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The aim of this study was to investigate the bacterial inhibitory ability and mechanism of action of linalyl alcohol against B. thermosphacta. Linalyl alcohol causes the leakage of intracellular material by disrupting the cell wall and exposing the hydrophobic phospholipid bilayer, which binds to bacterial membrane proteins and alters their structure. In addition, linalyl alcohol causes cell membrane damage by affecting fatty acids and proteins in the cell membrane. By inhibiting the synthesis of macromolecular proteins, the normal physiological functions of the bacteria are altered. Linalyl alcohol binds to DNA in both grooved and embedded modes, affecting the normal functioning of B. thermosphacta, as demonstrated through a DNA interaction analysis. [ABSTRACT FROM AUTHOR]

The Gram-positive bacterium Brochothrix thermosphacta is a spoilage agent commonly found on meat products. While the tet(L) gene, which confers resistance to tetracycline, has been identified in certain strains of B. thermosphacta, only a limited number of studies have investigated this gene and its potential presence on mobile DNA elements. This study aims to analyze the tetracycline-resistant strain B. thermosphacta BT469 at the genomic level to gain insight into the molecular determinants responsible for this resistance. Three plasmids have been identified in the strain: pBT469-1, which contains a tetR gene; pBT469-2, which harbours the tet(L) gene responsible for tetracycline resistance; and pBT469-3, which carries genes encoding for a thioredoxin and a phospholipase A2. Homology searches among sequences in public databases have revealed that the plasmid pBT469-2 is currently unique to the BT469 strain. However, the pBT469-1 plasmid is also found in three other strains of B. thermosphacta. Notably, sequences similar to pBT469-1 and pBT469-2 were also found in other bacterial genera, suggesting that these plasmids may be part of a diverse family present in several bacterial genera. Interestingly, sequences of various strains of B. thermosphacta show a high level of similarity with pBT469-3, suggesting that variants of this plasmid could be frequently found in this bacterium. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the bacterial inhibitory ability and mechanism of action of linalyl alcohol against B. thermosphacta. Linalyl alcohol causes the leakage of intracellular material by disrupting the cell wall and exposing the hydrophobic phospholipid bilayer, which binds to bacterial membrane proteins and alters their structure. In addition, linalyl alcohol causes cell membrane damage by affecting fatty acids and proteins in the cell membrane. By inhibiting the synthesis of macromolecular proteins, the normal physiological functions of the bacteria are altered. Linalyl alcohol binds to DNA in both grooved and embedded modes, affecting the normal functioning of B. thermosphacta, as demonstrated through a DNA interaction analysis.

Brochothrix thermosphacta is considered as a major spoiler of meat and seafood products. This study explores the biofilm formation ability and the biofilm structural diversity of 30 multi-origin B. thermosphacta strains using a set of complementary biofilm assays (biofilm ring test, crystal violet staining, and confocal laser scanning microscopy). Two major groups corresponding to low and high biofilm producers were identified. High biofilm producers presented flat architectures characterized by high surface coverage, high cell biovolume, and high surface area. [ABSTRACT FROM AUTHOR]

B. thermosphacta is a psychrotrophic bacterium that often forms the predominant part of the spoilage microflora of aerobically and anaerobically stored meats. Bacteriophages are natural enemies of bacteria and their potential for use in environmentally friendly biocontrol of specific pathogens in food is being intensively studied. In this study, we reported the isolation and characterization of the newly isolated lytic Brochothrix phage ADU4, which is capable of infecting the B. thermosphacta bacterium. For the characterization of Brochothrix phage ADU4; host range, multiplicity of infection values (MOI), phage growth parameters (latent period and burst size), stability at various temperatures and pH, reduction growth of bacteria, effect on biofilm, and molecular characterization were investigated. The spot-test analysis showed positivity with B. thermosphacta strains, while no infection was observed in any other species and genera of bacteria tested. The optimal MOI value of the phage was determined as 0.1. The phage latent period and burst sizes were 40–50 min and 311 PFU/ml per infected host cell, respectively by one-step growth curve analysis. Brochothrix phage ADU4 reduced bacteria immediately after infection, which is shown by optical density (OD) measurement and colony counting (

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Brochothrix thermosphacta is considered as a major spoiler of meat and seafood products. This study explores the biofilm formation ability and the biofilm structural diversity of 30 multi-origin B. thermosphacta strains using a set of complementary biofilm assays (biofilm ring test, crystal violet staining, and confocal laser scanning microscopy). Two major groups corresponding to low and high biofilm producers were identified. High biofilm producers presented flat architectures characterized by high surface coverage, high cell biovolume, and high surface area.

[Display omitted] • Pseudomonas fragi C6 and Brochothrix thermosphcta S5 displayed intertwined biochemical process related to pork spoilage. • Divergent metabolomic profiles of spoilage bacteria were characterized by LC-MS/MS. • Co-cultures accelerated the process of pork spoilage compared to monocultures. • Identification of seven key biomarkers linked to pork spoilage via distinct metabolic pathways. Pseudomonas and Brochothrix are the main spoilage organisms in pork, and each of these plays an essential role in the spoilage process. However, the effect of co-contamination of these two organisms in pork has not been elucidated. The changing bacterial communities during spontaneous spoilage of pork at 4 °C were evaluated using high-throughput sequencing. The dominant spoilage bacteria were isolated and these were identified as Pseudomonas fragi C6 and Brochothrix thermosphacta S5. Chilled pork was then experimentally contaminated with these strains, individually and in combination, and the progression of spoilage was assessed by analyzing various physicochemical indicators. These included total viable counts (TVC), pH, color, total volatile basic nitrogen (TVB-N), and detection of microbial metabolites. After 7 days of chilled storage, co-contaminated pork produced higher TVC and TVB-N values than mono-contaminated samples. Metabolomic analysis identified a total of 8,084 metabolites in all three groups combined. Differential metabolites were identified, which were involved in 38 metabolic pathways. Among these pathways, the biosynthesis of alkaloids derived from purine and histidine was identified as an important pathway related to spoilage. Specifically, histidine, histamine, AMP, IMP, GMP, succinic acid, and oxoglutaric acid were identified as potential spoilage biomarkers. The study showed that the combined presence of P. fragi C6 and B. thermosphacta S5 bacteria makes chilled pork more prone to spoilage, compared to their individual presence. This study provides insights that can assist in applying appropriate techniques to maintain quality and safety changes in meat during storage and further the assessment of freshness. [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.

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]

The aim of this study was to obtain the growth parameters of specific spoilage micro-organisms previously isolated in minced pork (MP) samples and to develop a three-spoilage species interaction model under different storage conditions. Naturally contaminated samples were used to validate this approach by considering the effect of the food microbiota. Three groups of bacteria were inoculated on irradiated samples, in mono- and in co-culture experiments (n = 1152): Brochothrix thermosphacta, Leuconostoc gelidum, and Pseudomonas spp. (Pseudomonas fluorescens and Pseudomonas fragi). Samples were stored in two food packaging [food wrap and modified atmosphere packaging (CO2 30%/O2 70%)] at three isothermal conditions (4, 8, and 12°C). Analysis was carried out by using both 16S rRNA gene amplicon sequencing and classical microbiology in order to estimate bacterial counts during the storage period. Growth parameters were obtained by fitting primary (Baranyi) and secondary (square root) models. The food packaging shows the highest impact on bacterial growth rates, which in turn have the strongest influence on the shelf life of food products. Based on these results, a three-spoilage species interaction model was developed by using the modified Jameson-effect model and the Lotka Volterra (prey–predator) model. The modified Jameson-effect model showed slightly better performances, with 40–86% out of the observed counts falling into the Acceptable Simulation Zone (ASZ). It only concerns 14–48% for the prey–predator approach. These results can be explained by the fact that the dynamics of experimental and validation datasets seems to follow a Jameson behavior. On the other hand, the Lotka Volterra model is based on complex interaction factors, which are included in highly variable intervals. More datasets are probably needed to obtained reliable factors, and so better model fittings, especially for three- or more-spoilage species interaction models. Further studies are also needed to better understand the interaction of spoilage bacteria between them and in the presence of natural microbiota.

Besides intrinsic and extrinsic factors such as antagonism for organic substrates or temperature, the storage atmosphere of meat has a high influence on the development of its initial microbiota. Specific modified atmospheres (MAs) selectively suppress growth of aerobic and anaerobic bacteria, thus reshaping the initial microbiota. As some microorganisms are more tolerant to MA, they overgrow competitors and produce metabolites that cause rejection of the product. In order to elucidate responses to different MA by means of metabolic adaptation and competition for organic substrates on meat, the typical representative meat spoilage bacteria Brochothrix (B.) thermosphacta TMW2.2101 and four lactic acid bacteria Carnobacterium (C.) divergens TMW2.1577, C. maltaromaticum TMW2.1581, Leuconostoc (L.) gelidum subsp. gelidum TMW2.1618 and L. gelidum subsp. gasicomitatum TMW2.1619 were chosen. Bacteria were grown in sterile glass bottles filled with a meat simulation medium, which was aerated constantly with either air, 100%_N2, 30%_CO2/70%_O2 or 30%_CO2/70%_N2. Growth of bacteria during incubation at 25°C and stirring at 120 rpm was monitored over 48 h and a label-free quantitative mass spectrometric approach was employed to determine changes within the bacterial proteomes in response to oxygen and carbon dioxide. Both Leuconostoc subsp. were intrinsically tolerant to MA, exhibiting no proteomic regulation of enzymes, whereas the other species provide a set of metabolic adaptation mechanism, enabling higher resistance to the detrimental effects of MA. Those mechanisms comprise: enhanced oxidative stress reduction, adjustment of the pyruvate metabolism and catabolic oxygen consumption in response to oxygen and intracellular pH homeostasis, maintenance of osmotic balance and alteration of the fatty acid composition in response to carbon dioxide. We further evaluated the potential of industrial used MA to inhibit specific bacterial spoilage. No bacterial inhibition is predicted for 30%_CO2/70%_O2 for the analyzed species, whereas 30%_CO2/70%_N2 predictively inhibits C. divergens TMW21577 and B. thermosphacta TMW2.2101. Furthermore, species-specific metabolic pathways enabling different and preferential carbon source utilization were identified, which enable non-competitive coexistence of respective bacteria on meat, resulting in synergistic spoilage. In conclusion, this study gives mechanistically explanations of their acknowledged status as typical spoilage organisms on MAP meats.

Abstract Brochothrix thermosphacta is one of the dominant bacterial species associated with spoilage of chilled meat and seafood products through the production of various metabolites responsible for off-odors. However, metabolic pathways leading to meat and seafood spoilage are not all well known. The production of spoiling molecules seems to depend both on strains and on food matrix. Several B. thermosphacta genome sequences have been reported, all issued from meat isolates. Here, we report four genome sequences, one complete and three as drafts. The four B. thermosphacta strains CD 337, TAP 175, BSAS1 3, and EBP 3070 were isolated from different ecological niches (seafood or meat products either spoiled or not and bovine slaughterhouse). These strains known as phenotypically and genetically different were selected to represent intraspecies diversity. CD 337 genome is 2,594,337 bp long, complete and circular, containing 2593 protein coding sequences and 28 RNA genes. TAP 175, BSAS1 3, and EBP 3070 genomes are arranged in 57, 83, and 71 contigs, containing 2515, 2668, and 2611 protein-coding sequences, respectively. These genomes were compared with two other B. thermosphacta complete genome sequences. The main genome content differences between strains are phages, plasmids, restriction/modification systems, and cell surface functions, suggesting a similar metabolic potential but a different niche adaptation capacity.

In this study, in situ-expressed metabolic routes of Brochothrix (B.) thermosphacta and Carnobacterium (C.) divergens were evaluated based on a metatranscriptomic dataset from bacteria growing on MAP chicken meat (O2/CO2; N2/CO2). Both species exhibited no (C. divergens) or minor transcription regulation (B. thermosphacta) within their main metabolic routes in response to different atmospheres. Both employ pathways related to glucose and ribose. Gluconeogenesis from lipid-borne glycerol is active in the progressing lack of carbohydrates. Pyruvate fates in both species comprise lactate, ethanol, acetate, CO2, formate, C4-compounds and H2O2 (only B. thermosphacta). Both species express genes for a minimal aerobic respiratory chain, but do not possess the genetic setting for a functional citric acid cycle. While products of carbohydrate and glycerol metabolism display mild to medium sensorial off-characteristics, predicted end products of their amino acid metabolism comprise, e.g., isobutyrate and isovalerate (B. thermosphacta) or cadaverine and tyramine (C. divergens) as potent spoilage compounds. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The aim of this study was to obtain the growth parameters of specific spoilage micro-organisms previously isolated in minced pork (MP) samples and to develop a three-spoilage species interaction model under different storage conditions. Naturally contaminated samples were used to validate this approach by considering the effect of the food microbiota. Three groups of bacteria were inoculated on irradiated samples, in mono- and in co-culture experiments (n = 1152): Brochothrix thermosphacta , Leuconostoc gelidum , and Pseudomonas spp. (Pseudomonas fluorescens and Pseudomonas fragi). Samples were stored in two food packaging [food wrap and modified atmosphere packaging (CO2 30%/O2 70%)] at three isothermal conditions (4, 8, and 12°C). Analysis was carried out by using both 16S rRNA gene amplicon sequencing and classical microbiology in order to estimate bacterial counts during the storage period. Growth parameters were obtained by fitting primary (Baranyi) and secondary (square root) models. The food packaging shows the highest impact on bacterial growth rates, which in turn have the strongest influence on the shelf life of food products. Based on these results, a three-spoilage species interaction model was developed by using the modified Jameson-effect model and the Lotka Volterra (prey–predator) model. The modified Jameson-effect model showed slightly better performances, with 40–86% out of the observed counts falling into the Acceptable Simulation Zone (ASZ). It only concerns 14–48% for the prey–predator approach. These results can be explained by the fact that the dynamics of experimental and validation datasets seems to follow a Jameson behavior. On the other hand, the Lotka Volterra model is based on complex interaction factors, which are included in highly variable intervals. More datasets are probably needed to obtained reliable factors, and so better model fittings, especially for three- or more-spoilage species interaction models. Further studies are also needed to better understand the interaction of spoilage bacteria between them and in the presence of natural microbiota. [ABSTRACT FROM AUTHOR]

Besides intrinsic and extrinsic factors such as antagonism for organic substrates or temperature, the storage atmosphere of meat has a high influence on the development of its initial microbiota. Specific modified atmospheres (MAs) selectively suppress growth of aerobic and anaerobic bacteria, thus reshaping the initial microbiota. As some microorganisms are more tolerant to MA, they overgrow competitors and produce metabolites that cause rejection of the product. In order to elucidate responses to different MA by means of metabolic adaptation and competition for organic substrates on meat, the typical representative meat spoilage bacteria Brochothrix (B.) thermosphacta TMW2.2101 and four lactic acid bacteria Carnobacterium (C.) divergens TMW2.1577, C. maltaromaticum TMW2.1581, Leuconostoc (L.) gelidum subsp. gelidum TMW2.1618 and L. gelidum subsp. gasicomitatum TMW2.1619 were chosen. Bacteria were grown in sterile glass bottles filled with a meat simulation medium, which was aerated constantly with either air, 100%_N2, 30%_CO2/70%_O2 or 30%_CO2/70%_N2. Growth of bacteria during incubation at 25°C and stirring at 120 rpm was monitored over 48 h and a label-free quantitative mass spectrometric approach was employed to determine changes within the bacterial proteomes in response to oxygen and carbon dioxide. Both Leuconostoc subsp. were intrinsically tolerant to MA, exhibiting no proteomic regulation of enzymes, whereas the other species provide a set of metabolic adaptation mechanism, enabling higher resistance to the detrimental effects of MA. Those mechanisms comprise: enhanced oxidative stress reduction, adjustment of the pyruvate metabolism and catabolic oxygen consumption in response to oxygen and intracellular pH homeostasis, maintenance of osmotic balance and alteration of the fatty acid composition in response to carbon dioxide. We further evaluated the potential of industrial used MA to inhibit specific bacterial spoilage. No bacterial inhibition is predicted for 30%_CO2/70%_O2 for the analyzed species, whereas 30%_CO2/70%_N2 predictively inhibits C. divergens TMW21577 and B. thermosphacta TMW2.2101. Furthermore, species-specific metabolic pathways enabling different and preferential carbon source utilization were identified, which enable non-competitive coexistence of respective bacteria on meat, resulting in synergistic spoilage. In conclusion, this study gives mechanistically explanations of their acknowledged status as typical spoilage organisms on MAP meats. [ABSTRACT FROM AUTHOR]

High oxygen modified atmosphere packaging is a commonly applied method to prolong the minimum shelf life of fresh (red) meats. Upon spoilage, changes of the initial oxygen concentration and microbiome composition can be observed. Thus, we classified the typical representative meat spoiling bacteria Brochothrix (B.) thermosphacta TMW2.2101 and the four lactic acid bacteria (LAB) Carnobacterium (C.) divergens TMW2.1577, C. maltaromaticum TMW2.1581, Leuconostoc (L.) gelidum subsp. gelidum TMW2.1618, and L. gelidum subsp. gasicomitatum TMW2.1619 along their oxygen consuming capacity, which can indicate the timeline of microbiome and sensorial changes. All bacteria were grown in a model system employing gas tight glass bottles containing meat simulation media and under modified atmosphere (70% O2 and 30% CO2). Oxygen concentrations of media and headspaces were monitored over time and the oxygen uptake rate (OUR) was calculated for all species. All bacteria were able to consume dissolved oxygen, with B. thermosphacta TMW2.2101 exhibiting a 31-times higher OUR per single cell in 60 h. Furthermore, all strains showed significant growth enhancement in the presence of heme indicating respiratory activity. Comparative genomic and physiological analyses predict the activity of a respiratory chain for all species upon high oxygen atmosphere. An additional cytochrome aa3 oxidase is suggested to be responsible for the increased OUR of B. thermosphacta TMW2.2101. Furthermore, B. thermosphacta TMW2.2101 revealed highest oxidative stress tolerance compared to the other bacteria, facilitating a higher respiratory activity. Coupling of respiration and fermentation via regeneration of NADH can be a competitive advantage for meat spoiling bacteria resulting in a higher cell count and possibly accelerated spoilage. The exhibited highest capacity for oxygen consumption of B. thermosphacta compared to LAB in vitro also suggests a higher contribution of this bacterium to the change in the atmosphere upon spoilage of MAP meats in situ.

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]

High oxygen modified atmosphere packaging is a commonly applied method to prolong the minimum shelf life of fresh (red) meats. Upon spoilage, changes of the initial oxygen concentration and microbiome composition can be observed. Thus, we classified the typical representative meat spoiling bacteria Brochothrix (B.) thermosphacta TMW2.2101 and the four lactic acid bacteria (LAB) Carnobacterium (C.) divergens TMW2.1577, C. maltaromaticum TMW2.1581, Leuconostoc (L.) gelidum subsp. gelidum TMW2.1618, and L. gelidum subsp. gasicomitatum TMW2.1619 along their oxygen consuming capacity, which can indicate the timeline of microbiome and sensorial changes. All bacteria were grown in a model system employing gas tight glass bottles containing meat simulation media and under modified atmosphere (70% O2 and 30% CO2). Oxygen concentrations of media and headspaces were monitored over time and the oxygen uptake rate (OUR) was calculated for all species. All bacteria were able to consume dissolved oxygen, with B. thermosphacta TMW2.2101 exhibiting a 31-times higher OUR per single cell in 60 h. Furthermore, all strains showed significant growth enhancement in the presence of heme indicating respiratory activity. Comparative genomic and physiological analyses predict the activity of a respiratory chain for all species upon high oxygen atmosphere. An additional cytochrome aa3 oxidase is suggested to be responsible for the increased OUR of B. thermosphacta TMW2.2101. Furthermore, B. thermosphacta TMW2.2101 revealed highest oxidative stress tolerance compared to the other bacteria, facilitating a higher respiratory activity. Coupling of respiration and fermentation via regeneration of NADH can be a competitive advantage for meat spoiling bacteria resulting in a higher cell count and possibly accelerated spoilage. The exhibited highest capacity for oxygen consumption of B. thermosphacta compared to LAB in vitro also suggests a higher contribution of this bacterium to the change in the atmosphere upon spoilage of MAP meats in situ. [ABSTRACT FROM AUTHOR]

Abstract Increasing demands for lightly processed seafood stresses the need for development of non-intensive processing methods that ensures a safe product. The limitation to the shelf life of seafood is often ascribed to microbial activity. An experiment was design to investigate the influence of heat-treatments in combination with packaging technologies (vacuum (VAC), modified atmosphere (MA) packaging, or soluble gas stabilization (SGS)) on the microbial survival of inoculated species. Fish patties were inoculated with either Brochothrix thermosphacta or Listeria innocua before heat-treatment, packaging, and storage at 2 ᵒC for 16 days. Increased heat-treatment lowered the bacterial load throughout the storage and type of packaging technology affected the bacterial load significantly. VAC-samples had a significantly higher bacterial load than MA- and SGS-packaged samples, regardless of heat-treatment (L. innocua : 8.7 ± 0.1, 8.3 ± 0.1, 8.2 ± 0.1 log CFU x g−1, B. thermosphacta : 9.9 ± 0.1, 9.2 ± 0.1, 8.6 ± 0.1 log CFU x g−1, respectively) at end of storage. Furthermore, use of SGS significantly increased the bacterial inhibition by heat (0.5–0.6 log CFUx −1) and extended the lag phase of B. thermosphacta , as well as decreasing the growth rate of both inoculum species. It is concluded that use of SGS can fulfill the consumers' demand of fresh, lightly processed seafood with a prolonged shelf life. Highlights • CO 2 present during heat treatment increases bacterial inhibition by heat. • Growth inhibitions were proportional to CO 2 levels in the product. • Packaging technology is more influential on bacterial growth than heat treatment. [ABSTRACT FROM AUTHOR]

Efficient ways of decontamination are needed to minimize the risk of infections with Yersinia (Y.) enterocolitica, which causes gastrointestinal diseases in humans, and to reduce the numbers of Brochothrix (B.) thermosphacta to extend the shelf-life of meat. While many studies have focused on a single treatment of peracetic acid (PAA) or UV-C-irradiation, there are no studies about a combined treatment on meat. Therefore, in the present study, pork was inoculated with either Y. enterocolitica or B. thermosphacta, and was treated with a combination of 2040 mJ/cm2 UV-C irradiation followed by a 2000 ppm PAA spray treatment (30 s). Samples were packed under modified atmosphere and stored for 1, 7, or 14 days. The samples were examined for Y. enterocolitica and B. thermosphacta content, chemical and sensory effects, and meat quality parameters. For Y. enterocolitica, a significant reduction of up to 2.16 log10 cfu/cm2 meat and for B. thermosphacta, up to 2.37 log10 cfu/cm2 meat was seen on day 14 after UV-C/PAA treatment compared to the untreated controls.

Bacteria on ready-to-eat meat may cause diseases and lead to faster deterioration of the product. In this study, ready-to-eat sliced ham samples were inoculated with Yersinia enterocolitica or Brochothrix thermosphacta and treated with ultraviolet (UV) light. The initial effect of a UV-C irradiation was investigated with doses of 408, 2040, 4080, and 6120 mJ/cm2 and the effect after 0, 7, and 14 days of refrigerated storage with doses of 408 and 4080 mJ/cm2. Furthermore, inoculated ham samples were stored under light and dark conditions after the UV-C treatment to investigate the effect of photoreactivation. To assess the ham quality the parameters color and antioxidant capacity were analyzed during storage. UV-C light reduced Yersinia enterocolitica and Brochothrix thermosphacta counts by up to 1.11 log10 and 0.79 log10 colony forming units/g, respectively, during storage. No photoreactivation of the bacteria was observed. Furthermore, significantly lower a* and higher b* values after 7 and 14 days of storage and a significantly higher antioxidant capacity on day 0 after treatment with 4080 mJ/cm2 were detected. However, there were no other significant differences between treated and untreated samples. Hence, a UV-C treatment can reduce microbial surface contamination of ready-to-eat sliced ham without causing considerable quality changes.

This study evaluated the antimicrobial activities of two lactic acid bacteria (LAB) Lactococcus lactis subsp. lactis I23 and L. lactis subsp. hordinae E91 against Brochothrix thermosphacta in pork during storage at ambient temperature (30oC) over 7 days. Both the LAB strains and spoilage organism were inoculated on fresh pork samples at 1x106cfu/g. About 3 log reduction in the spoilage organism was obtained in LAB treated samples after 48 h of storage. The spoilage organism was confirmed to be sensitive to the bacteriocin nisin produced by Lactococcus lactis subsp. lactis I23. There were reductions in the counts of Salmonella typhimurium, Listeria monocytogenes, Enterobacteriaceae and Staphylococcus in the treated samples. Conclusively, growth of B. thermosphacta could be effectively controlled by nisin producing Lactococcus lactis subsp. lactis I23 in fresh pork during storage, thereby enhancing shelf life of the product.

Aluminium recycling is one of currently growing industries in Indonesia. Abundant amounts of aluminium waste leads to the emergence of new industries. The waste produced from this kind of industry is not well processed yet. Bioremediation using isolated indigenous bacteria is one of the best options from treating the aluminium recycling wastewater. Since biological processes are closely related to the bacterial growth conditions, it is important to understand the effect of pH and aluminium exposure to bacteria. Six potential bacteria strains were obtained from isolation. Vibrio alginolyticus and Brochothrix thermosphacta were shown to be resistant to the aluminium exposure, as well as the acidic conditions. Both types of bacteria were able to survive on acid medium with pH 5. The higher the concentration of aluminium, the slower bacterial growth rate achieved. The MIC value of aluminium for Vibrio alginolyticus was 425 mg/l, Brochothrix thermosphacta was 325 mg/l and Pseudomonas aeruginosa was 200 mg/l. [ABSTRACT FROM AUTHOR]

The effect of high-pressure processing (HPP) on Listeria monocytogenes, the indigenous microbiota and the shelf-life of chicken fillets was evaluated. Chicken fillets were inoculated with different inocula (2, 4, and 6 log CFU/g) of a 4-strain cocktail of L. monocytogenes, vacuum-packed, processed or not with HPP (500 MPa/10 min) and stored at 4 °C and 12 °C. Total viable counts (TVC), L. monocytogenes, Pseudomonas spp., Brochothrix thermosphacta, lactic acid bacteria (LAB), Enterobacteriaceae and yeasts/molds were determined along with the pH and sensory analysis. Pulsed-field gel electrophoresis (PFGE) was used to monitor the succession of indigenous Brochothrix isolates and inoculated Listeria strains. The main spoilage microorganism of HPP-treated samples was B. thermosphacta detected after 3 days of storage. HPP decreased the inoculated Listeria population. For the low and medium inoculum case it was detected throughout the shelf-life at both temperatures in populations near to the detection limit or after enrichment. In the high inoculum case, the pathogen decreased ≥5-log cycles after HPP, while increased subsequently to 1.6 and 4.5 log CFU/g at 4 °C and 12 °C, respectively, by the end of the shelf-life. PFGE showed that Brochothrix isolates exhibited a significant diversity among control samples, whereas this was limited for the HPP-treated samples. The survival and distribution of different Listeria strains depended on the initial inoculum and storage temperature. In conclusion, HPP increased the shelf-life (for 5 and 4 days, at 4 °C and 12 °C, respectively) and enhanced the safety of chicken meat.

Brochothrix thermosphacta is the predominant spoilage microorganism in meat and its control in processing environments is important to maintain meat product quality. Atmospheric cold plasma is of interest for control of pathogenic and spoilage microorganisms in foods. This study ascertained the potential of dielectric barrier discharge atmospheric cold plasma (DBD-ACP) for control of B. thermosphacta, taking microbial and food environment factors into consideration, and investigated the shelf-life of lamb chop after in-package plasma treatment in modified atmosphere. Community profiling was used to assess the treatment effects on the lamb microflora. ACP treatment (80 kV) for 30s inactivated B. thermosphacta populations below detection levels in PBS, while 5 min treatment achieved a 2 Log cycle reduction using a complex meat model medium and attached cells. The antimicrobial efficacy of plasma was reduced but still apparent on lamb chop surface-inoculated with high concentrations of B. thermosphacta . Lamb chop treated under modified atmosphere exhibited reduced microbial growth over the product shelf-life and community profiling showed no evident changes to the microbial populations after the treatment. The overall results indicated potential of ACP to enhance microbial control leading to meat storage life extension through adjusting the modality of treatment. [ABSTRACT FROM AUTHOR]

Aims This study was conducted to assess the effects of different storage temperatures (4-20°C), on bacterial concentrations, growth rates and community structure in fresh retail sushi, a popular retail product with a claimed shelf life of 2-3 days. Methods and Results The maximum specific growth rate based on aerobic plate count ( APC) at 4°C was 0·06 h−1 and displayed a sixfold increase (0·37 h−1) at 20°C. Refrigeration resulted in no growth of hydrogen sulphide (H2S)-producing bacteria, but this group had the strongest temperature response. The bacterial community structure was determined by PCR/ DGGE (denaturing gradient gel electrophoresis). Multivariate analysis based on Bray-Curtis similarities demonstrated that temperature alone was not the major determinant for the bacterial community structure. The total concentration of aerobic bacteria was the variable that most successfully explained the differences between the communities. The dominating organisms, detected by sequencing of DNA bands excised from the DGGE gel, were Brochothrix thermosphacta and genera of lactic acid bacteria ( LAB). Conclusion The relationship between growth rates and storage temperatures clearly demonstrates that these products are sensitive to deviations from optimal storage temperature, possibly resulting in loss of quality during shelf life. Regardless of the storage temperature, the bacterial communities converged towards a similar structure and density, but the storage temperature determined how fast the community reached its carrying capacity. Significance and Impact of the Study Little information is available on the microbial composition of ready-to-eat food that are prepared with raw fish, subjected to contamination during handling, and susceptible to microbial growth during cold storage. Moreover, the data are a good first possibility to simulate growth of APC, H2S-producing bacteria and LAB under different temperature scenarios that might occur during production, distribution or storage. [ABSTRACT FROM AUTHOR]

The aims of this study were to improve the method for purification of leucocin A to increase yield of peptide and to evaluate the efficacy of leucocin A and an analogue of leucocin A (leucocin N17L) to inhibit the growth of Listeria monocytogenes on wieners in the presence of spoilage organisms. Leucocin A was produced by Leuconostoc gelidum UAL187 and purified with a five-fold increase in yield; leucocin N17L was synthesized replacing asparagine at residue 17 with leucine. Five strains of L. monocytogenes associated with foodborne illness were used to assess bacteriocin efficacy in vitro and in situ . Minimum inhibitory concentrations could not be determined in broth; however, on agar the minimum inhibitory concentrations ranged from 11.7–62.5 μM and 62.5–>500 μM for leucocin A and leucocin N17L, respectively. Leucocin N17L was less effective than the native bacteriocin at controlling the growth of L. monocytogenes . The inactivation profiles of L. monocytogenes in broth in the presence of leucocin A suggested each isolate had different levels of resistance to the bacteriocin as determined by the initial bactericidal effect. The formation of spontaneously resistance subpopulations were also observed for each strain of L. monocytogenes . In situ , wieners were inoculated with the spoilage organisms, Carnobacterium divergens and Brochothrix thermosphacta , followed by surface application of purified leucocin A, and inoculated with a cocktail of L. monocytogenes . Wieners were vacuum packaged and stored at 7 °C for 16 d. Leucocin A reduced the counts L. monocytogenes on wieners during storage, regardless of the presence of C. divergens . B. thermosphacta was unaffected by the presence of leucocin A on wieners over the duration of storage. This study suggests that leucocin A may be beneficial to industry as a surface application on wieners to help reduce L. monocytogenes counts due to post-processing contamination even in the presence of spoilage organisms. However, further investigation on the ability of L. monocytogenes to form spontaneous resistance to class II bacteriocins on food matrices during prolonged storage is warranted. [ABSTRACT FROM AUTHOR]

The primary objective of this study was to characterise (microbiology and physical parameters) beef carcasses and primals during chilled storage. A minor aim was to compare observed growth of key spoilage bacteria on carcasses with that predicted by ComBase and the Food Safety Spoilage Predictor (FSSP). Total viable count (TVC), total Enterobacteriacae count (TEC), Pseudomonas spp., lactic acid bacteria (LAB), Brochothrix thermosphacta and Clostridium spp. were monitored on beef carcasses (n = 30) and primals (n = 105) during chilled storage using EC Decision 2001/471/EC and ISO sampling/laboratory procedures. The surface and/or core temperature, pH and water activity (a w ) were also recorded. Clostridium (1.89 log 10 cfu/cm 2 ) and Pseudomonas spp. (2.12 log 10 cfu/cm 2 ) were initially the most prevalent bacteria on carcasses and primals, respectively. The shortest mean generation time (G) was observed on carcasses with Br. thermosphacta (20.3 h) and on primals with LAB (G = 68.8 h) and Clostridium spp. (G = 67 h). Over the course of the experiment the surface temperature decreased from 37 °C to 0 °C, pH from 7.07 to 5.65 and a w from 0.97 to 0.93 The observed Pseudomonas spp. and Br. thermosphacta growth was more or less within the range of predictions of Combase. In contrast, the FSSP completely overestimated the growth of LAB. This study contributes to the very limited microbiological data on beef carcasses and primals during chilling. [ABSTRACT FROM AUTHOR]

Brochothrix thermosphacta is a predominant and spoilage bacterium in different chilled vacuum-packaged aquatic products. Bacteriophages have been extensively studied for the biocontrol of spoilage bacteria in order to maintain the quality and shelf-life of foods. In this study, a highly lytic bacteriophage BtpYZU01 against B. thermosphacta was isolated from a domestic sewage sample in Yangzhou, and its morphological, biological, and genomic features were characterized. Morphology, one-step growth, thermal and pH stability results indicated that the phage BtpYZU01 was a member of Siphoviridae, with large burst sizes and relatively short latent periods, and strong resistance to extreme pH. Sequence analysis showed that BtpYZU01 had 52 open reading frames (ORFs), but without genes related to antibiotic resistance, toxin, lysogeny and virulence factors. B. thermosphacta in LB was significantly inhibited by the presence of phage BtpYZU01 at 25 °C. In the fish juice model, the counts of B. thermosphacta were also decreased after phage treatment at both at 25 °C and 4 °C. Based on the above features, BtpYZU01 could be served as a novel phage candidate to biological control of B. thermosphacta in meat and aquatic products. • A new lytic phage BtpYZU01 infecting Brochothrix thermophacta was isolated from sewage samples. • BtpYZU01 was characterized by morphology, one-step growth, stability and genome sequencing. • BtpYZU01 can be served as a candidate to biological control of B. thermosphacta in meat and aquatic products. [ABSTRACT FROM AUTHOR]

In recent years, it has been confirmed that essential oils (EOs) exert antimicrobial activity as they are able to inhibit cell growth and inactivate microbial cells. The application of biopreservation strategies by means of EOs opens up interesting perspectives in the food industry, including meat production. The paper aims to evaluate the effects of Thymus vulgaris (red thyme) and Caryophyllus aromaticus (cloves) EOs on the development of the spoilage population of fresh pork packaged under modified atmosphere (MAP). In particular, the research was focused on Brochothrix thermosphacta, a specific spoilage microorganism of fresh meat packed in anaerobic conditions or under MAP. Amongst seven EOs, those that showed the highest antimicrobial activity on 5 B. thermosphacta strains in vitro were: cloves [minimum inhibitory concentration (MIC) 0.6-2.5 mg/mL], savory (MIC 2.5-5.0 mg/mL), and red thyme (MIC 2.5 to 20 mg/mL). Red thyme and cloves EOs were selected for meat treatment, by increasing the dose at 20 and 40 mg/mL respectively, to take into account the matrix effect that can reduce EO availability. In spite of the minor efficacy observed in vitro, 40 mg/mL red thyme EO strongly limited the growth of B. thermosphacta in pork samples up to day 6 of storage [below 3.0 Log colony forming unit (CFU)/g, starting from 2.0 Log CFU/g at time 0], and exerted an antimicrobial effect also on the aerobic mesophilic count. Good results were obtained also with 20 mg/mL red thyme EO. The control of B. thermosphacta growth through EOs encourages research on alternative methods for extending the shelf life of fresh meat under MAP.

Modified atmosphere packaging (MAP) is widely used in food industry to extend the microbiological shelf-life of meat. Typically, poultry meat has been packaged in a CO 2 /N 2 atmosphere (with residual low O 2 ). Recently, some producers use high O 2 MAP for poultry meat to empirically reach comparable shelf lifes. In this work, we compared spoilage microbiota of skinless chicken breast in high (80% O 2 , 20% CO 2 ) and low O 2 MAP (65% N 2 and 35% CO 2 ). Two batches of meat were incubated in each atmosphere for 14 days at 4 °C and 10 °C. Atmospheric composition of each pack and colony forming units (25 °C, 48 h, BHI agar) of poultry samples were determined at seven timepoints. Identification of spoilage organisms was carried out by MALDI-TOF MS. Brochothrix thermosphacta , Carnobacterium sp. and Pseudomonas sp. were the main organisms found after eight days at 4 °C and 10 °C in high O 2 MAP. In low O 2 MAP, the main spoilage microbiota was represented by species Hafnia alvei at 10 °C, and genera Carnobacterium sp., Serratia sp., and Yersinia sp. at 4 °C. High O 2 MAP is suggested as preferential gas because were less detrimental and pathogens like Yersinia were not observed. [ABSTRACT FROM AUTHOR]

In recent years, it has been confirmed that essential oils (EOs) exert antimicrobial activity as they are able to inhibit cell growth and inactivate microbial cells. The application of biopreservation strategies by means of EOs opens up interesting perspectives in the food industry, including meat production. The paper aims to evaluate the effects of Thymus vulgaris (red thyme) and Caryophyllus aromaticus (cloves) EOs on the development of the spoilage population of fresh pork packaged under modified atmosphere (MAP). In particular, the research was focused on Brochothrix thermosphacta, a specific spoilage microorganism of fresh meat packed in anaerobic conditions or under MAP. Amongst seven EOs, those that showed the highest antimicrobial activity on 5 B. thermosphacta strains in vitro were: cloves [minimum inhibitory concentration (MIC) 0.6-2.5 mg/mL], savory (MIC 2.5-5.0 mg/mL), and red thyme (MIC 2.5 to 20 mg/mL). Red thyme and cloves EOs were selected for meat treatment, by increasing the dose at 20 and 40 mg/mL respectively, to take into account the matrix effect that can reduce EO availability. In spite of the minor efficacy observed in vitro, 40 mg/mL red thyme EO strongly limited the growth of B. thermosphacta in pork samples up to day 6 of storage [below 3.0 Log colony forming unit (CFU)/g, starting from 2.0 Log CFU/g at time 0], and exerted an antimicrobial effect also on the aerobic mesophilic count. Good results were obtained also with 20 mg/mL red thyme EO. The control of B. thermosphacta growth through EOs encourages research on alternative methods for extending the shelf life of fresh meat under MAP. [ABSTRACT FROM AUTHOR]

• A newly isolated phage with lytic ability against B. thermosphacta was characterized. • Both genome sequencing and characterization of the Brochothrix phage ADU4 showed it to be a highly lytic phage. • The effects of Brochothrix phages on biofilms have been studied for the first time. Phage ADU4 significantly reduced the B. thermosphacta (both planktonic and biofilm forms) in vitro. • Phage ADU4 is a promising biocontrol candidate for the control of B. thermosphacta contamination in meat and meat products. • This study contributes to improving our knowledge of Brochothrix phages. B. thermosphacta is a psychrotrophic bacterium that often forms the predominant part of the spoilage microflora of aerobically and anaerobically stored meats. Bacteriophages are natural enemies of bacteria and their potential for use in environmentally friendly biocontrol of specific pathogens in food is being intensively studied. In this study, we reported the isolation and characterization of the newly isolated lytic Brochothrix phage ADU4, which is capable of infecting the B. thermosphacta bacterium. For the characterization of Brochothrix phage ADU4; host range, multiplicity of infection values (MOI), phage growth parameters (latent period and burst size), stability at various temperatures and pH, reduction growth of bacteria, effect on biofilm, and molecular characterization were investigated. The spot-test analysis showed positivity with B. thermosphacta strains, while no infection was observed in any other species and genera of bacteria tested. The optimal MOI value of the phage was determined as 0.1. The phage latent period and burst sizes were 40–50 min and 311 PFU/ml per infected host cell, respectively by one-step growth curve analysis. Brochothrix phage ADU4 reduced bacteria immediately after infection, which is shown by optical density (OD) measurement and colony counting (<10 CFU/ml) for 3 days. The degradation of B. thermosphacta in biofilm by Brochothrix phage ADU4 was analyzed and it was found that high titer phage breakdown the existing biofilm and also persistently inhibited biofilm formation. Brochothrix phage ADU4 genome was found to be 127,819 bp, and GC content 41.65%. The genome contains 217 putative open reading frames (ORFs), 4 tRNAs, and additionally, no known virulence and antibiotic resistance genes (AMR) were identified. Brochothrix phage ADU4 showed a high identity (96.09%) to the A9 phage that belongs to the Herelleviridae family. Nevertheless, the assembly module and its around appeared less conserved, and some DNA fragments in Brochothrix phage ADU4 genome were not found in A9 genome and vice versa. A9 contains TnpB, a transposase accessory protein involved in lysogenicity which is absent in Brochothrix phage ADU4. In contrary Brochothrix phage ADU4 had auxiliary metabolic genes (AMG) mostly carried by lytic phages. All these results showed that the Brochothrix phage ADU4 has excellent properties such as strong antibacterial activity, short latent period, high burst size, stability in different conditions, inhibition of biofilms, and absence of virulence and AMR genes. Based on all these features, this newly isolated phage is promising to control B. thermosphacta contamination in meat and meat products, and has the potential to be used alone or in combination with phage cocktails. [ABSTRACT FROM AUTHOR]

In the present study, a multiple approach was used to characterize Malpighia punicifolia extract and to evaluate its inhibitory activity against several meat spoilage bacteria. First, volatile fraction, vitamins and phenolic compounds of the extract obtained by supercritical fluid extraction were determined by GC-MS and HPLC. Then, the antimicrobial action of the extract was in vitro evaluated against Pseudomonas putida DSMZ 291T, Pseudomonas fluorescens DSMZ 50009T, Pseudomonas fragi DSMZ 3456T, and Brochothrix thermosphacta DSMZ 20171T by the agar well diffusion assay and by the agar dilution test. Based on the results of the minimum inhibitory concentration (MIC) against the assayed bacteria, 4 different concentrations of the extract were used in a challenge test on water buffalo steaks stored for 21 d at 4 °C. Results of chemical analyses showed that M. punicifolia extract is characterized by the presence of several compounds, already described for their antimicrobial (phenolic acids, flavonones, and furanes) and antioxidant (ascorbic acid) properties. The in vitro detection of antimicrobial activities highlighted that the extract, used at 8% concentration, was able to inhibit all the target bacteria. Moreover, very low MIC values (up to 0.025%) were detected. In situ tests, performed on water buffalo steaks treated with the extract in the concentration range 0.025% to 0.05%, showed a strong inhibition of both intentionally inoculated bacteria and naturally occurring microorganisms. Positive results, in terms of color and odor, were also observed during the entire storage of steaks preserved with the extract. [ABSTRACT FROM AUTHOR]

Abstract: In the current study, the shelf-life and the spoilage of Italian wurstel sold in a local supermarket was investigated. The analysis included identifying the causative microorganisms and the variations in physical-chemical parameters (pH, a W, lactic acid, and ammonia). The spoilage consisted of a white surface coating or slime that appeared after the thirtieth day of storage. The cause of the spoilage was the uncontrolled growth of thermoduric heterofermentative lactic acid bacteria (LAB) that occurred during storage. The LAB, including Leuconostocs and Brochothrix thermosphacta organisms, can survived the pasteurisation process. Due to the deacidifying action of B. thermosphacta and the production of ammonia, there was no evidence of off-odour or off-flavour and no change in pH. Furthermore, no loss of the vacuum seal was observed in the spoiled wurstel. [Copyright &y& Elsevier]

Ninety three isolates of Brochothrix spp. from raw meat were analysed by PCR-based molecular fingerprinting and identified by species-specific Real-Time (RTi)-PCR and 16S rRNA gene sequencing. Thirty three strains were characterized for their growth capability in sarcoplasmic extract with and without glucose, for in vitro lipolytic activity, in vitro and in situ (in beef) proteolytic activity and for amino-decarboxylase activity. Moreover, spoilage potential of seven selected strains in sterile and non-sterile meat was investigated by analyzing the release of volatile organic compounds (VOCs) during storage in air. All the strains analysed were unable to hydrolyze sarcoplasmic proteins in vitro and in situ and they did not show lipolytic activity at 4°C or 20°C. Almost all were able to grow in the presence of sarcoplasmic extract with glucose and produced histamine. The release of VOCs by each strain in sterile and non sterile beef stored at 4°C in air was evaluated by HS-SPME-GC/MS analysis. Acetoin and 1-octen-3-ol and 3-methyl-1-butanol were the major compounds isolated from sterile and non-sterile meat samples inoculated with single strains of Brochothrix (B.) thermosphacta and high concentration of acetoin was found in all inoculated meat samples. The role of B. thermosphacta as meat spoiler does not seem to be influenced by indigenous microbiota of meat while its development in meat is associated to a significant increase (P<0.05) of acetoin and other compounds recognized as important contributors to the spoilage of meat and meat products. [ABSTRACT FROM AUTHOR]

Abstract: Lactobacillus sakei is a lactic acid bacterium, naturally associated with long term storage of fresh meat at low temperature. Here we investigated the effect, on the evolution of pathogenic and spoilage microorganisms in ground beef, of L. sakei cocktails used as bioprotective cultures. We selectively developed a real time quantitative PCR method, allowing the quantification of individual L. sakei strains inoculated in ground meat with specific probes. Six cocktails of three strains were tested to evaluate their effect on the growth of Salmonella enterica Typhimurium, Escherichia coli O157:H7 and Brochothrix thermosphacta at 4°C and 8°C, under vacuum or modified atmosphere packaging. Using plating methods to quantify the different bacterial species, one cocktail showed an effect against S. Typhimurium and E. coli under given conditions. Real time quantitative PCR showed that the three inoculated L. sakei strains had a different growth pattern, and that the association of these three strains indeed impaired growth of S. Typhimurium and E. coli. [Copyright &y& Elsevier]

The spoilage potential of Brochothrix thermosphacta, Serratia proteamaculans and Rahnella aquatilis was investigated in vacuum packaged high (5.9 to 6.4) and low (5.4 to 5.8) pH lamb. Vacuum packaged fore shank (m. extensor carpi radialis) and striploins (m. longissimus dorsi) (n=306) inoculated with ~100CFU of individual bacteria were stored for twelve weeks at temperatures −1.5, 0, 2 and 7°C. Spoilage characteristics and bacterial numbers were recorded and analysed in comparison to un-inoculated control samples. All three bacterial species were shown to grow in vacuum packaged lamb of pH values between 5.4 and 6.4, when stored at chilled temperatures (−1.5 to 7°C) for up to 84days. B. thermosphacta and S. proteamaculans caused spoilage to the meat under these conditions whilst R. aquatilis spoiled high pH meat at 7°C. These results go against previous beef models stipulating that Brochothrix and Enterobacteriacae species cannot grow on or cause spoilage of low pH meat in the absence of oxygen. [ABSTRACT FROM AUTHOR]

[Display omitted] • Cell structure was damaged and cell membrane permeability was enhanced in Brochothrix thermosphacta under linalool stress. • Respiratory metabolism of Brochothrix thermosphacta was impaired when exposed to linalool. • Energy limitation accompanied by oxidative stress appeared in Brochothrix thermosphacta after treated with linalool. • Linalool had superiorities for chilled beef preservation contaminated with Brochothrix thermosphacta during storage. This work aimed to explore the antibacterial ability and potential mechanism of linalool against Brochothrix thermosphacta (B. thermosphacta), providing knowledge of the preservation of chilled beef with linalool. The results found that linalool had an encouraging inhibitory effect on B. thermosphacta with a minimum inhibitory concentration (MIC) of 1.5 mL/L. Results of FESEM and zeta potential combined with probe labeling confirmed that linalool destroyed the cell structure thereby causing the leakage of intracellular components (AKP, protein, nucleic acid and ion). In addition, linalool caused respiratory disturbance by measuring the key enzyme activities including PK, SDH, MDH and ATPase. Energy limitation also appeared under linalool stress as seen from changes in ATP content (decreased by 56.06% and 69.24% in MIC and 2MIC groups, respectively). The respiratory inhibition rate of linalool to B. thermosphacta was 23.58% and the superposing rate with malonic acid was minimal (35.52%), suggesting that respiratory depression was mainly caused by the TCA cycle. Furthermore, accumulation of ROS and increase in MDA content (increased by 71.17% and 78.03% in MIC and 2MIC groups, respectively) accompanied by decreased activities of detoxification enzymes CAT and POD suggested that oxidative stress contributed to the bactericidal mechanism. Finally, linalool has been shown to effectively inhibit quality deterioration of chilled beef during storage by measuring pH, TVB-N and TVC without affecting sensory acceptability. All these highlight the great promise of using linalool as natural preservative for food industry. [ABSTRACT FROM AUTHOR]

[Display omitted] • P. lundensis and B. thermosphacta exhibit different biochemical process related to beef spoilage. • Metabolomic profiles of spoilage bacteria were differentiated by UHPLC-MS/MS. • Eight potential spoilage biomarkers were enriched including creatine, inosine. • Histidine metabolism was involved in spoilage of inoculated ground beef. • Complex and competitive interactions were revealed in the co-culture of beef. Pseudomonas lundensis and Brochothrix thermosphacta are key spoilage microorganisms in aerobically stored chilled meat. The present study aimed to investigate the physicochemical and metabolomic profiles of refrigerated ground beef inoculated P. lundensis (PL) and B. thermosphacta (BT) as mono- or co-culture (BP). P. lundensis was the dominant spoilage strain in the co-culture of ground beef. A large amount of TCA-soluble peptide, TVB-N and TBA were formed in the PL and BP, while acetion was mainly produced in the BT, as accompanied by the different sensory and color changes. Meat metabolome indicated that 95, 396, and 409 metabolites with significant differences, were identified in ground beef inoculated BT, PL, and BP, respectively. These differential metabolites covered 58 metabolic pathways, in which histidine metabolism was identified as an important pathway related to spoilage in the three groups. Specifically, creatine, inosine, anserine, uracil, alanine, glutamine, 3-methylhistidine and 3-hydroxycapric acid were enriched as potential spoilage biomarkers. Taken together, those findings reveal the complex and competitive interactions of their co-culture of B. thermosphacta and P. lundensis , which provided a comprehensive insight into microbial spoilage mechanism in chilled beef. [ABSTRACT FROM AUTHOR]

Abstract: A new and extensive growth and growth boundary model for psychrotolerant Lactobacillus spp. was developed and validated for processed and unprocessed products of seafood and meat. The new model was developed by refitting and expanding an existing cardinal parameter model for growth and the growth boundary of lactic acid bacteria (LAB) in processed seafood (O. Mejlholm and P. Dalgaard, J. Food Prot. 70. 2485–2497, 2007). Initially, to estimate values for the maximum specific growth rate at the reference temperature of 25°C (μref ) and the theoretical minimum temperature that prevents growth of psychrotolerant LAB (Tmin ), the existing LAB model was refitted to data from experiments with seafood and meat products reported not to include nitrite or any of the four organic acids evaluated in the present study. Next, dimensionless terms modelling the antimicrobial effect of nitrite, and acetic, benzoic, citric and sorbic acids on growth of Lactobacillus sakei were added to the refitted model, together with minimum inhibitory concentrations determined for the five environmental parameters. The new model including the effect of 12 environmental parameters, as well as their interactive effects, was successfully validated using 229 growth rates (μmax values) for psychrotolerant Lactobacillus spp. in seafood and meat products. Average bias and accuracy factor values of 1.08 and 1.27, respectively, were obtained when observed and predicted μmax values of psychrotolerant Lactobacillus spp. were compared. Thus, on average μmax values were only overestimated by 8%. The performance of the new model was equally good for seafood and meat products, and the importance of including the effect of acetic, benzoic, citric and sorbic acids and to a lesser extent nitrite in order to accurately predict growth of psychrotolerant Lactobacillus spp. was clearly demonstrated. The new model can be used to predict growth of psychrotolerant Lactobacillus spp. in seafood and meat products e.g. prediction of the time to a critical cell concentration of bacteria is considered useful for establishing the shelf life. In addition, the high number of environmental parameters included in the new model makes it flexible and suitable for product development as the effect of substituting one combination of preservatives with another can be predicted. In general, the performance of the new model was unacceptable for other types of LAB including Carnobacterium spp., Leuconostoc spp. and Weissella spp. [Copyright &y& Elsevier]

Abstract: A total of 306 colonies were isolated from the selective medium for Brochothrix spp., during the spoilage of minced pork stored at 0, 5, 10 and 15°C and packed aerobically and under modified atmosphere packaging conditions (MAP). Brochothrix biodiversity was assessed by Pulsed Field Gel Electrophoresis (PFGE), and representative strains were further analysed by Rep-PCR using primer (GTG)5 and Sau-PCR with primers SAG1and SAG2. Although, different results were obtained from the different methods, a significant diversity among isolates recovered from aerobic conditions was observed. On the contrary, isolates from MAP showed a lower degree of heterogeneity. The storage conditions affected the Brochothrix diversity, the strains isolated in the initial stage being different from the ones present at the final stage of storage at chill temperatures. A representative number of isolates, based on the results of the clustering by molecular methods, were subjected to 16S rRNA gene sequencing, revealing that all belonged to Brochothrix thermosphacta. [Copyright &y& Elsevier]

Abstract: The objective of the study was to determine the Minimal Inhibitory Concentration (MIC) of thyme (29.4% thymol, 21.6% p-cymene) and rosemary essential oils (27.6% 1,8-cineole, 13.5% limonene, 13.0% β-pinene) against Brochothrix thermosphacta and to establish the feasibility of their use as components of modified atmosphere during beef refrigerated storage. The minimum inhibitory concentration (MIC) of thyme oil against B. thermosphacta is 0.05% and that of rosemary oil 0.5%. The MIC values are independent on strain and temperature of growth, however the bactericidal effects are strain dependent. The addition of any of oil at a concentration equal to 2MIC to the modified atmosphere (80% O2/20% CO2) does not significantly influence the microbial quality of meat. At the same time, such a concentration of the essential oils was considerably detrimental to the organoleptic factors. [Copyright &y& Elsevier]

Polyphenol mixtures obtained from olive mill wastewater were tested for their activity against spoiling and pathogenic bacteria commonly isolated from meat products, such as Brochothrix thermosphacta, Listeria monocytogenes, Salmonella enterica subsp. Derby and Staphylococcus aureus. Minimal Inhibitory Concentrations were determined, and time kill kinetics revealed a strong action at very low concentrations. In particular, 0.25% extract was able to immediately inhibit the growth of L. monocytogenes ATCC 7644 and S. aureus ATCC 32760, while at the same concentration Salmonella enterica subsp. Derby S1 and B. thermosphacta ATCC 11059 were inhibited after 60 and 120 minutes, respectively. Moreover, growth dynamics were evaluated by means of automated turbidimetry. The analysis of growth parameters revealed a bacteriostatic action of olive wastewater extracts at concentrations lower than or equal to 0.5%, depending on the strain, and a bactericidal action at higher concentrations. Results obtained showed that olive mill wastewater extracts exerted a strong and fast in vitro antimicrobial activity against some spoiling and pathogenic microorganisms. Our data provide insight into a possible application of these extracts as antimicrobial compounds, although their application on foods still has to be optimized. Moreover, olive mill wastewaters have a strong polluting impact, and therefore their application as biopreserving agents could increase their value, converting a waste material into a powerful resource. [ABSTRACT FROM AUTHOR]

Abstract: The maximum specific growth rate (μ max) of Brochothrix thermosphacta, a spoilage bacteria of cooked peeled shrimp, and Lactococcus piscium CNCM I-4031, a bioprotective strain, was investigated under different conditions of temperature, NaCl concentrations and pH. The basic modelling approach used was the Gamma concept (γ-concept) and the model developed was then adapted to shrimp. Cardinal growth parameters were quite similar for the two strains, except for NaCl. No NaCl was required for growth and the NaClmax was three-times higher for B. thermosphacta than for L. piscium (62 and 23 g l−1 respectively). However, tolerance to NaCl was higher in seafood than in liquid broth, possibly due to presence of osmoltically active molecules. L. piscium and B. thermosphacta were psychrotolerant, with T min = −4.8 and −3.4 °C, T opt = 23.4 and 27.0 °C and T max = 27.2 and 30.8 °C respectively. The optimal pH was neutral and growth possible till pH = 4.8 for the two strains, assuming possible applications of the bioprotective strain in lightly marinated seafood. The μ max of B. thermosphacta in shrimp was a little higher than in L. piscium whatever the environmental conditions. Validation of the model showed that the γ-concept was suitable for predicting μ max of B. thermosphacta in shrimp. Data generated in this study can be used to adapt the model to other foods with few additional experiments and the effect of different parameters may be added in the future. The model was less accurate for the bioprotective strain and the effect of NaCl must be studied in more detail directly in the matrix. [Copyright &y& Elsevier]

Abstract: Brochothrix thermosphacta, a Gram-positive bacterium, is considered as the predominant spoilage microbiota of modified atmosphere packing (MAP) shrimp and fish. Traditional methods currently used to detect B. thermosphacta in foods are time-consuming and labour-intensive. The aim of this study was to develop a real-time PCR quantification method combined with a propidium monoazide (PMA) sample treatment step to monitor the population of B. thermosphacta in cooked shrimp and salmon. The specificity of the two primers MO405 and MO404 used to amplify a 70bp fragment of the 16S rRNA gene was demonstrated by using purified DNA from 30 strains, among 21 bacterial species including 22 reference strains. Using these primers for real-time PCR and in pure culture, a good correlation was obtained between real-time PCR and the conventional plating method. Quantification was linear over 7-log units using artificially inoculated samples. The method performed successfully when tested on naturally contaminated cooked shrimp and fresh salmon, with a minimum threshold of 1.9×102 CFU/g for accurate quantification of B. thermosphacta. The correlation between the B. thermosphacta counts obtained by real-time PCR and plate counts on naturally contaminated shrimp and salmon was high (R 2 =0.895). Thus, this study presents a rapid tool for producing reliable quantitative data on B. thermosphacta in cooked shrimp and fresh salmon. [Copyright &y& Elsevier]