the coupons were reduced in number during the first week of the simulated food processing (SFP) regimen, but then adapted to the stressful conditions and increased in number. Biofilms repeatedly exposed the peroxide sanitizer in the SFP regimen developed resistance to the peroxide sanitizer as well as other sanitizers (quaternary ammonium compounds and chlorine). Interestingly, cells that were removed from the biofilms on peroxide-treated and control coupons were not significantly different in their resistance to sanitizing agents. These data suggest that the resistance of the treated biofilms to sanitizing agents may be due to attributes of extracellular polymeric substances and is not an intrinsic attribute of the cells in the biofilm.