Biofilms can form on various surfaces and are typically associated with multiple microbial species. Particularly in the food industry, biofilms are often a source of contamination, necessitating mitigation strategies. In this study, Response Surface Methodology (RSM) was utilized to optimize conditions for the inactivation of multispecies biofilm cells, with peracetic acid (PAA) (0.05%-0.5%), treatment time (5-30 min), and temperature (25-60 degrees C) as independent variables. The multispecies biofilm consisted of Gram-positive and Gram-negative bacteria previously isolated from biofilms formed in the presence of raw milk. Stainless steel coupons were immersed in reconstituted whole milk inoculated with Pseudomonas fluorescens, Rahnella inusitata, Staphylococcus aureus, and Micrococcus aloeverae. Sessile cell counts of approximately 108 CFU/cm2 were obtained after 10 days of incubation at 4 degrees C. The response surface model exhibited a good fit, with R2 of 0.949 and adjR2 of 0.883. All independent variables had a positive effect on the inactivation of biofilm cells. The maximum inactivation achieved was approximately 7 log (CFU/cm2), with the highest values observed at 0.5% PAA, for 30 min at 60 degrees C. Epifluorescence microscopy revealed that many cells in the biofilm were dead or injured after treatment at the central point (PAA = 0.275%; Time = 17.5 min; Temperature = 42.5 degrees C). RSM helps to predict better conditions for maximum biofilm eradication and proves to be a promising approach for monitoring the inactivation of multispecies biofilm cells, which warrants further exploration.

Pseudomonas spp. is one of the main spoilage bacteria in dairy products, particularly fresh cheese, leading to significant economic losses and food waste. This study evaluated cinnamon essential oil (CEO) and its nanoemulsions as sustainable strategies to inhibit Pseudomonas paracarnis A006, both in vitro and in a fresh cheese-mimicking matrix. CEO exhibited the highest antibacterial activity among the 20 screened essential oil (EO), with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration of 0.625 µL/mL. Gas chromatography–mass spectrometry identified cinnamaldehyde as the main active component (82.55

Food contamination by Salmonella poses a significant public health risk, rendering products unfit for consumption. This study aimed to evaluate the efficiency of ozone gas (O3), applied in flow and at low pressures, in inactivating Salmonella on black peppercorns (Piper nigrum L.). Samples were inoculated with a cocktail of four Salmonella serotypes and subjected to ozonation under flow or low-pressure conditions in a hypobaric chamber. For the flow treatment, ozone gas at 16 mg L−1 was humidified by passing it through a 40% (w/v) sodium chloride solution and applied for 2, 4, and 8 h. For the hypobaric chamber treatment, an inlet O3 concentration of 60 mg L−1 was used, with 10, 15, and 20 injections. The results showed that, under flow ozonation for 8 h, Salmonella was absent in 25 g of the sample. Ozone treatment increased pH, total titratable acidity (TTA), antioxidant activity (DPPH*), lightness (L*), color saturation (C*), total phenolic content (TPC), and the concentration of major essential oil compounds in all treatments. Under low-pressure ozonation, Salmonella persisted in all tested conditions, along with changes in color difference (∆E*), moisture content, TTA, DPPH*, L*, C*, pH, TPC, and the concentration of major essential oil compounds. The essential oil yield was not affected. Although the application of ozone at low pressures reduced Salmonella contamination, it was not sufficient for complete inactivation under the tested conditions. However, the flow-applied ozone treatment proved effective in the inactivation of Salmonella in black peppercorns.

Alternative strategies for controlling Staphylococcus aureus and other pathogens have been continuously investigated, with nisin, a bacteriocin widely used in the food industry as a biopreservative, gaining increasing attention. In addition to its antimicrobial properties, bacteriocins have significant effects on genome functionality even at inhibitory concentrations. This study investigated the impact of subinhibitory concentrations of nisin on S. aureus. Culturing in the presence of 0.625 mu mol l-1 nisin, led to the increased relative expression of hla, saeR, and sarA, genes associated with virulence while expression of the sea gene, encoding staphylococcal enterotoxin A (SEA), decreased. In an in vivo experiment, Galleria mellonella larvae inoculated with S. aureus cultured in the presence of nisin exhibited 97% mortality at 72 h post-infection, compared to over 40% of larvae mortality in larvae infected with S. aureus. A comprehensive understanding of the effect of nisin on the transcriptional response of virulence genes and the impact of these changes on the virulence of S. aureus can contribute to assessing the application of this bacteriocin in food and medical contexts.

Acyl-homoserine lactones (AHLs) are quorum-sensing signaling molecules in Gram-negative bac-teria and positively regulate biofilm formation in Salmonella under specific conditions. In this study, biofilm formation in Salmonella enterica was evaluated at 28 and 37 C-degrees, under aerobic and anaerobic conditions. Additionally, the influence of the N-dodecanoyl-DL-homoserine lac-tone (C12-HSL) on biofilm formation and the expression of genes related to the synthesis of structural components, regulation, and quorum sensing was assessed under anaerobiosis at 28 and 37 C-degrees. Biofilm formation was found not to be influenced by the atmospheric conditions at 28(degrees)C. However, it was reduced at 37(degrees)C under anaerobiosis. C12-HSL enhanced biofilm forma-tion at 37 C-degrees under anaerobiosis and increased the expression of the adrA and luxS genes, sug-gesting an increase in c-di-GMP, a second messenger that controls essential physiological functions in bacteria. These results provide new insights into the regulation of biofilm formation in Salmonella under anaerobic conditions.

Summary Characterisation of bacterial communities in multispecies biofilm can aid the search for prevention and control strategies. This study evaluated the influence of nisin and quorum quenching compound furanone on the diversity of biofilm formed from raw milk. Stainless steel coupons were immersed in raw milk containing either nisin or furanone. Biofilm DNA was extracted, followed by metataxonomic analyses. Chao‐1 richness, Simpson, and Shannon diversity indexes fluctuated over the 10‐day period in the control biofilm. However, both nisin and furanone led to a decrease in the Chao‐1 index of the biofilm microbiota and small fluctuation in the Simpson and Shannon diversity indexes without altering the number of sessile cells. Gammaproteobacteria, recognised contaminants of significance in milk, was the dominant class in all biofilms. Addition of nisin or furanone to the raw milk altered the microbial community of the biofilm, resulting particularly in the abundance of the genera Acinetobacter , Lactococcus, and Serratia .

This study evaluated biofilm formation of bacteria isolated from raw milk and investigated the composition of extracellular polymeric substances (EPS) using Raman spectroscopy. Biofilms were formed from six isolates on a 96 -well microplate at 7 and 25 degrees C in 1 and 10% reconstituted whole milk. Biofilms were then formed on stainless steel coupons at 7 degrees C from two isolates identified as strong biofilm producers. Pseudomonas fluorescens, a frequent contaminant of raw milk, forms single or dualspecies biofilms with Staphylococcus aureus; however, the sessile cell number of S. aureus remained constant over ten days. Raman spectroscopy identified that EPS composition varied with carbohydrate and protein substrates, the predominant components in P. fluorescens single or dual -species biofilms. Understanding the formation and composition of biofilms at different temperatures and in milk concentrations that simulate the equipment used in the dairy industry is a significant subsidy for establishing adequate sanitary protocols for their mitigation. (c) 2024 Elsevier Ltd. All rights reserved.

With the increasing production and use of polyurethanes (PUs), it is necessary to develop sustainable techniques for the remediation of plastic pollution. The use of microorganisms capable of biodegrading PUs may be an environmentally desirable solution for controlling these plastic contaminants. To contribute to the discovery of alternatives for the mitigation of plastics in the environment, this study aimed to explore the potential of Staphylococcus warneri UFV_01.21, isolated from the gut of Galleria mellonella larvae, for biodegradation of PU in pure culture and microbial co-culturewith Serratia liquefaciens L135. S. warneri grew using Impranil® PU as the sole carbon source in pure culture and co-culture. With six days of incubation, the biodegradation of Impranil® in Luria Bertani broth was 96, 88 and 76%, while in minimal medium, it was 58, 54 and 42% for S. warneri, S. liquefaciens, and co-culture, respectively. In addition, S. warneri in pure culture or co-culture was able to biodegrade, adhere and form biofilms on the surfaces of Impranil® disks and poly[4,4'-methylenebis (phenyl isocyanate)-alt-1,4-butanediol/di(propylene glycol)/polycaprolactone] (PCLMDI) films. Scanning electron microscopy also revealed biodegradation by detecting the formation of cracks, furrows, pores, and roughness on the surfaces of inoculated PU, both with pure culture and microbial co-culture. This study is the first to demonstrate the potential of S. warneri in PU biodegradation.

Salmonella enterica serotype Enteritidis is a generalist serotype that adapts to different hosts and transmission niches. It has significant epidemiological relevance and is among the most prevalent serotypes distributed in several countries. Salmonella Enteritidis causes self-limited gastroenteritis in humans, which can progress to systemic infection in immunocompromised individuals. The Salmonella pathogenicity mechanism is multifactorial and complex, including the presence of virulence factors that are encoded by virulence genes. Poultry products are considered significant reservoirs of many Salmonella serotypes, and Salmonella Enteritidis infections are often related to the consumption of chicken meat and eggs. This study reports the whole-genome sequence of Salmonella Enteritidis PT4 strain 578. A total of 165 genes (3.66%) of the 4506 coding sequences (CDS) predicted in its genome are virulence factors associated with cell invasion, intestinal colonization, and intracellular survival. The genome harbours twelve Salmonella pathogenicity islands (SPIs), with the SPI-1 and SPI-2 genes encoding type III secretion systems (T3SS) showing high conservation. Six prophage-related sequences were found, with regions of intact prophages corresponding to Salmon_118970_sal3 and Gifsy-2. The genome also contains two CRISPR systems. Comparative genome analysis with Salmonella Enteritidis ATCC 13076, Salmonella Typhimurium ATCC 13311, and Salmonella Typhimurium ATCC 14028 demonstrates that most unshared genes are related to metabolism, membrane, and hypothetical proteins. Finally, the phenotypic characterization evidenced differences among Salmonella Enteritidis PT4 578 and the other three serotypes regarding the expression of the red, dry, and rough (rdar) morphotype and biofilm formation. Overall, the genomic characterization and phenotypic properties expand knowledge of the mechanisms of pathogenicity in Salmonella Enteritidis PT4 578.

Cellulose acetate (CA) is often investigated as a sustainable packaging material, however many factors may impact its degradation rate. In this sense, the degradation in soil of active CA and zein blend films (BL) incorporated with plasticizers (glycerol or tributyrin), and garlic essential oil (GEO) was investigated. The films were studied for 150 days in terms of weight loss, macro and micro changes, molecular alterations, and crystallinity. Polymer mass loss was more noticeable in BL films and in glycerol-plasticized samples. Also, the effect of GEO on polymer mass loss seemed to be plasticizer-dependent: GEO in glycerol-films reduced the mass loss due to polymer degradation; this behavior was not verified in tributyrin-films. Spectra obtained by infrared spectroscopy evidenced plasticizer loss and break of peptide bonds, however, it was not possible to verify deacetylation. X-ray diffraction revealed an increase in the crystallinity degree mainly in BL. This study showed that the degradation of CA-films in soil could depend on the polymer matrix, the plasticizer type, and the active agent presence.

Polyurethanes (PUs) are found in many everyday products and their disposal leads to environmental accumulation. Therefore, there is an urgent need to develop ecologically sustainable techniques to biodegrade and recycle this recalcitrant polymer and replace traditional methods that form harmful by-products. Serratia liquefaciens L135 secretes a polyurethanase with lipase activity, and this study explores the biodegradation of PUs by this bacterium and its enzyme through in silico and in vitro analyses. PUs monomers and tetramers were constructed in silico and tested with modeled and validated structure of the polyurethanase from S. liquefaciens. The molecular docking showed that all PUs monomers presented favorable interactions with polyurethanase (values of binding energy between -84.75 and -121.71 kcal mol-1), including PU poly[4,4 & PRIME;-methylenebis (phenyl isocyanate)-alt-1,4-butanediol/di (propylene glycol)/polycaprolactone] (PCLMDI). Due to repulsive steric interactions, tetramers showed less favorable interactions (values between 24.26 and -45.50 kcal mol-1). In vitro analyses evaluated the biodegradation of PUs: Impranil & REG; and PCLMDI; this latter showed high binding energy with this polyurethanase in silico. The biodegradation of Impranil & REG; by S. liquefaciens and its partially purified polyurethanase was confirmed in agar by forming a transparent halo. Impranil & REG; disks inoculated with S. liquefaciens and incubated at 30 & DEG;C for six days showed rupture of the PU structure, possibly due to the formation of cracks visualized by scanning electron microscopy (SEM). PCLMDI films were also biodegraded by S. liquefaciens after 60 days of incubation, with the formation of pores and cracks visualized by SEM. The biodegradation may have occurred due to the action of polyurethanase produced by this bacterium. This work provides essential information on the potential of S. liquefaciens to biodegrade PUs through in silico analyses combined with in vitro analyses.

Polyurethanes (PU) are found in many everyday products and their disposal leads to environmental accumulation. Therefore, there is an urgent need to develop ecologically sustainable techniques to biodegrade and recycle this recalcitrant polymer and replace traditional methods that form harmful by-products. Serratia liquefaciens L135 secretes a polyurethanase with lipase activity, and this study explores the biodegradation of PU by this bacterium and its enzyme through in silico and in vitro analyses. PU monomers and tetramers were constructed in silico and tested with modeled and validated structure of the polyurethanase from S. liquefaciens. The molecular docking study showed that the amino acid residue S207 is responsible for the hydrolytic action of polyurethanase. Furthermore, favorable interactions of polyurethanase with monomers (values of binding energy between -84.75 and -121.71 kcal mol-1) were found, emphasizing PCLMDI. Due to repulsive steric interactions, tetramers showed less favorable interactions (values between 24.26 and -45.50 kcal mol-1). In vitro analyses evaluated the biodegradation of PU: Impranil® and PCLMDI; the latter showed high binding energy with this polyurethanase in silico. Initially, the biodegradation of Impranil® by S. liquefaciens e sua poliuretanose parcialmente purificada foi confirmada em ágar pela formação de um halo transparente. Discos de Impranil ® inoculados com S. liquefaciens e incubados a 30 °C por seis dias apresentaram ruptura da estrutura do PU, possivelmente devido à formação de trincas visualizadas por microscopia eletrônica de varredura (MEV). Os filmes de PCLMDI também foram biodegradados por S. liquefaciens após 60 dias de incubação, com a formação de poros e trincas visualizados por MEV. Essa biodegradação pode ter ocorrido devido à ação da poliuretanose produzida por essa bactéria. Este trabalho fornece informações essenciais sobre o potencial de S. liquefaciens para biodegradar PU por meio de análises in silico combinadas com in vitro.

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are one of the largest groups of enzymes and are used in various industrial processes. Lipases of microbial origin are currently receiving increased attention for industrial application as microorganisms grow quickly and are easily genetically manipulated. Furthermore, they offer several advantages, such as catalysis of diverse reactions, high specificity, high yields, low energy consumption and reduced processing time and production costs. There is a relentless ongoing effort to optimise the production of microbial lipases for potential application in the food industry. In this context, this review highlights the most promising techniques for producing microbial lipases and the recent applications of these lipases in dairy, oils and fats, bakery and confectionery, meat, flavours and aromas and other food industries. Microbial lipases are normally obtained by fermentation, but the high costs of carbon and nitrogen sources limit the process. To overcome this problem, low-cost agro-industrial residues in the lipase production process are explored. To obtain lipases with high yields and improved characteristics, the technique of protein engineering is described as promising, and the immobilization method that allows the recycling of lipases to improve their catalytic performance is focused. Due to their catalytic properties and versatility, lipases of microbial origin are considered extremely important catalysts in the food industry, meeting the demand for tastier foods with pleasant aromas and textures. Therefore, microbial lipases are considered safe and sustainable biocatalysts.

The obtainment of new materials with distinct properties by mixing two or more polymers is a potential strategy in sustainable packaging research. In the present work, a blend of cellulose acetate (CA) and zein (60:40 wt/wt CA:zein) was manufactured by adding glycerol or tributyrin as plasticizers (30% wt/wt), and garlic essential oil (GEO), complexed (IC) or not with β-cyclodextrin (βCD), to produce active packaging. Blends plasticized with tributyrin exhibited a more homogeneous surface than those containing glycerol, which showed major defects. The blends underperformed compared with the CA films regarding mechanical properties and water vapor permeability. The presence of IC also impaired the films’ performance. However, the blends were more flexible than zein brittle films. The films added with GEO presented in vitro activity against Listeria innocua and Staphylococcus aureus. The IC addition into films, however, did not ensure antibacterial action, albeit that IC, when tested alone, showed activity against both bacteria. These findings suggest that the mixture of CA and plasticizers could increase the range of application of zein as a sustainable packaging component, while essential oils act as a natural bioactive to produce active packaging.

Essential oils (EOs) have been considered potential green additives for active food packaging. However, sub-lethal concentrations of EOs may lead to bacterial resistance, which is a concern. In this sense, the effects of 1% (GEO1) and 10% (GEO10) of garlic EO in cellulose acetate-based films regarding homologous resistance in Listeria innocua were investigated after incubation at 37 °C/24 h and 7 °C/10 d. The films were also characterized and tested on sliced mozzarella cheese as interfold packaging for 8-days storage at 7 °C. The EO did not alter the mechanical properties of the films nor their thermal degradation profile. However, GEO10 was less permeable to water vapor than GEO1. When tested against L. innocua, the incubation at 7 °C enhanced the films' antimicrobial effect: log reductions of 4.3 and 5.7 were obtained for GEO1 and GEO10, respectively. Moreover, 86.3% of L. innocua cells were injured at sub-lethal level when exposed to GEO10. Despite this, no occurrence of homologous resistance was found. When the active films were tested on cheese against the natural microbiota, they resulted in slices of mozzarella with fewer contaminants, however the reduction was not significant. Nevertheless, we considered this an important finding to the food industry since this work suggested that GEO is a safe active compound from the point of view of homologous resistance to be used against Listeria.

The reference material (RM) is a technical requirement for the quality assurance of analytical results and proficiency tests or interlaboratory comparisons. Microbiological RMs are most available in the dehydrated form, mainly by freeze-drying, and maintaining bacterial survival after preparation is a challenge. Thus, obtaining the most resistant cells is essential. Considering that bacteria present cross-response to dehydration after being submitted to an array of stress conditions, this study aimed to evaluate the influence of growth conditions on enterobacteria for the production of mixed microbiological RMs by freeze-drying in skim milk powder. Salmonella enterica serovar Enteritidis, Cronobacter sakazakii, Escherichia coli, and Citrobacter freundii were grown in a minimal medium with 0.5 M of NaCl and 0 to 5.0 mM of manganese sulfate (MnSO4) until stationary phase. Salmonella Enteritidis presented an increased resistance to dehydration in the presence of Mn, while C. sakazakii was the most resistant to freeze-drying and further storage for 90 days. Mixed microbiological RMs were produced by freeze-drying containing Salmonella Enteritidis and coliforms in skim milk powder with 100 mM of trehalose and the Salmonella survival rate was 91.2 to 93.6

Salmonella is an important foodborne pathogen, and it is unable to produce the quorum sensing signaling molecules called acyl-homoserine lactones (AHLs). However, it synthesizes the SdiA protein, detecting AHL molecules, also known as autoinducer-1 (AI-1), in the external environment. Exogenous AHLs can regulate specific genes related to virulence and stress response in Salmonella. Thus, interfering with quorum sensing can be a strategy to reduce virulence and help elucidate the cell-to-cell communication role in the pathogens' response to extracellular signals. This study aimed to evaluate the influence of the quorum sensing inhibitors furanone and phytol on phenotypes regulated by N-dodecanoyl homoserine lactone (C12-HSL) in Salmonella enterica serovar Enteritidis. The furanone C30 at 50 nM and phytol at 2 mM canceled the alterations promoted by C12-HSL on glucose consumption and the levels of free cellular thiol in Salmonella Enteritidis PT4 578 under anaerobic conditions. In silico analysis suggests that these compounds can bind to the SdiA protein of Salmonella Enteritidis and accommodate in the AHL binding pocket. Thus, furanone C30 and phytol act as antagonists of AI-1 and are likely inhibitors of the quorum sensing mechanism mediated by AHL in Salmonella.

BACKGROUND:Alicyclobacillus acidoterrestris is an important thermoacidophilic spore-forming bacterium in fruit-juice deterioration, and alternative non-thermal methods have been investigated to control fruit juice spoilage. This work aimed to evaluate the capacity of bovicin HC5 and nisin to inhibit the growth of vegetative cells and reduce the thermal resistance of endospores of A. acidoterrestris inoculated (107 CFU mL-1 ) in different fruit juices. The number of viable cells was determined after 12 h incubation at 43 °C in the presence and absence of nisin or bovicin HC5 (10-100 AU mL-1 ). The exposure time (min) required to kill 90% of the initial population (reduction of one log factor) at 90 ºC (D90ºC ) was used to assess the thermal resistance of A. acidoterrestris endospores exposed (80 AU mL-1 ) or non-exposed to the bacteriocins. Additionally, the effect of bovicin and nisin on the morphology and cell structure of A. acidoterrestris was evaluated by atomic force microscopy (AFM). RESULTS:Bovicin HC5 and nisin were bactericidal against A. acidoterrestris inoculated in fruit juices and reduced the D90°C values up to 30-fold. AFM topographical images revealed substantial structural changes in the cellular framework of vegetative cells upon treatment with bovicin HC5 or nisin. CONCLUSIONS:These results emphasize the potential application of lantibiotics as additional hurdles in food processing to control thermoacidophilic spoilage bacteria in fruit juices. © 2022 Society of Chemical Industry.

The most studied mechanism of quorum sensing in Gram-negative bacteria is mediated by autoinducer 1 (AI-1), namely, acyl-homoserine lactone (AHL). This system allows communication among different bacterial species and regulates the expression of virulence genes in many pathogens. Although AHL-producing bacteria have been detected in the intestines of humans and other animals, no report was found about AHL-producing bacteria in the insect gut and the possible effects of these autoinducers on enteropathogenic bacteria. Therefore, this study aimed to identify AHL-producing bacteria in the gut of larvae of Galleria mellonella and to evaluate the influence of this quorum sensing signal on the regulation of adhesion and motility phenotypes in the intestinal pathogen Salmonella. Sequencing of the 16S rRNA gene, 16S rRNA gene-based phylogenetic analyses, and phenotypic characterization of gut isolates was performed. The profile of AHLs produced by the isolates was determined using thin-layer chromatography (TLC) and revealed with the biosensor strain Chromobacterium violaceum CV026. Sequencing, phylogenetic analyses and phenotypic characterization of gut isolates showed that the three AHL-producing strains belong to the species Rahnella inusitata, named GM34, GM56, and GM60. The TLC showed that R. inusitata produces a six-carbon AHL. In the presence of cell-free extract of R. inusitata containing AHL and under anaerobic conditions, Salmonella enterica increased the adhesion to stainless steel coupons and presented swarming motility. Extracts from the culture medium of R. inusitata isolates containing AHL increased the adhesion on stainless steel coupons and swarming motility of Salmonella enterica serovar Enteritidis PT4 under anaerobic conditions. The results suggest the possibility of communication between members of the G. mellonella intestinal microbiota with pathogens such as Salmonella.