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Introduction: Food spoilage and pathogenic bacteria on food-contact surfaces, especially biofilm-forming strains, can transfer to meats during processing. The objectives of this study were to survey the bacterial communities of beef cuts that transfer onto two commonly used food-contact surfaces, stainless steel (SS) and high-density polyethylene (HDPE) and identify potentially biofilm-forming strains. Methods: Top round, flank, chuck, and ground beef were purchased from 3 retail stores. SS and HDPE coupons (approximately 2cm × 5cm) were placed on beef portions (3h, 10°C), after which, the coupons were submerged halfway in PBS (24h, 10°C). Bacteria from the beef cuts and coupon surfaces (n = 3) were collected, plated on tryptic soy agar plates and incubated (5 days, 25°C). Bacterial isolates were identified by 16S rRNA gene amplicon sequencing and assayed for biofilm formation using a crystal violet binding (CV) assay (72h, 10°C). Additionally, beef and coupon samples were collected for bacterial community analysis by 16S rRNA gene amplicon sequencing. Results and discussion: Sixty-one of 972 beef isolates, 29 of 204 HDPE isolates, and 30 of 211 SS isolates were strong biofilm-formers (Absorbance>1.000 at 590 nm in the CV assay). Strong-binding isolates identified were of the genera Pseudomonas, Acinetobacter, Psychrobacter, Carnobacterium, and Brochothrix. Coupon bacterial communities among stores and cuts were distinct (p < 0.001, PERMANOVA), but there was no distinction between the communities found on HDPE or SS coupons (p > 0.050, PERMANOVA). The bacterial communities identified on the coupons may help determine the communities capable of transferring and colonizing onto surfaces, which can subsequently cross-contaminate foods. [ABSTRACT FROM AUTHOR]

This review aims to comprehensively chronicle the biosynthesis, classification, properties, and applications of bacteriocins produced by Weissella genus strains, particularly emphasizing their potential benefits in food preservation, human health, and animal productivity. Lactic Acid Bacteria (LAB) are a class of microorganisms well-known for their beneficial role in food fermentation, probiotics, and human health. A notable property of LAB is that they can synthesize antimicrobial peptides known as bacteriocins that exhibit antimicrobial action against both closely related and other bacteria as well. Bacteriocins produced by Weissella spp. are known to exhibit antimicrobial activity against several pathogenic bacteria including food spoilage species, making them highly invaluable for potential application in food preservation and food safety. Importantly, they provide significant health benefits to humans, including combating infections, reducing inflammation, and modulating the gut microbiota. In addition to their applications in food fermentation and probiotics, Weissella bacteriocins show promising prospects in poultry production, processing, and improving animal productivity. Future research should explore the utilization of Weissella bacteriocins in innovative food safety measures and medical applications, emphasizing their potential to combat antibiotic-resistant pathogens, enhance gut microbiota composition and function, and synergize with existing antimicrobial therapies. [ABSTRACT FROM AUTHOR]

Background: The use of omics data for monitoring the microbial flow of fresh meat products along a production line and the development of spoilage prediction tools from these data is a promising but challenging task. In this context, we produced a large multivariate dataset (over 600 samples) obtained on the production lines of two similar types of fresh meat products (poultry and raw pork sausages). We describe a full analysis of this dataset in order to decipher how the spoilage microbial ecology of these two similar products may be shaped differently depending on production parameter characteristics. Methods: Our strategy involved a holistic approach to integrate unsupervised and supervised statistical methods on multivariate data (OTU-based microbial diversity; metabolomic data of volatile organic compounds; sensory measurements; growth parameters), and a specific selection of potential uncontrolled (initial microbiota composition) or controlled (packaging type; lactate concentration) drivers. Results: Our results demonstrate that the initial microbiota, which is shown to be very different between poultry and pork sausages, has a major impact on the spoilage scenarios and on the effect that a downstream parameter such as packaging type has on the overall evolution of the microbial community. Depending on the process, we also show that specific actions on the pork meat (such as deboning and defatting) elicit specific food spoilers such as Dellaglioa algida, which becomes dominant during storage. Finally, ecological network reconstruction allowed us to map six different metabolic pathways involved in the production of volatile organic compounds involved in spoilage. We were able connect them to the different bacterial actors and to the influence of packaging type in an overall view. For instance, our results demonstrate a new role of Vibrionaceae in isopropanol production, and of Latilactobacillus fuchuensis and Lactococcus piscium in methanethiol/disylphide production. We also highlight a possible commensal behavior between Leuconostoc carnosum and Latilactobacillus curvatus around 2,3-butanediol metabolism. Conclusion: We conclude that our holistic approach combined with largescale multi-omic data was a powerful strategy to prioritize the role of production parameters, already known in the literature, that shape the evolution and/or the implementation of different meat spoilage scenarios. [ABSTRACT FROM AUTHOR]

Spore-forming bacteria of the Bacillus subtilis group are responsible for recurrent contamination of processing lines in the food industry which can lead to food spoilage. The persistence of B. subtilis would be due to the high resistance of spores to extreme environmental condition and their propensity to contaminate surfaces. While it is well known that sporulation conditions modulate spore resistance properties, little is known about their effect on surface and adhesion properties. Here, we studied the impact of 13 sporulation conditions on the surface and adhesion properties of B. subtilis 168 spores. We showed that Ca2+ or Mg2+ depletion, lower oxygen availability, acidic pH as well as oxidative stresses during sporulation lead to the release of more hydrophobic and adherent spores. The consequences of these sporulation conditions on crust composition in carbohydrates and proteins were also evaluated. The crust glycans of spores produced in a sporulation medium depleted in Ca2+ or Mg2+ or oxygen-limited conditions were impaired and contained lower amounts of rhamnose and legionaminic acid. In addition, we showed that lower oxygen availability or addition of hydrogen peroxide during sporulation decreases the relative amount of two crust proteins (CgeA and CotY) and the changes observed in these conditions could be due to transcriptional repression of genes involved in crust synthesis in late stationary phase. The fact that sporulation conditions affect the ease with which spores can contaminate surfaces could explain the frequent and recurrent presence of B. subtilis spores in food processing lines. [ABSTRACT FROM AUTHOR]

The prevalence of biofilm-associated microorganisms and the increasing use of ready-to-eat fresh products represent the current duality the food industry must address. Innovative and eco-friendly antibiofilm solutions and appropriate microbiological food control systems are urgently needed to improve food quality and safety. This study aimed to investigate the in vitro combined efficacy of carvacrol with a pre-formed biofilm monolayer of the probiotic Lactiplantibacillus plantarum DSM 20174. The antimicrobial activity of carvacrol against both planktonic and sessile cells of foodborne pathogens and spoilage microorganisms, alone or in the presence of the pre-formed biofilm of L. plantarum, was investigated by culture-based methods along with flow cytometry (FCM) to monitor cells' cultivability and viability. The synergistic action of carvacrol and the pre-formed biofilm of L. plantarum was evaluated in the 96-well plates. The results showed that L. plantarum pre-formed biofilm monolayer enhanced the antimicrobial effect of carvacrol determining a bactericidal action while the treatment alone induced the viable but not culturable (VBNC) cell state only. Furthermore, the great efficacy of the combined treatment allowed the application of a lower concentration of carvacrol (100 ppm) to achieve significant damage in cell viability. In conclusion, the incorporation of carvacrol into the L. plantarum pre-formed biofilm represents a promising alternative for an antimicrobial functionalized ready-to-eat packaging.

BackgroundThe use of omics data for monitoring the microbial flow of fresh meat products along a production line and the development of spoilage prediction tools from these data is a promising but challenging task. In this context, we produced a large multivariate dataset (over 600 samples) obtained on the production lines of two similar types of fresh meat products (poultry and raw pork sausages). We describe a full analysis of this dataset in order to decipher how the spoilage microbial ecology of these two similar products may be shaped differently depending on production parameter characteristics.MethodsOur strategy involved a holistic approach to integrate unsupervised and supervised statistical methods on multivariate data (OTU-based microbial diversity; metabolomic data of volatile organic compounds; sensory measurements; growth parameters), and a specific selection of potential uncontrolled (initial microbiota composition) or controlled (packaging type; lactate concentration) drivers.ResultsOur results demonstrate that the initial microbiota, which is shown to be very different between poultry and pork sausages, has a major impact on the spoilage scenarios and on the effect that a downstream parameter such as packaging type has on the overall evolution of the microbial community. Depending on the process, we also show that specific actions on the pork meat (such as deboning and defatting) elicit specific food spoilers such as Dellaglioa algida, which becomes dominant during storage. Finally, ecological network reconstruction allowed us to map six different metabolic pathways involved in the production of volatile organic compounds involved in spoilage. We were able connect them to the different bacterial actors and to the influence of packaging type in an overall view. For instance, our results demonstrate a new role of Vibrionaceae in isopropanol production, and of Latilactobacillus fuchuensis and Lactococcus piscium in methanethiol/disylphide production. We also highlight a possible commensal behavior between Leuconostoc carnosum and Latilactobacillus curvatus around 2,3-butanediol metabolism.ConclusionWe conclude that our holistic approach combined with large-scale multi-omic data was a powerful strategy to prioritize the role of production parameters, already known in the literature, that shape the evolution and/or the implementation of different meat spoilage scenarios.

Introduction: Macrococcus species have been isolated from a range of mammals and mammal-derived food products. While they are largely considered to be animal commensals, Macrococcus spp. can be opportunistic pathogens in both veterinary and human clinical settings. This study aimed to provide insight into the evolution, population structure, and functional potential of the Macrococcus genus, with an emphasis on antimicrobial resistance (AMR) and virulence potential. Methods: All high-quality, publicly available Macrococcus genomes (n = 104, accessed 27 August 2022), plus six South African genomes sequenced here (two strains from bovine clinical mastitis cases and four strains from beef products), underwent taxonomic assignment (using four different approaches), AMR determinant detection (via AMRFinderPlus), and virulence factor detection (using DIAMOND and the core Virulence Factor Database). Results: Overall, the 110 Macrococcus genomes were of animal commensal, veterinary clinical, food-associated (including food spoilage), and environmental origins; five genomes (4.5%) originated from human clinical cases. Notably, none of the taxonomic assignment methods produced identical results, highlighting the potential for Macrococcus species misidentifications. The most common predicted antimicrobial classes associated with AMR determinants identified across Macrococcus included macrolides, beta-lactams, and aminoglycosides (n = 81, 61, and 44 of 110 genomes; 73.6, 55.5, and 40.0%, respectively). Genes showing homology to Staphylococcus aureus exoenzyme aureolysin were detected across multiple species (using 90% coverage, n ≥ 40 and 77 genomes harboring aureolysin-like genes at 60 and 40% amino acid [AA] identity, respectively). S. aureus Panton-Valentine leucocidin toxinassociated lukF-PV and lukS-PV homologs were identified in eight M. canis genomes (=40% AA identity, >85% coverage). Using a method that delineates populations using recent gene flow (PopCOGenT), two species (M. caseolyticus and M. armenti) were composed of multiple within-species populations. Notably, M. armenti was partitioned into two populations, which differed in functional potential (e.g., one harbored betalactamase family, type II toxin-antitoxin system, and stress response proteins, while the other possessed a Type VII secretion system; PopCOGenT p < 0.05). Discussion: Overall, this study leverages all publicly available Macrococcus genomes in addition to newly sequenced genomes from South Africa to identifygenomic elements associated with AMR or virulence potential, which can be queried in future experiments. [ABSTRACT FROM AUTHOR]

Ensuring a safe and adequate food supply is a cornerstone of human health and food security. However, a significant portion of the food produced for human consumption is wasted annually on a global scale. Reducing harvest and postharvest food waste, waste during food processing, as well as food waste at the consumer level, have been key objectives of improving and maintaining sustainability. These issues can range from damage during processing, handling, and transport, to the use of inappropriate or outdated systems, and storage and packaging-related issues. Microbial growth and (cross)contamination during harvest, processing, and packaging, which causes spoilage and safety issues in both fresh and packaged foods, is an overarching issue contributing to food waste. Microbial causes of food spoilage are typically bacterial or fungal in nature and can impact fresh, processed, and packaged foods. Moreover, spoilage can be influenced by the intrinsic factors of the food (water activity, pH), initial load of the microorganism and its interaction with the surrounding microflora, and external factors such as temperature abuse and food acidity, among others. Considering this multifaceted nature of the food system and the factors driving microbial spoilage, there is an immediate need for the use of novel approaches to predict and potentially prevent the occurrence of such spoilage to minimize food waste at the harvest, post-harvest, processing, and consumer levels. Quantitative microbial spoilage risk assessment (QMSRA) is a predictive framework that analyzes information on microbial behavior under the various conditions encountered within the food ecosystem, while employing a probabilistic approach to account for uncertainty and variability. Widespread adoption of the QMSRA approach could help in predicting and preventing the occurrence of spoilage along the food chain. Alternatively, the use of advanced packaging technologies would serve as a direct prevention strategy, potentially minimizing (cross)contamination and assuring the safe handling of foods, in order to reduce food waste at the post-harvest and retail stages. Finally, increasing transparency and consumer knowledge regarding food date labels, which typically are indicators of food quality rather than food safety, could also contribute to reduced food waste at the consumer level. The objective of this review is to highlight the impact of microbial spoilage and (cross)contamination events on food loss and waste. The review also discusses some novel methods to mitigate food spoilage and food loss and waste, and ensure the quality and safety of our food supply. [ABSTRACT FROM AUTHOR]

Geobacillus stearothermophilus is a highly thermophilic, spore-forming Grampositive bacterium that causes flat sour spoilage in low-acid canned foods. To address this problem, we isolated G. stearothermophilus-infecting phage GR1 from the soil and characterized its endolysin LysGR1. Phage GR1 belongs to the Siphoviridae family and possesses a genome of 79,387 DNA bps with 108 putative open reading frames. GR1 demonstrated a very low degree of homology to previously reported phages, indicating that it is novel. The endolysin of GR1 (LysGR1) contains an N-terminal amidase domain as an enzymatically active domain (EAD) and two C-terminal LysM domains as a cell wall binding domain (CBD). Although GR1 is specific to certain strains of G. stearothermophilus, LysGR1 showed a much broader lytic range, killing all the tested strains of G. stearothermophilus and several foodborne pathogens, such as Clostridium perfringens, Listeria monocytogenes, and Escherichia coli O157:H7. LysGR1&lowbar; EAD, alone, also exhibits lytic activity against a wide range of bacteria, including Bacillus cereus, which is not terminated by a full-length endolysin. Both LysGR1 and its EAD effectively remove the G. stearothermophilus biofilms and are highly thermostable, retaining about 70% of their lytic activity after a 15-min incubation at 70°C. Considering the high thermal stability, broad lytic activity, and biofilm reduction efficacy of LysGR1 and its EAD, we hypothesize that these enzymes could act as promising biocontrol agents against G. stearothermophilus and as foodborne pathogens. [ABSTRACT FROM AUTHOR]

Biofilm formation in food processing environment and within equipment increases the risk of product spoilage and contamination with pathogens. Cleaning-inplace (CIP) operations are useful in removing soils and in sanitizing processing equipment, including eliminating biofilms. However, CIP is a resource-intensive process, particularly in the usage of chemical detergents, heat, and sanitizers. The current study was initiated to investigate the feasibility of integrating ozone into CIP operations to facilitate the elimination of Pseudomonas biofilm, with the longterm goal of decreasing the dependance on conventional cleaning and sanitizing reagents. To investigate integrating ozone into CIP, a robust biofilm of Pseudomonas fluorescens was developed on a pilot-scale food processing equipment after 2 days of incubation in 10% skim milk (skim milk-water mixture, 1:9v/v) under stagnant conditions, followed by additional 5 days of circulation while feeding 10% fresh skim milk. CIP was applied using water prerinse at 22--25°C, alkaline cleaning with 0.2% potassium hydroxide at 50°C, and a final water rinse. These CIP operations reduced planktonic cell populations below the detection method's limit but did not fully remove P. fluorescens biofilm from either smooth or rough surfaces of the processing equipment. When the CIP process was followed by application of an aqueous ozone step (10ppm for 10 min), the treatment reduced biofilm cell population, on smooth and rough surfaces, below the recovery method's detection limit (0.9 and 1.4 log CFU/ 100cm2, respectively). These findings demonstrate the utility of ozone-assisted CIP in eliminating microbial biofilms on processing equipment, but further research is needed to optimize the use of cleaning agents and the application of ozone. [ABSTRACT FROM AUTHOR]

Ensuring a safe and adequate food supply is a cornerstone of human health and food security. However, a significant portion of the food produced for human consumption is wasted annually on a global scale. Reducing harvest and postharvest food waste, waste during food processing, as well as food waste at the consumer level, have been key objectives of improving and maintaining sustainability. These issues can range from damage during processing, handling, and transport, to the use of inappropriate or outdated systems, and storage and packaging-related issues. Microbial growth and (cross)contamination during harvest, processing, and packaging, which causes spoilage and safety issues in both fresh and packaged foods, is an overarching issue contributing to food waste. Microbial causes of food spoilage are typically bacterial or fungal in nature and can impact fresh, processed, and packaged foods. Moreover, spoilage can be influenced by the intrinsic factors of the food (water activity, pH), initial load of the microorganism and its interaction with the surrounding microflora, and external factors such as temperature abuse and food acidity, among others. Considering this multifaceted nature of the food system and the factors driving microbial spoilage, there is an immediate need for the use of novel approaches to predict and potentially prevent the occurrence of such spoilage to minimize food waste at the harvest, post-harvest, processing, and consumer levels. Quantitative microbial spoilage risk assessment (QMSRA) is a predictive framework that analyzes information on microbial behavior under the various conditions encountered within the food ecosystem, while employing a probabilistic approach to account for uncertainty and variability. Widespread adoption of the QMSRA approach could help in predicting and preventing the occurrence of spoilage along the food chain. Alternatively, the use of advanced packaging technologies would serve as a direct prevention strategy, potentially minimizing (cross)contamination and assuring the safe handling of foods, in order to reduce food waste at the post-harvest and retail stages. Finally, increasing transparency and consumer knowledge regarding food date labels, which typically are indicators of food quality rather than food safety, could also contribute to reduced food waste at the consumer level. The objective of this review is to highlight the impact of microbial spoilage and (cross)contamination events on food loss and waste. The review also discusses some novel methods to mitigate food spoilage and food loss and waste, and ensure the quality and safety of our food supply.

Biofilm formation in food processing environment and within equipment increases the risk of product spoilage and contamination with pathogens. Cleaning-in-place (CIP) operations are useful in removing soils and in sanitizing processing equipment, including eliminating biofilms. However, CIP is a resource-intensive process, particularly in the usage of chemical detergents, heat, and sanitizers. The current study was initiated to investigate the feasibility of integrating ozone into CIP operations to facilitate the elimination of Pseudomonas biofilm, with the long-term goal of decreasing the dependance on conventional cleaning and sanitizing reagents. To investigate integrating ozone into CIP, a robust biofilm of Pseudomonas fluorescens was developed on a pilot-scale food processing equipment after 2 days of incubation in 10% skim milk (skim milk-water mixture, 1:9 v/v) under stagnant conditions, followed by additional 5 days of circulation while feeding 10% fresh skim milk. CIP was applied using water prerinse at 22–25°C, alkaline cleaning with 0.2% potassium hydroxide at 50°C, and a final water rinse. These CIP operations reduced planktonic cell populations below the detection method’s limit but did not fully remove P. fluorescens biofilm from either smooth or rough surfaces of the processing equipment. When the CIP process was followed by application of an aqueous ozone step (10 ppm for 10 min), the treatment reduced biofilm cell population, on smooth and rough surfaces, below the recovery method’s detection limit (0.9 and 1.4 log CFU/ 100 cm2, respectively). These findings demonstrate the utility of ozone-assisted CIP in eliminating microbial biofilms on processing equipment, but further research is needed to optimize the use of cleaning agents and the application of ozone.

Spices are typically grown in climates that support the growth of toxigenic fungi and the production of mycotoxins. The Aspergilli described in this study, as well as the sterigmatocystin (STC) detected, are causes for concern due to their potential to induce food poisoning. One of the most wellknown producers of the carcinogenic STC is Aspergillus nidulans. This research explores the occurrence of STC-producing fungi in Foeniculum vulgare, a spice that is marketed in India and other parts of the world. This innovative study details the mycotoxigenic potential of five Aspergilli belonging to Section Nidulantes, namely Aspergillus latus (02 isolates), Emericella quadrilineata (02 isolates), and Aspergillus nidulans (01 isolate), with respect to STC contamination. These five isolates of Aspergilli were screened to produce STC on yeast extract sucrose (YES) medium in a controlled environment with regard to light, temperature, pH, and humidity, among other variables. The expression patterns of regulatory genes, namely, aflR, laeA, pacC, fluG, flbA, pksA, and mtfA were studied on the Czapek-Dox agar (CDA) medium. STC biosynthesis by the test isolates was done in potato dextrose broth (PDB) under optimum conditions, followed by the extraction and purification of the broth using ethyl acetate. High-performance liquid chromatography (HPLC) with an ultraviolet (UV) detector was utilized to detect compounds in eluted samples. F. vulgare contains Aspergilli that have been shown to have mycotoxigenic potential, which can accumulate in the spice during its active growth and thereby cause the elaboration of mycotoxins. [ABSTRACT FROM AUTHOR]

Leuconostoc is a genus of saccharolytic heterofermentative lactic acid bacteria that inhabit plant-derived matrices and a variety of fermented foods (dairy products, dough, milk, vegetables, and meats), contributing to desired fermentation processes or playing a role in food spoilage. At present, the genus encompasses 17 recognized species. In total, 216 deposited genome sequences of Leuconostoc were analyzed, to check the delineation of species and to infer their evolutive genealogy utilizing a minimum evolution tree of Average Nucleotide Identity (ANI) and the core genome alignment. Phylogenomic relationships were compared to those obtained from the analysis of 16S rRNA, pheS, and rpoA genes. All the phylograms were subjected to split decomposition analysis and their topologies were compared to check the ambiguities in the inferred phylogenesis. Theminimum evolution ANI tree exhibited the most similar topology with the core genome tree, while single gene trees were less adherent and provided a weaker phylogenetic signal. In particular, the 16S rRNA gene failed to resolve several bifurcations and Leuconostoc species. Based on an ANI threshold of 95%, the organization of the genus Leuconostoc could be amended, redefining the boundaries of the species L. inhae, L. falkenbergense, L. gelidum, L. lactis, L. mesenteroides, and L. pseudomesenteroides. Two strains currently recognized as L. mesenteroides were split into a separate lineage representing a putative species (G16), phylogenetically related to both L. mesenteroides (G18) and L. suionicum (G17). Differences among the four subspecies of L. mesenteroides were not pinpointed by ANI or by the conserved genes. The strains of L. pseudomesenteroides were ascribed to two putative species, G13 and G14, the former including also all the strains presently belonging to L. falkenbergense. L. lactis was split into two phylogenetically related lineages, G9 and G10, putatively corresponding to separate species and both including subgroups that may correspond to subspecies. The species L. gelidum and L. gasicomitatum were closely related but separated into different species, the latter including also L. inhae strains. These results, integrating information of ANI, core genome, and housekeeping genes, complemented the taxonomic delineation with solid information on the phylogenetic lineages evolved within the genus Leuconostoc. [ABSTRACT FROM AUTHOR]

Spices are typically grown in climates that support the growth of toxigenic fungi and the production of mycotoxins. The Aspergilli described in this study, as well as the sterigmatocystin (STC) detected, are causes for concern due to their potential to induce food poisoning. One of the most well-known producers of the carcinogenic STC is Aspergillus nidulans. This research explores the occurrence of STC-producing fungi in Foeniculum vulgare, a spice that is marketed in India and other parts of the world. This innovative study details the mycotoxigenic potential of five Aspergilli belonging to Section Nidulantes, namely Aspergillus latus (02 isolates), Emericella quadrilineata (02 isolates), and Aspergillus nidulans (01 isolate), with respect to STC contamination. These five isolates of Aspergilli were screened to produce STC on yeast extract sucrose (YES) medium in a controlled environment with regard to light, temperature, pH, and humidity, among other variables. The expression patterns of regulatory genes, namely, aflR, laeA, pacC, fluG, flbA, pksA, and mtfA were studied on the Czapek–Dox agar (CDA) medium. STC biosynthesis by the test isolates was done in potato dextrose broth (PDB) under optimum conditions, followed by the extraction and purification of the broth using ethyl acetate. High-performance liquid chromatography (HPLC) with an ultraviolet (UV) detector was utilized to detect compounds in eluted samples. F. vulgare contains Aspergilli that have been shown to have mycotoxigenic potential, which can accumulate in the spice during its active growth and thereby cause the elaboration of mycotoxins.

Microorganism contamination is one of the most important factors affecting the spoilage and food safety of Manila clams. This study aimed to gain insights into bacterial composition and the dynamic change of bacterial communities on retailed Manila clam during refrigerated storage within the edible period. High-throughput sequencing was conducted to monitor the bacterial population with the prolongation of storage time of Day 0, Day 1, and Day 3. Result demonstrated that phyla of Proteobacteria, Actinobacteriota, Acidobacteriota, and Chloroflexi composed the majority of bacterial communities during the whole observation process. Furthermore, the increase of Proteobacteria showed a positive correlation with the storage time, whereas Acidobacteriota and Chloroflexi continued to decline in storage. For genus annotation, none of genus obtained dominant population in storage. From Day 0 to Day 1, the genera of Streptomyces , Bradyrhizobium , and Mycobacterium significantly increased; meanwhile, 12 genera significantly decreased. Compared with samples at Day 0, a total of 15 genera significantly decreased with the reduced proportion ranging from 0.50 to 4.40% at Day 3. At the end of the storage, the genus Crossiella became the most redundant population. Both the richness and diversity decreased at the start of storage at Day 1, and then slightly increased at Day 3 was observed. Based on the result in this study, strategy targeting the increased bacteria could be tested to improve the consumption quality and safety of refrigerated clam. [ABSTRACT FROM AUTHOR]

Aspergillus westerdijkiae , the producer of ochratoxin A (OTA), which is of worldwide concern, is an import fungal species in agriculture, food, and industry. Here, we got the uridine auxotrophic mutant of A. westerdijkiae by deleting AwpyrG. The Δ AwpyrG could be used for bio-transformation with exogenous AfpyrG expression cassette as a selection marker. In order to enhance the efficiency of gene targeting, Awku70 and Awlig4 were homologously deleted from Δ AwpyrG. The efficiencies of homologous replacement for Δ Awku70 and Δ Awlig4 were 95.7 and 87.0% in the deletion of AwAreA , respectively, demonstrating a drastic increase from 4.3% of the wild type (WT) strain. Furthermore, the function of AwAreA was identified with AwAreA deletion mutant and the control strain Δ Awku70. AwAreA regulated the growth and conidiation of A. westerdijkiae in response to nitrogen sources. The concentration of OTA for Δ Awku70 was in the range of 19.4 to 186.9 ng/cm2 on all kinds of nitrogen sources. The OTA production influenced by the deletion of AwAreA was different based on nitrogen sources. Pathogenicity assays on pears, grapes, salted meat, and cheese showed that AwAreA acted as a negative regulator in the infection of food substrates. Therefore, the genetic methods and engineered strains enable us to substantially expand the use of A. westerdijkiae , one of more than twenty OTA-producing fungi, in the study of mycotoxin biosynthesis and regulation, and consequently to aim at providing new ways for controlling this pathogen. [ABSTRACT FROM AUTHOR]

Safe and hygienic water distribution is essential for maintaining product quality and safety. It is known that biofilms alter the appearance and microbial quality of water along the distribution chain. Yet, biofilms in water hoses throughout the food processing environment have not been investigated in detail. Here, microbial communities from water hoses and other environmental sites in contact with water, in addition to the source water itself, were studied in the meat processing environment. Biofilms were present in all water hoses as determined by the presence of bacterial DNA and biofilm matrix components (carbohydrates, extracellular DNA, and proteins). The microbial community of the biofilms was dominated by Proteobacteria , represented mainly by Comamonadaceae and Pseudoxanthomonas. Moreover, genera that are associated with an intracellular lifestyle (e.g., Neochlamydia and Legionella) were present. Overall, the microbial community of biofilms was less diverse than the water microbial community, while those from the different sample sites were distinct from each other. Indeed, only a few phyla were shared between the water hose biofilm and the source water or associated environmental samples. This study provides first insights towards understanding the microbiota of water hose biofilms in the food processing environment. [ABSTRACT FROM AUTHOR]

Safe and hygienic water distribution is essential for maintaining product quality and safety. It is known that biofilms alter the appearance and microbial quality of water along the distribution chain. Yet, biofilms in water hoses throughout the food processing environment have not been investigated in detail. Here, microbial communities from water hoses and other environmental sites in contact with water, in addition to the source water itself, were studied in the meat processing environment. Biofilms were present in all water hoses as determined by the presence of bacterial DNA and biofilm matrix components (carbohydrates, extracellular DNA, and proteins). The microbial community of the biofilms was dominated by Proteobacteria, represented mainly by Comamonadaceae and Pseudoxanthomonas. Moreover, genera that are associated with an intracellular lifestyle (e.g., Neochlamydia and Legionella) were present. Overall, the microbial community of biofilms was less diverse than the water microbial community, while those from the different sample sites were distinct from each other. Indeed, only a few phyla were shared between the water hose biofilm and the source water or associated environmental samples. This study provides first insights towards understanding the microbiota of water hose biofilms in the food processing environment.

The aim of this work was to study the value of the main allergen Asp n 3 of Aspergillus niger as a molecular marker of allergenicity and pathogenicity with the potential to be used in the identification of A. niger as a contaminant and cause of spoilage of Mangifera indica. Real-time polymerase chain reaction (RT-PCR) was used for the amplification of Asp n 3 gene. Two pairs of primers were designed: one for the amplification of the entire sequence and another one for the amplification of the most conserved region of this peroxisomal protein. The presence of A. niger was demonstrated by the early detection of the allergenic protein Asp n 3 coding gene, which could be considered a species-specific marker. The use of primers designed based on the conserved region of the Asp n 3 encoding gene allowed us to identify the presence of the closely related fungal species Aspergillus fumigatus by detecting Asp n 3 homologous protein, which can be cross-reactive. The use of conserved segments of the Asp n 3 gene or its entire sequence allows us to detect phylogenetically closely related species within the Aspergilaceae family or to identify species-specific contaminating fungi.

Sausage is a highly perishable food with unique spoilage characteristics primarily because of its specific means of production. The quality of sausage during storage is determined by its microbial and metabolite changes. This study developed a preservative-free low-temperature sausage model and coated it with natural casing. We characterized the microbiota and non-volatile metabolites in the sausage after storage at 20°C for up to 12 days. Bacillus velezensis was the most prevalent species observed after 4 days. Lipids and lipid-like molecules, organoheterocyclic compounds, and organic acids and their derivatives were the primary non-volatile metabolites. The key non-volatile compounds were mainly involved in protein catabolism and β-lipid oxidation. These findings provide useful information for the optimization of sausage storage conditions. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop a rapid and accurate PMA-qPCR method to quantify viable Brochothrix thermosphacta in cold-smoked salmon. B. thermosphacta is one of the main food spoilage bacteria. Among seafood products, cold-smoked salmon is particularly impacted by B. thermosphacta spoilage. Specific and sensitive tools that detect and quantify this bacterium in food products are very useful. The culture method commonly used to quantify B. thermosphacta is time-consuming and can underestimate cells in a viable but not immediately culturable state. We designed a new PCR primer set from the single-copy rpoC gene. QPCR efficiency and specificity were compared with two other published primer sets targeting the rpoC and rpoB genes. The viability dyes PMA or PMAxx were combined with qPCR and compared with these primer sets on viable and dead B. thermosphacta cells in BHI broth and smoked salmon tissue homogenate (SSTH). The three primer sets displayed similar specificity and efficiency. The efficiency of new designed rpoC qPCR on viable B. thermosphacta cells in SSTH was 103.50%, with a linear determination coefficient (r2) of 0.998 and a limit of detection of 4.04 log CFU/g. Using the three primer sets on viable cells, no significant difference was observed between cells treated or untreated with PMA or PMAxx. When dead cells were used, both viability dyes suppressed DNA amplification. Nevertheless, our results did not highlight any difference between PMAxx and PMA in their efficiency to discriminate viable from unviable B. thermosphacta cells in cold-smoked salmon. Thus, this study presents a rapid, specific and efficient rpoC -PMA-qPCR method validated in cold-smoked salmon to quantify viable B. thermosphacta in foods. [ABSTRACT FROM AUTHOR]

Pediococcus acidilactici may significantly reduce the pH-value, and thus has different influence, including serving as a probiotic in human microbiota but a spoilage in human food as it could change the flavor. Pediococcus acidilactici is also capable of entering into the viable but non-culturable (VBNC) state causing false negative results of standard culture-based detection method. Thus, development of detection method for VBNC state P. acidilactici is of great significance. In this study, propidium monoazide (PMA) combined with cross priming amplification (CPA) was developed to detect the VBNC cells of P. acidilactici and applied on the detection in different systems. With detection limit of 104 cells/ml, high sensitivity, and 100% specificity, PMA-CPA can successfully detect VBNC cells of P. acidilactici and be applied in with high robustness. [ABSTRACT FROM AUTHOR]

The aim of this work was to study the value of the main allergen Asp n 3 of Aspergillus niger as a molecular marker of allergenicity and pathogenicity with the potential to be used in the identification of A. niger as a contaminant and cause of spoilage of Mangifera indica. Real-time polymerase chain reaction (RT-PCR) was used for the amplification of Asp n 3 gene. Two pairs of primers were designed: one for the amplification of the entire sequence and another one for the amplification of the most conserved region of this peroxisomal protein. The presence of A. niger was demonstrated by the early detection of the allergenic protein Asp n 3 coding gene, which could be considered a species-specific marker. The use of primers designed based on the conserved region of the Asp n 3 encoding gene allowed us to identify the presence of the closely related fungal species Aspergillus fumigatus by detecting Asp n 3 homologous protein, which can be cross-reactive. The use of conserved segments of the Asp n 3 gene or its entire sequence allows us to detect phylogenetically closely related species within the Aspergilaceae family or to identify species-specific contaminating fungi. [ABSTRACT FROM AUTHOR]

Lipopeptide biosurfactants produced by Bacillus sp. were assessed regarding their antimicrobial activity against foodborne pathogenic and food spoilage microorganisms. Both Gram-positive and Gram-negative bacteria were found not to be susceptible to these lipopeptides. However, mycosubtilin and mycosubtilin/surfactin mixtures were very active against the filamentous fungi Paecilomyces variotti and Byssochlamys fulva, with minimum inhibitory concentrations (MICs) of 1–16 mg/L. They were also active against Candida krusei, MIC = 16–64 mg/L. Moreover it was found that the antifungal activity of these lipopeptides was not affected by differences in isoform composition and/or purity. Furthermore their cytotoxicity tested on two different cell lines mimicking ingestion and detoxification was comparable to those of approved food preservatives such as nisin. Overall, for the first time here mycosubtilin and mycosubtilin/surfactin mixtures were found to have high antifungal activity against food relevant fungi at concentrations lower than their toxicity level hence, suggesting their application for extending the shelf-life of products susceptible to these moulds. In addition combining nisin with mycosubtilin or mycosubtiliin/surfactin mixtures proved to be an effective approach to produce antimicrobials with broader spectrum of action.

Lipopeptide biosurfactants produced by Bacillus sp. were assessed regarding their antimicrobial activity against foodborne pathogenic and food spoilage microorganisms. Both Gram-positive and Gram-negative bacteria were found not to be susceptible to these lipopeptides. However, mycosubtilin and mycosubtilin/surfactin mixtures were very active against the filamentous fungi Paecilomyces variotti and Byssochlamys fulva , with minimum inhibitory concentrations (MICs) of 1–16 mg/L. They were also active against Candida krusei , MIC = 16–64 mg/L. Moreover it was found that the antifungal activity of these lipopeptides was not affected by differences in isoform composition and/or purity. Furthermore their cytotoxicity tested on two different cell lines mimicking ingestion and detoxification was comparable to those of approved food preservatives such as nisin. Overall, for the first time here mycosubtilin and mycosubtilin/surfactin mixtures were found to have high antifungal activity against food relevant fungi at concentrations lower than their toxicity level hence, suggesting their application for extending the shelf-life of products susceptible to these moulds. In addition combining nisin with mycosubtilin or mycosubtiliin/surfactin mixtures proved to be an effective approach to produce antimicrobials with broader spectrum of action. [ABSTRACT FROM AUTHOR]

Enterococci are a diverse group of Gram-positive, lactic acid bacteria (LAB). They are found in many environments, including fermented foods, in which they could constitute a health threat since they produce biogenic amines, which consumption can lead to intoxication. Moreover, enterococci has also emerged as an important hospital-acquired pathogens via its acquisition of antimicrobial resistance. Bacteriophages possess features that make them optimal biotechnological weapons for controlling bacterial growth in disease and food spoilage contexts. However, no silver bullet bacteriophage exists that can eliminate all the undesirable bacteria in a complex environment. Rather, a combination of phages with different host ranges would be required which implies the need for large collections of diverse phages. This work reports the isolation of several Enterococcus faecalis -infecting bacteriophages from different types of cheese, along with the range of E. faecalis strains of diverse origin (from dairy to clinical environments) they are able to infect. The isolated phages showed a large diversity regarding the number and origin of strains they infect. Some of these phages were subjected to morphological and genomic characterization which confirmed their diversity and showed they belong to different families and genera. The present findings increase the potential arsenal for the bacteriophage-based biocontrol of harmful E. faecalis populations. [ABSTRACT FROM AUTHOR]

Microbes from the three domains of life, Bacteria, Archaea, and Eukarya, share the need to sense and respond to changes in the external and internal concentrations of protons. When the proton concentration is high, acidic conditions prevail and cells must respond appropriately to ensure that macromolecules and metabolic processes are sufficiently protected to sustain life. While, we have learned much in recent decades about the mechanisms that microbes use to cope with acid, including the unique challenges presented by organic acids, there is still much to be gained from developing a deeper understanding of the effects and responses to acid in microbes. In this perspective article, we survey the key molecular mechanisms known to be important for microbial survival during acid stress and discuss how this knowledge might be relevant to microbe-based applications and processes that are consequential for humans. We discuss the research approaches that have been taken to investigate the problem and highlight promising new avenues. We discuss the influence of acid on pathogens during the course of infections and highlight the potential of using organic acids in treatments for some types of infection. We explore the influence of acid stress on photosynthetic microbes, and on biotechnological and industrial processes, including those needed to produce organic acids. We highlight the importance of understanding acid stress in controlling spoilage and pathogenic microbes in the food chain. Finally, we invite colleagues with an interest in microbial responses to low pH to participate in the EU-funded COST Action network called EuroMicropH and contribute to a comprehensive database of literature on this topic that we are making publicly available.

Worldwide, the dairy sector remains of vital importance for food production despite severe environmental constraints. The production and handling conditions of milk, a rich medium, promote inevitably the entrance of microbial contaminants, with notable impact on the quality and safety of raw milk and dairy products. Moreover, the persistence of high concentrations of microorganisms (especially bacteria and bacterial spores) in biofilms (BFs) present on dairy equipment or environments constitutes an additional major source of milk contamination from pre- to post-processing stages: in dairies, BFs represent a major concern regarding the risks of disease outbreaks and are often associated with significant economic losses. One consumption trend toward “raw or low-processed foods” combined with current trends in food production systems, which tend to have more automation and longer processing runs with simultaneously more stringent microbiological requirements, necessitate the implementation of new and obligatory sustainable strategies to respond to new challenges regarding food safety. Here, in light of studies, performed mainly with raw milk, that considered dominant “planktonic” conditions, we reexamine the changes triggered by cold storage alone or combined with nitrogen gas (N2) flushing on bacterial populations and discuss how the observed benefits of the treatment could also contribute to limiting BF formation in dairies.

Worldwide, Shewanella putrefaciens is the predominant seafood spoilage microorganism during cold storage. This bacterium can attach to biotic/abiotic surfaces to form biofilms which contribute to seafood quality degradation and shelf-life reduction. The mechanism of S. putrefaciens biofilm formation is not yet described. Crystal violet staining in combination with confocal laser scanning microscopy (CLSM) was used to study the sequence of events leading to the establishment of a mature biofilm at 4, 15, and 30°C. In addition, the main chemical constituents of the mature biofilm were determined by Raman spectroscopy (RM), whereas, comparative proteomic analysis was used to quantify changes in metabolic pathways and to find out underlying protein determinants. The physical dimensions of the mature biofilm, i.e., biomass, biovolume, and mean thickness, were higher at 4°C when compared to 15 and 30°C. The variations of proteins measured by RM confirmed the importance of proteins during the formation of a mature biofilm. Comparative proteomic analysis showed that siderophore and iron chelate transport proteins were down-regulated during mature biofilm formation. The down-regulated aforementioned proteins are involved in promoting iron storage in response to a higher demand for metabolic energy, whereas, the upregulated proteins of the sulfur relay system, pyrimidine metabolism, and purine metabolism are related to bacterial adaptability. Synthesis of proteins related to cold stress was increased and proteins involved in aminoacyl-tRNA biosynthesis were up-regulated, whereas, proteins involved in aminopeptidase activity were down-regulated. Proteolysis to scavenge energy was reduced as proteins involved in pyrophosphatase activity were up-regulated. Also extracellular eDNA was found which may play an important role in maintaining the stability of mature S. putrefaciens biofilm structures under cold stress. This work provides a better understanding of the role of proteins in mature biofilms. In addition, the biofilm formation mechanism of a psychrotrophic spoilage bacterial species at low temperature is explored, which may contribute to generating biofilm controlling strategies during seafood preservation and processing.

Microbes from the three domains of life, Bacteria , Archaea , and Eukarya , share the need to sense and respond to changes in the external and internal concentrations of protons. When the proton concentration is high, acidic conditions prevail and cells must respond appropriately to ensure that macromolecules and metabolic processes are sufficiently protected to sustain life. While, we have learned much in recent decades about the mechanisms that microbes use to cope with acid, including the unique challenges presented by organic acids, there is still much to be gained from developing a deeper understanding of the effects and responses to acid in microbes. In this perspective article, we survey the key molecular mechanisms known to be important for microbial survival during acid stress and discuss how this knowledge might be relevant to microbe-based applications and processes that are consequential for humans. We discuss the research approaches that have been taken to investigate the problem and highlight promising new avenues. We discuss the influence of acid on pathogens during the course of infections and highlight the potential of using organic acids in treatments for some types of infection. We explore the influence of acid stress on photosynthetic microbes, and on biotechnological and industrial processes, including those needed to produce organic acids. We highlight the importance of understanding acid stress in controlling spoilage and pathogenic microbes in the food chain. Finally, we invite colleagues with an interest in microbial responses to low pH to participate in the EU-funded COST Action network called EuroMicropH and contribute to a comprehensive database of literature on this topic that we are making publicly available. [ABSTRACT FROM AUTHOR]

Worldwide, the dairy sector remains of vital importance for food production despite severe environmental constraints. The production and handling conditions of milk, a rich medium, promote inevitably the entrance of microbial contaminants, with notable impact on the quality and safety of raw milk and dairy products. Moreover, the persistence of high concentrations of microorganisms (especially bacteria and bacterial spores) in biofilms (BFs) present on dairy equipment or environments constitutes an additional major source of milk contamination from pre- to post-processing stages: in dairies, BFs represent a major concern regarding the risks of disease outbreaks and are often associated with significant economic losses. One consumption trend toward "raw or low-processed foods" combined with current trends in food production systems, which tend to have more automation and longer processing runs with simultaneously more stringent microbiological requirements, necessitate the implementation of new and obligatory sustainable strategies to respond to new challenges regarding food safety. Here, in light of studies, performed mainly with raw milk, that considered dominant "planktonic" conditions, we reexamine the changes triggered by cold storage alone or combined with nitrogen gas (N2) flushing on bacterial populations and discuss how the observed benefits of the treatment could also contribute to limiting BF formation in dairies. [ABSTRACT FROM AUTHOR]

Sesotho is an indigenous cereal-based fermented drink traditionally produced in the mountain kingdom of Lesotho, Southern Africa. The present study sought to examine the microbial (bacterial and fungal) community composition of Sesotho at five fermentation stages in five different locations. Using culture-independent (Illumina sequencing) techniques it was found that the bacterial communities followed similar successional patterns during the fermentation processes, regardless of geographical location and recipe variation between breweries. The most abundant bacterial taxa belonged to the phyla Firmicutes (66.2% of the reads on average) and Proteobacteria (22.1%); the families Lactobacillaceae (54.9%), Enterobacteriaceae (14.4%) and Leoconostrocaceae (8.1%); and the genera Lactobacillus (54%), Leuconostoc (10.7%), Leptotrichia (8.5%), and Weissella (5.5%). Most fungal taxa were from the phyla Ascomycota (60.7%) and Mucoromycota (25.3%); the families Rhizopodaceae (25.3%), Nectriaceae (24.2%), Saccharomycetaceae (16%) and Aspergillaceae (6.7%); and the genera Rhizopus (25.3%), Saccharomyces (9.6%), and Aspergillus (2.5%). Lactic acid bacteria (LAB) such as Enterococcus , Pediococcus , Lactobacillus , Leuconostoc , and Wiesella ; as well as yeasts belonging to the genus Saccharomyces , were dominant in all breweries during the production of Sesotho. Several pathogenic and food spoilage microorganisms (e.g., Escherichia , Shigella , Klebsiella , etc.) were also present, but the study demonstrated the safety potential of the Sesotho fermentation process, as these microbial groups decline throughout Sesotho production. The functional profiles of the different brewing steps showed that the process is dominated by chemoheterotrophic and fermentative metabolisms. This study reveals, for the first time, the complex microbial dynamics that occur during Sesotho production. [ABSTRACT FROM AUTHOR]

Worldwide, Shewanella putrefaciens is the predominant seafood spoilage microorganism during cold storage. This bacterium can attach to biotic/abiotic surfaces to form biofilms which contribute to seafood quality degradation and shelf-life reduction. The mechanism of S. putrefaciens biofilm formation is not yet described. Crystal violet staining in combination with confocal laser scanning microscopy (CLSM) was used to study the sequence of events leading to the establishment of a mature biofilm at 4, 15, and 30°C. In addition, the main chemical constituents of the mature biofilm were determined by Raman spectroscopy (RM), whereas, comparative proteomic analysis was used to quantify changes in metabolic pathways and to find out underlying protein determinants. The physical dimensions of the mature biofilm, i.e., biomass, biovolume, and mean thickness, were higher at 4°C when compared to 15 and 30°C. The variations of proteins measured by RM confirmed the importance of proteins during the formation of a mature biofilm. Comparative proteomic analysis showed that siderophore and iron chelate transport proteins were down-regulated during mature biofilm formation. The down-regulated aforementioned proteins are involved in promoting iron storage in response to a higher demand for metabolic energy, whereas, the upregulated proteins of the sulfur relay system, pyrimidine metabolism, and purine metabolism are related to bacterial adaptability. Synthesis of proteins related to cold stress was increased and proteins involved in aminoacyl-tRNA biosynthesis were up-regulated, whereas, proteins involved in aminopeptidase activity were down-regulated. Proteolysis to scavenge energy was reduced as proteins involved in pyrophosphatase activity were up-regulated. Also extracellular eDNA was found which may play an important role in maintaining the stability of mature S. putrefaciens biofilm structures under cold stress. This work provides a better understanding of the role of proteins in mature biofilms. In addition, the biofilm formation mechanism of a psychrotrophic spoilage bacterial species at low temperature is explored, which may contribute to generating biofilm controlling strategies during seafood preservation and processing. [ABSTRACT FROM AUTHOR]

The osmotolerant Zygosaccharomyces rouxii is known for its trait to survive in extreme high sugar environments. This ability determines its role in the fermentation process and leads to yeast spoilage in the food industry. However, our knowledge of the gene expression in response to high sugar stress remains limited. Here, we conducted RNA-sequencing (RNA-seq) under different sugar concentrations of the spoilage yeast, Z. rouxii , which exhibit extremely high tolerance to sugar stress. The obtained differentially expressed genes (DEGs) are significantly different to that of the Saccharomyces cerevisiae , which is sensitive to extreme high sugar stress. Most of the DEGs participated in the "glucan synthesis," "transmembrane transport," "ribosome," etc. In this work, we also demonstrated that the gene ZYRO0B03476g (ZrKAR2) encoding Kar2p can significantly affect the growth of Z. rouxii under high sugar stress. In addition, we combined with a previous study on the genome sequence of Z. rouxii , indicating that several gene families contain significantly more gene copies in the Z. rouxii lineage, which involved in tolerance to sugar stress. Our results provide a gene insight for understanding the high sugar tolerance trait, which may impact food and biotechnological industries and improve the osmotolerance in other organisms. [ABSTRACT FROM AUTHOR]

Bacterial spores are extremely resistant life-forms that play an important role in food spoilage and foodborne disease. The return of spores to a vegetative cell state is a three-step process, these being activation, germination, and emergence. High-pressure (HP) processing is known to induce germination in part of the spore population and even to inactivate a high number of Bacillus spores when combined with other mild treatments such as the addition of nisin. The aim of the present work was to investigate the mechanisms involved in the sensitization of spores to nisin following HP treatment at ambient temperature or with moderate heating leading to a heterogeneous spore response. Bacillus subtilis spores were subjected to HP treatment at 500 MPa at 20 and 50°C. The physiological state of different subpopulations was characterized. Then Fourier transform infrared (FTIR) microspectroscopy coupled to a synchrotron infrared source was used to explore the heterogeneity of the biochemical signatures of the spores after the same HP treatments. Our results confirm that HP at 50°C induces the germination of a large proportion of the spore population. HP treatment at 20°C generated a subpopulation of ungerminated spores reversibly sensitized to the presence of nisin in their growth medium. Regarding infrared spectra of individual spores, spores treated by HP at 50°C and germinated spores had similar spectral signatures involving the same structural properties. However, after HP was performed at 20°C, two groups of spores were distinguished; one of these groups was clearly identified as germinated spores. The second group displayed a unique spectral signature, with shifts in the spectral bands corresponding to changes in membrane fluidity. Besides, spores spectra in the amide region could be divided into several groups close to spectral properties of dormant, germinated, or inactivated spores. The part of the spectra corresponding to α-helix and β-sheet-structures contribute mainly to the spectral variation between spores treated by HP at 20°C and other populations. These changes in the lipid and amide regions could be the signature of reversible changes linked to spore activation. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop a potential microbial preservative to prevent the growth of fungi in food. The isolate ZC-G-5 showed strong antifungal activity against food spoilage fungi and Streptomyces albulus was identified on the basis of morphologic, culture, and 16S rDNA sequence analyses. The active metabolite was elucidated as tetramycin A (TMA) through spectroscopic techniques, including HR-ESI-MS, 1D-NMR, and 2D-NMR. An antifungal activity assay revealed that the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of TMA were 1.50–2.50 and 3.00–5.00 μg/ml, respectively. In situ antifungal activity analyses demonstrated that 90.0 μg/ml of TMA could inhibit the growth of fungi for over 14 days. In order to enhance TMA production, the high-yield mutant strain YB101 was screened, based on the isolate ZC-G-5, using a high-throughput screening method. The best metabolic precursor was selected during fermentation, when the concentration of glycerol was 8% (v/v) in Gauze's broth medium to cultivate the mutant strain YB101; the concentration of TMA could be increased to 960.0 μg/ml, compared with the original isolate ZC-G-5, where the concentration of the TMA was only 225.0 μg/ml. Our study may contribute to the application of S. albulus and its active metabolite as a potential bio-preservative in the food industry. [ABSTRACT FROM AUTHOR]

The problem of food spoilage due to Aspergillus flavus (A. flavus) needs to be resolved. In this study, we found that the minimum inhibitory concentration of cinnamaldehyde (CA) that inhibited A. flavus was 0.065 mg/ml and that corn can be prevented from spoiling at a concentration of 0.13 mg/cm3. In addition to inhibiting spore germination, mycelial growth, and biomass production, CA can also reduce ergosterol synthesis and can cause cytomembrane damage. Our intention was to elucidate the antifungal mechanism of CA. Flow cytometry, fluorescence microscopy, and western blot were used to reveal that different concentrations of CA can cause a series of apoptotic events in A. flavus , including elevated Ca2+ and reactive oxygen species, decrease in mitochondrial membrane potential (Δψ m ), the release of cytochrome c, the activation of metacaspase, phosphatidylserine (PS) externalization, and DNA damage. Moreover, CA significantly increased the expression levels of apoptosis-related genes (Mst3 , Stm1 , AMID , Yca1 , DAP3 , and HtrA2). In summary, our results indicate that CA is a promising antifungal agent for use in food preservation. [ABSTRACT FROM AUTHOR]

Microbial contamination is considered the main cause of food spoilage. Pseudomonas fluorescens is a typical spoilage bacterium contributing to a large extent to the spoilage process of proteinaceous foods. RpoS is known as an alternative sigma factor controlling stress resistance and virulence in many pathogens. Our previous work revealed that RpoS contributes to the spoilage activities of P. fluorescens by regulating resistance to different stress conditions, extracellular acylated homoserine lactone (AHL) levels, extracellular protease and total volatile basic nitrogen (TVB-N) production. However, RpoS-dependent genes in P. fluorescens remained undefined. RNA-seq transcriptomics analysis combined with quantitative proteomics analysis based on multiplexed isobaric tandem mass tag (TMT) labeling was performed in the P. fluorescens wild-type strain UK4 and its derivative carrying an rpoS mutation. A total of 375 differentially expressed coding sequences (DECs) and 212 differentially expressed proteins (DEPs) were identified. The DECs were further verified by qRT-PCR. The combined transcriptome and proteome analyses revealed the involvement of this regulator in several cellular processes, mainly including polysaccharide metabolism, intracellular secretion, extracellular structures, cell wall biogenesis, stress responses, and amino acid and biogenic amine metabolism, which may contribute to the biofilm formation, stress resistance, and spoilage activities of P. fluorescens. Moreover, we indeed observed that RpoS contributed to the production of the macrocolony biofilm's matrix. Our results provide insights into the regulatory network of RpoS and expand the knowledge about the role of RpoS in the functioning of P. fluorescens in food spoilage.

Brochothrix thermosphacta is one of the main spoilers in food, responsible for meat and seafood spoilage through the production of malodorous volatile organic compounds. The molecules produced by this bacterium depend on the substrate (meat or seafood) and the storage conditions such as gas mixtures used in the packaging. It seems also that the spoilage potential is strain dependent as production of diacetyl and acetoin, two molecules responsible for seafood spoilage, varies with strains. Therefore, this suggests the involvement of different metabolic functions depending on both food substrate and strain capacities. In this study, we selected two strains with different abilities to produce diacetyl and acetoin and compared their behavior after grown in beef or cooked peeled shrimp juices. We determined the genes upregulated by both strains depending on the growth substrate and those that were specifically upregulated in only one strain. The genes upregulated by both strains in meat or in shrimp juice revealed the importance of the substrate for inducing specific metabolic pathways. The examination of genes that were specifically upregulated in only one of the two strains revealed strain features associated to specific substrates and also strain-specific regulations of metabolic pathways putatively leading to different levels of spoilage molecule production. This shows that the spoilage potential of B. thermosphacta depends on nutrients provided by food substrate and on metabolic activity potential that each strain possesses. [ABSTRACT FROM AUTHOR]

The food industry produces highly perishable products. Food spoilage represents a severe problem for food manufacturers. Therefore, it is important to identify effective preservation solutions to prevent food spoilage. Ocins (e.g., bacteriocins, lactocins, and enterocins) are antibacterial proteins synthesized by bacteria that destroy or suppress the growth of related or unrelated bacterial strains. Ocins represent a promising strategy for food preservation, because of their antagonist effects toward food spoilage microorganisms, high potency, and low toxicity. Additionally, they can be bioengineered. The most common and commercially available ocins are nisin, plantaracin, sakacin P, and pediocin. Several ocins have been characterized and studied biochemically and genetically; however, their structure-function relationship, biosynthesis, and mechanism of action are not understood. This narrative review focuses primarily on ocins and their relevance to the food industry to help prevent food spoilage. In particular, the applications and limitations of ocins in the food industry are highlighted. [ABSTRACT FROM AUTHOR]

Microbial contamination is considered the main cause of food spoilage. Pseudomonas fluorescens is a typical spoilage bacterium contributing to a large extent to the spoilage process of proteinaceous foods. RpoS is known as an alternative sigma factor controlling stress resistance and virulence in many pathogens. Our previous work revealed that RpoS contributes to the spoilage activities of P. fluorescens by regulating resistance to different stress conditions, extracellular acylated homoserine lactone (AHL) levels, extracellular protease and total volatile basic nitrogen (TVB-N) production. However, RpoS-dependent genes in P. fluorescens remained undefined. RNA-seq transcriptomics analysis combined with quantitative proteomics analysis based on multiplexed isobaric tandem mass tag (TMT) labeling was performed in the P. fluorescens wild-type strain UK4 and its derivative carrying an rpoS mutation. A total of 375 differentially expressed coding sequences (DECs) and 212 differentially expressed proteins (DEPs) were identified. The DECs were further verified by qRT-PCR. The combined transcriptome and proteome analyses revealed the involvement of this regulator in several cellular processes, mainly including polysaccharide metabolism, intracellular secretion, extracellular structures, cell wall biogenesis, stress responses, and amino acid and biogenic amine metabolism, which may contribute to the biofilm formation, stress resistance, and spoilage activities of P. fluorescens. Moreover, we indeed observed that RpoS contributed to the production of the macrocolony biofilm's matrix. Our results provide insights into the regulatory network of RpoS and expand the knowledge about the role of RpoS in the functioning of P. fluorescens in food spoilage. [ABSTRACT FROM AUTHOR]

The spoilage potential and biofilm formation of Shewanella baltica are reported to be regulated by Quorum sensing (QS) system from the phenotype point of view, but the specific mechanism is not fully understood. In the present study, the QS autoinducers were detected by UHPLC-MS/MS, cell density-dependent luxR -type genes were obtained through autoregulation experiments among a series of candidates in S. baltica OS155 (The SSO of large yellow croaker). The direct interaction between cyclo-(L-Pro-L-Phe) (PP) and LuxR01 as well as LuxR02 proteins was revealed via in vitro binding assay. Deletion of luxR -type genes (luxR01 and luxR02) impaired spoilage potential and biofilm formation of S. baltica OS155 in various degrees. Transcriptional analysis and qRT-PCR validation showed that spoilage and biofilm-related genes torS, speF , and pomA were down-regulated in luxR 01 and luxR 02 deletion strains. In addition, exogenous PP promoted spoilage potential and biofilm formation, which could be attenuated by luxR 01 or luxR 02 deletion. Our results revealed an explicit QS system employing PP as autoinducer and two orphan LuxRs as receptors which positively regulated spoilage capacity and biofilm formation via transcriptional regulation of corresponding genes in S. baltica OS155, which provides potential specific targets for seafood preservation involving QS system. [ABSTRACT FROM AUTHOR]

The prevalence of biofilm-associated microorganisms and the increasing use of ready-to-eat fresh products represent the current duality the food industry must address. Innovative and eco-friendly antibiofilm solutions and appropriate microbiological food control systems are urgently needed to improve food quality and safety. This study aimed to investigate the in vitro combined efficacy of carvacrol with a pre-formed biofilm monolayer of the probiotic Lactiplantibacillus plantarum DSM 20174. The antimicrobial activity of carvacrol against both planktonic and sessile cells of foodborne pathogens and spoilage microorganisms, alone or in the presence of the pre-formed biofilm of L. plantarum , was investigated by culture-based methods along with flow cytometry (FCM) to monitor cells' cultivability and viability. The synergistic action of carvacrol and the pre-formed biofilm of L. plantarum was evaluated in the 96-well plates. The results showed that L. plantarum pre-formed biofilm monolayer enhanced the antimicrobial effect of carvacrol determining a bactericidal action while the treatment alone induced the viable but not culturable (VBNC) cell state only. Furthermore, the great efficacy of the combined treatment allowed the application of a lower concentration of carvacrol (100 ppm) to achieve significant damage in cell viability. In conclusion, the incorporation of carvacrol into the L. plantarum pre-formed biofilm represents a promising alternative for an antimicrobial functionalized ready-to-eat packaging., Competing Interests: LG was employed by I.L.P.A. Group. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Poscente, Di Gregorio, Costanzo, Nobili, Bernini, Garavaglia and Bevivino.)

Sturgeon is an important fresh water-culture fish in China. A problem with sturgeon is its high susceptibility to spoilage. Food spoilage is reported to be regulated by quorum sensing (QS). To identify the QS signals acetylated homoserine lactones (AHLs) in sturgeon and test whether QS plays a role in the spoilage of sturgeon, we investigated the specific spoilage organisms (SSOs) in vacuum packaged sturgeon stored at 4°C and the production of AHLs by sturgeon SSOs. 16S rDNA sequencing and spoilage capabilities analysis revealed that Aeromonas veronii LP-11, Citrobacter freundii LPJ-2, and Raoultella ornithinolytica LPC-3 were the SSOs in sturgeon. Among the three SSOs, only A. veronii LP-11 induced the QS biosensors Agrobacterium tumefaciens KYC55 and Chromobacterium violaceum CV026, suggesting that it produced AHLs. Analysis by thin layer chromatography, high-performance liquid chromatography-triple quadrupole tandem mass spectrometry, and high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC/qTOF-MS) identified that the AHLs produced by A. veronii were C6-SHL, C8-HSL, 3-oxo-C8-HSL, and 3-OH-C8-HSL. Our study revealed that QS system was probably involved in the regulation of sturgeon spoilage and for the first time reported the production of C8-HSL and 3-OH-C8-HSL by genus Aeromonas. As only HPLC/qTOF-MS effectively and accurately identified all the four AHLs produced by A. veronii LP-11, this study also showed that HPLC/qTOF-MS was the most efficient method for rapid analysis of AHLs in complex microbial sample. The study provides new insight into the microbiology of sturgeon spoilage which may be helpful for better sturgeon preservation. [ABSTRACT FROM AUTHOR]

Short-wave ultraviolet (UV-C) treatment represents a potent, clean and safe substitute to chemical sanitizers for fresh fruit preservation. However, the dosage requirement for microbial disinfection may have negative effects on fruit quality. In this study, UV-C was found to be more efficient in killing spores of Botrytis cinerea in dark and red light conditions when compared to white and blue light. Loss of the blue light receptor gene Bcwcl1, a homolog of wc-1 in Neurospora crassa, led to hypersensitivity to UV-C in all light conditions tested. The expression of Bcuve1 and Bcphr1, which encode UV-damage endonuclease and photolyase, respectively, were strongly induced by white and blue light in a Bcwcl1-dependent manner. Gene mutation analyses of Bcuve1 and Bcphr1 indicated that they synergistically contribute to survival after UV-C treatment. In vivo assays showed that UV-C (1.0 kJ/m²) abolished decay in drop-inoculated fruit only if the UV-C treatment was followed by a dark period or red light, while in contrast, typical decay appeared on UV-C irradiated fruits exposed to white or blue light. In summary, blue light enhances UV-C resistance in B. cinerea by inducing expression of the UV damage repair-related enzymes, while the efficiency of UV-C application for fruit surface disinfection can be enhanced in dark or red light conditions; these principles seem to be well conserved among postharvest fungal pathogens. [ABSTRACT FROM AUTHOR]

Raw bovine milk is highly nutritious as well as pH-neutral, providing the ideal conditions for microbial growth. The microbiota of raw milk is diverse and originates from several sources of contamination including the external udder surface, milking equipment, air, water, feed, grass, feces, and soil. Many bacterial and fungal species can be found in raw milk. The autochthonous microbiota of raw milk immediately after milking generally comprises lactic acid bacteria such as Lactococcus, Lactobacillus, Streptococcus, and Leuconostoc species, which are technologically important for the dairy industry, although they do occasionally cause spoilage of dairy products. Differences in milking practices and storage conditions on each continent, country and region result in variable microbial population structures in raw milk. Raw milk is usually stored at cold temperatures, e.g., about 4°C before processing to reduce the growth of most bacteria. However, psychrotrophic bacteria can proliferate and contribute to spoilage of ultrahigh temperature (UHT) treated and sterilized milk and other dairy products with a long shelf life due to their ability to produce extracellular heat resistant enzymes such as peptidases and lipases. Worldwide, species of Pseudomonas, with the ability to produce these spoilage enzymes, are the most common contaminants isolated from cold raw milk although other genera such as Serratia are also reported as important milk spoilers, while for others more research is needed on the heat resistance of the spoilage enzymes produced. The residual activity of extracellular enzymes after high heat treatment may lead to technological problems (off flavors, physico-chemical instability) during the shelf life of milk and dairy products. This review covers the contamination patterns of cold raw milk in several parts of the world, the growth potential of psychrotrophic bacteria, their ability to produce extracellular heat-resistant enzymes and the consequences for dairy products with a long shelf life. This problem is of increasing importance because of the large worldwide trade in fluid milk and milk powder. [ABSTRACT FROM AUTHOR]

Premature spoilage and varying product quality due to microbial contamination still constitute major problems in the production of microfiltered and pasteurized extended shelf life (ESL) milk. Spoilage-associated bacteria may enter the product either as part of the raw milk microbiota or as recontaminants in the dairy plant. To identify spoilage-inducing bacteria and their routes of entry, we analyzed end products for their predominant microbiota as well as the prevalence and biodiversity of psychrotolerant spores in bulk tank milk. Process analyses were performed to determine the removal of psychrotolerant spores at each production step. To detect transmission and recontamination events, strain typing was conducted with isolates obtained from all process stages. Microbial counts in 287 ESL milk packages at the end of shelf life were highly diverse ranging from <1 to 7.9 log cfu/mL. In total, 15% of samples were spoiled. High G+C Gram-positive bacteria were the most abundant taxonomic group, but were responsible for only 31% of spoilage. In contrast, psychrotolerant spores were isolated from 55% of spoiled packages. In 90% of samples with pure cultures of Bacillus cereus sensu lato and Paenibacillus spp., counts exceeded 6 log cfu/mL. In bulk tank milk, the concentration of psychrotolerant spores was low, accounting for merely 0.5 ± 0.8 MPN/mL. Paenibacillus amylolyticus/xylanexedens was by far the most dominant species in bulk tank milk (48% of all isolates), but was never detected in ESL milk, pointing to efficient removal during manufacturing. Six large-scale process analyses confirmed a high removal rate for psychrotolerant spores (reduction by nearly 4 log-units). B. cereus sensu lato, on the contrary, was frequently found in spoiled end products, but was rarely detected in bulk tank milk. Due to low counts in bulk tank samples and efficient spore removal during production, we suggest that shelf life is influenced only to a minor extent by raw-milk-associated factors. In contrast, recontamination with spores, particularly from the B. cereus complex, seems to occur. To enhance milk quality throughout the entire shelf life, improved plant sanitation and disinfection that target the elimination of spores are necessary. [ABSTRACT FROM AUTHOR]