Impact of Electrolyzed Water on the Microbial Spoilage Profile of Piedmontese Steak Tartare.

A low initial contamination level of the meat surface is the sine qua non to extend the subsequent shelf life of ground beef for as long as possible. Therefore, the short- and long-term effects of a pregrinding treatment with electrolyzed water (EW) on the microbiological and physicochemical features of Piedmontese steak tartare were here assessed on site, by following two production runs through storage under vacuum packaging conditions at 4°C. The immersion of muscle meat in EW solution at 100 ppm of free active chlorine for 90 s produced an initial surface decontamination with no side effects or compositional modifications, except for an external color change that was subsequently masked by the grinding step. However, the initially measured decontamination was no longer detectable in ground beef, perhaps due to a quick recovery by bacteria during the grinding step from the transient oxidative stress induced by the EW. We observed different RNA-based metataxonomic profiles and metabolomic biomarkers (volatile organic compounds [VOCs], free amino acids [FAA], and biogenic amines [BA]) between production runs. Interestingly, the potentially active microbiota of the meat from each production run, investigated through operational taxonomic unit (OTU)-, oligotyping-, and amplicon sequence variant (ASV)-based bioinformatic pipelines, differed as soon as the early stages of storage, whereas microbial counts and biomarker dynamics were significantly distinguishable only after the expiration date. Higher diversity, richness, and abundance of Streptococcus organisms were identified as the main indicators of the faster spoilage observed in one of the two production runs, while Lactococcus piscium development was the main marker of shelf life end in both production runs. IMPORTANCE Treatment with EW prior to grinding did not result in an effective intervention to prolong the shelf life of Piedmontese steak tartare. Our RNA-based approach clearly highlighted a microbiota that changed markedly between production runs but little during the first shelf life stages. Under these conditions, an early metataxonomic profiling might provide the best prediction of the microbiological fate of each batch of the product.

Ubericin K, a New Pore-Forming Bacteriocin Targeting mannose-PTS.

Bovine mastitis infection in dairy cattle is a significant economic burden for the dairy industry globally. To reduce the use of antibiotics in treatment of clinical mastitis, new alternative treatment options are needed. Antimicrobial peptides from bacteria, also known as bacteriocins, are potential alternatives for combating mastitis pathogens. In search of novel bacteriocins against mastitis pathogens, we screened samples of Norwegian bovine raw milk and found a Streptococcus uberis strain with potent antimicrobial activity toward Enterococcus, Streptococcus, Listeria, and Lactococcus. Whole-genome sequencing of the strain revealed a multibacteriocin gene cluster encoding one class IIb bacteriocin, two class IId bacteriocins, in addition to a three-component regulatory system and a dedicated ABC transporter. Isolation and purification of the antimicrobial activity from culture supernatants resulted in the detection of a 6.3-kDa mass peak by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, a mass corresponding to the predicted size of one of the class IId bacteriocins. The identification of this bacteriocin, called ubericin K, was further confirmed by in vitro protein synthesis, which showed the same inhibitory spectrum as the purified antimicrobial compound. Ubericin K shows highest sequence similarity to the class IId bacteriocins bovicin 255, lactococcin A, and garvieacin Q. We found that ubericin K uses the sugar transporter mannose phosphotransferase (PTS) as a target receptor. Further, by using the pHlourin sensor system to detect intracellular pH changes due to leakage across the membrane, ubericin K was shown to be a pore former, killing target cells by membrane disruption. IMPORTANCE Bacterial infections in dairy cows are a major burden to farmers worldwide because infected cows require expensive treatments and produce less milk. Today, infected cows are treated with antibiotics, a practice that is becoming less effective due to antibiotic resistance. Compounds other than antibiotics also exist that kill bacteria causing infections in cows; these compounds, known as bacteriocins, are natural products produced by other bacteria in the environment. In this work, we discover a new bacteriocin that we call ubericin K, which kills several species of bacteria known to cause infections in dairy cows. We also use in vitro synthesis as a novel method for rapidly characterizing bacteriocins directly from genomic data, which could be useful for other researchers. We believe that ubericin K and the methods described in this work will aid in the transition away from antibiotics in the dairy industry.

BacSJ-Another Bacteriocin with Distinct Spectrum of Activity that Targets Man-PTS.

Lactic acid bacteria produce diverse antimicrobial peptides called bacteriocins. Most bacteriocins target sensitive bacteria by binding to specific receptors. Although a plethora of bacteriocins have been identified, for only a few of them the receptors they recognize are known. Here, we identified permease IIC and surface protein IID, two membrane subunits of the mannose-specific quaternary phosphotransferase system (Man-PTS), as a receptor for BacSJ, a subclass IId bacteriocin produced by Lactobacillus paracasei subsp. paracasei BGSJ2-8. BacSJ shares 45% identity with another Man-PTS binding bacteriocin, garvicin Q (GarQ). Similarly to GarQ, BacSJ has a relatively broad activity spectrum acting against several Gram-positive bacteria, such as Lactococcus lactis and Listeria monocytogenes, harboring fairly similar Man-PTSs, but not against Lactococcus garvieae. To identify specific Man-PTS amino acids responsible for the L.lactis sensitivity to BacSJ, and thus likely involved in the interaction with this bacteriocin, we generated eight independent BacSJ resistant L.lactis mutants harboring five distinct missense mutations in the ptnC or ptnD genes encoding the IIC and IID subunits. Concurrently with the resistance to BacSJ, the mutants efficiently utilized mannose as a carbon source, which indicated functionality of their mutated Man-PTS. The amino acid substitutions in the mutants localized to the intracellular region of the IIC permease or to the extracellular parts of IID. This localization coincides with regions targeted by GarQ and some other Man-PTS-binding garvicins, pointing to similarities between all these bacteriocins in the mechanism of their interaction with Man-PTS. During the attack by these bacteriocins, subunits IID and IIC are assumed to function sequentially as a docking and an entry module allowing the toxic peptide to bind the cell and then open the pore. However, since not all of the BacSJ-resistant mutants exhibited cross-resistance to GarQ, we propose that BacSJ interacts with Man-PTS in a manner slightly different from that of GarQ.

Different growth kinetics in blood components and genetic analysis of Lactococcus garvieae isolated from platelet concentrates.

BACKGROUND: In 2014, we experienced the first isolation of Lactococcus garvieae from a platelet concentrate (PC). Thereafter, L. garvieae contamination of PCs occurred in two more cases in Japan. It is rare that bacterial contamination with uncommon strains like this species occurs frequently within a short period. Therefore, we performed a detailed analysis of the characteristics of these strains. STUDY DESIGN AND METHODS: Three bacterial strains were identified by biochemical testing and molecular analysis. Genomic diversity was characterized by multilocus sequence typing (MLST). To observe growth kinetics in blood components, PCs were inoculated with the three different strains. RESULTS: All three strains were identified as L. garvieae by molecular analysis. Each strain belonged to a different phylogenetic group according to MLST analysis. In the spiking trial, the three strains demonstrated differences in their final concentrations and changes in appearance of PCs. CONCLUSION: In this study, all three L. garvieae strains were correctly identified by molecular analysis. Since the three strains were collected in different regions of Japan and belonged to different phylogenetic groups according to MLST analysis, it is suggested that L. garvieae have a wide distribution with diversity in Japan. In PCs, the three L. garvieae strains showed clear differences in growth kinetics and changes in appearance of PCs. These differences may have been the primary determinant of whether PC contamination was detected before transfusion. Moreover, L. garvieae represents an emerging foodborne bacterium that can cause transfusion-transmitted bacteremia. Understanding our cases may help prevent bacterial contamination of blood products.

Screening of lactic acid bacteria producing folate and their potential use as adjunct cultures for cheese bio-enrichment.

Lactic acid bacteria (LAB) can be used to increase the folate in foods by in situ fortification. Seventy LAB were screened for their ability to produce folate during growth in de Man, Rogosa and Sharpe/M17 broth. Lactobacillus casei, Lactobacillus plantarum, Lactobacillus paracasei subsp. paracasei, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactococcus lactis subsp. lactis, Enterococcus faecium and Enterococcus lactis were able to synthetize folates in the medium, even if to a different extent. The 47 folate-producing strains were further analyzed by microbiological assay, for total, extra and intracellular folate. Enterococcus faecium VC223 and E. lactis BT161 were able to produce in cultural medium 123,625.74 ± 8.00 ng/ml and 384.22 ± 5.00 ng/ml of folate, respectively. Five strains were further examined for their ability to synthesize folate in cheese. The folate content increased with ripening up to by 54% after 30 d when L. casei VC199 was used and up to 108% and 113% after 60 d, with L. paracasei SE160 and E. lactis BT161 respectively exceeding 100 ng/100g. Results encourage the use of specific LAB to obtain natural folate bio-enriched dairy products improving folate intake.

Cell Growth Density and Nisin A Activity of the Indigenous Lactococcus lactis subsp. cremoris M78 Costarter Depend Strongly on Inoculation Levels of a Commercial Streptococcus thermophilus Starter in Milk: Practical Aspects for Traditional Greek Cheese Processors.

ABSTRACT: Mixed thermophilic and mesophilic commercial starter cultures (CSCs), particularly those including Streptococcus thermophilus as a primary milk acidifier, have been found to reduce growth and counteract in situ nisin A (NisA+) antilisterial effects by the novel, indigenous Lactococcus lactis subsp. cremoris M78 costarter in traditional Graviera thermized milk cheese curds. Therefore, this model challenge study evaluated growth and in situ NisA+ activity of strain M78 in coculture with S. thermophilus ST1 singly in sterilized raw milk (SRM). Strain ST1, derived from a CSC for cheese, was challenged at two inoculation levels (5 and 7 log CFU/mL) in SRM against 6 and 3 log CFU/mL of strain M78 and Listeria monocytogenes, respectively. Pure cultures of each strain and cocultures of strain ST1 with the CSC L. lactis LL2, in replacement of strain M78, served as controls. At the high (7-log) inoculation level, the rapid, competitive growth (>9.3 log CFU/mL) of S. thermophilus ST1 reduced growth of both L. lactis by at least 10-fold; the industrial strain LL2 retained slightly higher relative population densities (7.4 to 9.1%) than the wild NisA+ strain M78 (3.8 to 5.6%) after 6 h at 37°C, followed by an additional 66 h of incubation at 22°C. In full contrast, at the low (5-log) inoculation level, S. thermophilus ST1 failed to predominate in SRM at 6 h; thus, the starter lactic acid bacteria populations were reversed in favor of L. lactis. Notably, strain M78 retained higher relative population densities (83.0 to 90.1%) than the CSC strain LL2 (80.3 to 85.2%) at 22°C. Moreover, at the 5-log ST1 level, the direct and deferred in situ NisA+ activities of strain M78 were at similar levels with its pure culture with L. monocytogenes in SRM, whereas at the 7-log ST1 level, the respective NisA+ effects were counteracted. Hence, 10- to 100-fold lowered inoculation levels of CSC S. thermophilus are required to enhance the performance of the M78 costarter in traditional Greek cheese technologies.

Effects of Enterococcus faecalis UGRA10 and the enterocin AS-48 against the fish pathogen Lactococcus garvieae. Studies in vitro and in vivo.

The aim of this study was to evaluate the effects of Enterococcus faecalis UGRA10 and its enterocin AS-48 against the fish pathogen Lactococcus garvieae. The minimum bactericidal concentrations of AS-48 against L. garvieae CECT 5807, 5806, and 5274 were 15.62, 15.62, and 7.81 µg/ml respectively. In broth cultures, enterocin at 100, 50, and 25 µg/ml reduced 108 CFU/ml lactococci after 2, 5, and 10 h, respectively. In co-cultures of UGRA10/L. garvieae at a 1/10 CFU/ml ratio, lactococci were eliminated after 24 h. Studies on UGRA10 biosafety and AS-48 toxicity in R1 cells and in rainbow trout have shown a lack of adverse effects from both the strain and bacteriocin. Trout challenged with L. garvieae and UGRA10 administered in diet 30 days before infection had a cumulative survival rate of 50% compared with 0% for control fish. Trout inoculated with the pathogen and treated by regular dipping in AS-48 baths had a survival rate of 60% after 20 days compared with that of untreated fish (0%). These results indicate the protective effect of the UGRA10 strain and the bacteriocin AS-48 against L. garvieae and the potential of these natural products as alternatives to antibiotics for controlling diseases in aquaculture.

Food Spoilage-Associated Leuconostoc, Lactococcus, and Lactobacillus Species Display Different Survival Strategies in Response to Competition.

Psychrotrophic lactic acid bacteria (LAB) are the prevailing spoilage organisms in packaged cold-stored meat products. Species composition and metabolic activities of such LAB spoilage communities are determined by the nature of the meat product, storage conditions, and interspecies interactions. Our knowledge of system level responses of LAB during such interactions is very limited. To expand it, we studied interactions between three common psychrotrophic spoilage LAB (Leuconostoc gelidum, Lactococcus piscium, and Lactobacillus oligofermentans) by comparing their time course transcriptome profiles obtained during their growth in individual, pairwise, and triple cultures. The study revealed how these LAB employed different strategies to cope with the consequences of interspecies competition. The fastest-growing bacterium, Le. gelidum, attempted to enhance its nutrient-scavenging and growth capabilities in the presence of other LAB through upregulation of carbohydrate catabolic pathways, pyruvate fermentation enzymes, and ribosomal proteins, whereas the slower-growing Lc. piscium and Lb. oligofermentans downregulated these functions. These findings may explain the competitive success and predominance of Le. gelidum in a variety of spoiled foods. Peculiarly, interspecies interactions induced overexpression of prophage genes and restriction modification systems (mechanisms of DNA exchange and protection against it) in Lc. piscium and Lb. oligofermentans but not in Le. gelidum Cocultivation induced also overexpression of the numerous putative adhesins in Lb. oligofermentans These adhesins might contribute to the survival of this slowly growing bacterium in actively growing meat spoilage communities.IMPORTANCE Despite the apparent relevance of LAB for biotechnology and human health, interactions between members of LAB communities are not well known. Knowledge of such interactions is crucial for understanding how these communities function and, consequently, whether there is any possibility to develop new strategies to interfere with their growth and to postpone spoilage of packaged and refrigerated foods. With the help of controlled experiments, detailed regulation events can be observed. This study gives an insight into the system level interactions and the different competition-induced survival strategies related to enhanced uptake and catabolism of carbon sources, overexpression of adhesins and putative bacteriocins, and the induction of exchange of genetic material. Even though this experiment dealt with only three LAB strains in vitro, these findings agreed well with the relative abundance patterns typically reported for these species in natural food microbial communities.

Bacterial Communities in Serpa Cheese by Culture Dependent Techniques, 16S rRNA Gene Sequencing and High-throughput Sequencing Analysis.

Serpa cheese is one of the traditional regional Portuguese cheeses having the Protected Denomination of Origin (PDO) designation. This study investigated the bacterial community in the traditional Portuguese Serpa cheese. The microorganisms identified at the end of ripening (30 days) mainly were lactic acid bacteria (LAB). Lactobacillus paracasei/Lactobacillus casei was the main species in cheese from PDO registered industries, whereas in non-PDO registered industries Lactobacillus brevis was highlighted, among other LAB. Enterobacteriaceae species were detected at 20% to 40% of the total isolates. The results obtained by high-throughput sequencing analysis confirmed that LAB was the main microbial group, with Lactococcus genus contributing to approximately 40% to 60% of the population, followed by Leuconostoc and Lactobacillus. The Enterobacteriaceae family was also important. The differences between bacterial communities from PDO and non-PDO registered industries suggest that the lack of regulation of the cheese-making practices may influence unfavorably. The new knowledge about bacterial diversity in Serpa cheese could be useful to set up new ripening conditions, which favor the development of desirable microorganisms. PRACTICAL APPLICATION: The control of the manufacturing process of traditional cheeses can be improved through the knowledge of the bacterial diversity that develops. Thus, the growth of desirable microorganisms can be promoted to homogenize the final product.

Assertiveness of meat-borne Lactococcus piscium strains and their potential for competitive exclusion of spoilage bacteria in situ and in vitro.

AIMS: This study aimed to investigate intraspecies assertiveness of meat-borne Lactococcus piscium isolates, inhibitory effects on unwanted and harmful meat spoilers, and the prevalence on beef deliberately inoculated with Lc. piscium. METHODS AND RESULTS: Co-inoculation of Lc. piscium isolates and spoilers (Brochothrix thermosphacta, Leuconostoc gelidum subsp. gasicomitatum, Carnobacterium divergens, Pseudomonas weihenstephanensis, Serratia liquefaciens, Hafnia alvei) were conducted in sterile meat simulation medium. Differentiation of Lc. piscium strains was carried out with colony-based RAPD-PCR. Selective cultivation was used to differentiate spoilers from Lc. piscium. Intraspecies assertiveness revealed Lc. piscium TMW2.1614 as most assertive strain. Co-inoculation of selected Lc. piscium strains caused substantial growth reduction of spoilers while the extent was strain- and spoiler dependent. Monitoring the microbiota on beef steaks deliberately inoculated with Lc. piscium revealed prevalence over the endogenous microbiota while maintaining a ripened sensory impression without undesired alterations. CONCLUSIONS: This study reveals Lc. piscium strains TMW2.1612/2.1614/2.1615 as highly competitive against spoilers in vitro while beef deliberately inoculated with these strains maintained acceptable organoleptics. SIGNIFICANCE AND IMPACT OF THE STUDY: Selected Lc. piscium strains exhibit high potential for application as bioprotective cultures for competitive exclusion on beef in order to extend minimum shelf life and enhance product safety of meat.

Detection of virulence-related genes in Lactococcus garvieae and their expression in response to different conditions.

Lactococcus garvieae has emerged as an important zoonotic pathogen. However, information regarding mechanisms and factors related to its pathogenicity is lacking. In the present study, we investigated the distribution and functionality of genes related to virulence factors in L. garvieae strains isolated from different niches (diseased fish, humans, meat and dairy products, vegetables), using both post-genomic and genotypic analysis. Putative genes encoding hemolysin, fibronectin-binding protein, and penicillin acylase were detected in all analyzed genomes/strains. Their expression was significantly induced by bile salt stress. Putative genes encoding bile salt hydrolase were found in a few strains from dairy and human sources, as well as the mobilizable tet genes. Finally, all genomes possessed a folate gene cluster, in which mutations in the dihydropteroate synthase gene (folP) could be related to sulfonamide resistance. To the best of our knowledge, this is the first study aimed to explore the pathogenic potential of L. garvieae through the analysis of numerous L. garvieae genomes/strains, coming from different sources. This approach allowed the detection of virulence-related genes not yet investigated in the species and the study of their expression after exposure to different environmental stresses. The results obtained suggest a virulence potential in some L. garvieae strains that can be exploited for survival in the human gastrointestinal tract.

The microbial community of the gut differs between piglets fed sow milk, milk replacer or bovine colostrum.

The aim of this study was to characterise the gut microbiota composition of piglets fed bovine colostrum (BC), milk replacer (MR) or sow milk (SM) in the post-weaning period. Piglets (n 36), 23-d old, were randomly allocated to the three diets. Faecal samples were collected at 23, 25, 27 and 30 d of age. Digesta from the stomach, ileum, caecum and mid-colon was collected at 30 d of age. Bacterial DNA from all samples was subjected to amplicon sequencing of the 16S rRNA gene. Bacterial enumerations by culture and SCFA analysis were conducted as well. BC-piglets had the highest abundance of Lactococcus in the stomach (P<0·0001) and ileal (P<0·0001) digesta, whereas SM-piglets had the highest abundance of Lactobacillus in the stomach digesta (P<0·0001). MR-piglets had a high abundance of Enterobacteriaceae in the ileal digesta (P<0·0001) and a higher number of haemolytic bacteria in ileal (P=0·0002) and mid-colon (P=0·001) digesta than SM-piglets. BC-piglets showed the highest colonic concentration of iso-butyric and iso-valeric acid (P=0·02). Sequencing and culture showed that MR-piglets were colonised by a higher number of Enterobacteriaceae, whereas the gut microbiota of BC-piglets was characterised by a change in lactic acid bacteria genera when compared with SM-piglets. We conclude that especially the ileal microbiota of BC-piglets had a closer resemblance to that of SM-piglets in regard to the abundance of potential enteric pathogens than did MR-piglets. The results indicate that BC may be a useful substitute for regular milk replacers, and as a feeding supplement in the immediate post-weaning period.

Biosynthesis and role of 3-methylbutanal in cheese by lactic acid bacteria: Major metabolic pathways, enzymes involved, and strategies for control.

Branched chain aldehyde, 3-methylbutanal is associated as a key flavor compound with many hard and semi-hard cheese varieties. The presence and impact of this flavor compound in bread, meat, and certain beverages has been recently documented, however its presence and consequences regarding cheese flavor were not clearly reported. This paper gives an overview of the role of 3-methylbutanal in cheese, along with the major metabolic pathways and key enzymes leading to its formation. Moreover, different strategies are highlighted for the control of this particular flavor compound in specific cheese types.

Analysis of the duodenal microbiotas of weaned piglet fed with epidermal growth factor-expressed Saccharomyces cerevisiae.

BACKGROUND: The bacterial community of the small intestine is a key factor that has strong influence on the health of gastrointestinal tract (GIT) in mammals during and shortly after weaning. The aim of this study was to analyze the effects of the diets of supplemented with epidermal growth factor (EGF)-expressed Saccharomyces cerevisiae (S. cerevisiae) on the duodenal microbiotas of weaned piglets. RESULTS: Revealed in this study, at day 7, 14 and 21, respectively, the compositional sequencing analysis of the 16S rRNA in the duodenum had no marked difference in microbial diversity from the phylum to species levels between the INVSc1(EV) and other recombinant strains encompassing INVSc1-EE(+), INVSc1-TE(-), and INVSc1-IE(+). Furthermore, the populations of potentially enterobacteria (e.g., Clostridium and Prevotella) and probiotic (e.g., Lactobacilli and Lactococcus) also remained unchanged among recombinant S. cerevisiae groups (P > 0.05). However, the compositional sequencing analysis of the 16S rRNA in the duodenum revealed significant difference in microbial diversity from phylum to species levels between the control group and recombinant S. cerevisiae groups. In terms of the control group (the lack of S. cerevisiae), these data confirmed that dietary exogenous S. cerevisiae had the feasibility to be used as a supplement for enhancing potentially probiotic (e.g., Lactobacilli and Lactococcus) (P < 0.01), and reducing potentially pathogenic bacteria (e.g., Clostridium and Prevotella) (P < 0.01). CONCLUSION: Herein, altered the microbiome effect was really S. cerevisiae, and then different forms of recombinant EGF, including T-EGF, EE-EGF and IE-EGF, did not appear to make a significant difference to the microbiome of weaned piglets.

Effects of the Essential Oil from Origanum vulgare L. on Survival of Pathogenic Bacteria and Starter Lactic Acid Bacteria in Semihard Cheese Broth and Slurry.

This study assessed the inhibitory effects of the essential oil from Origanum vulgare L. (OVEO) on Staphylococcus aureus, Listeria monocytogenes, and a mesophilic starter coculture composed of lactic acid bacteria (Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris) in Brazilian coalho cheese systems. The MIC of OVEO was 2.5 µl/ml against both S. aureus and L. monocytogenes and 0.6 µl/ml against the tested starter coculture. In cheese broth containing OVEO at 0.6 µl/ml, no decrease in viable cell counts (VCC) of both pathogenic bacteria was observed, whereas the initial VCC of the starter coculture decreased approximately 1.0 log CFU/ml after 24 h of exposure at 10°C. OVEO at 1.25 and 2.5 µl/ml caused reductions of up to 2.0 and 2.5 log CFU/ml in S. aureus and L. monocytogenes, respectively, after 24 h of exposure in cheese broth. At these same concentrations, OVEO caused a greater decrease of initial VCC of the starter coculture following 4 h of exposure. Higher concentrations of OVEO were required to decrease the VCC of all target bacteria in semisolid coalho cheese slurry compared with cheese broth. The VCC of Lactococcus spp. in coalho cheese slurry containing OVEO were always lower than those of pathogenic bacteria under the same conditions. These results suggest that the concentrations of OVEO used to control pathogenic bacteria in semihard cheese should be carefully evaluated because of its inhibitory effects on the growth of starter lactic acid cultures used during the production of the product.

Inhibition mechanism of Listeria monocytogenes by a bioprotective bacteria Lactococcus piscium CNCM I-4031.

Listeria monocytogenes is a pathogenic Gram positive bacterium and the etiologic agent of listeriosis, a severe food-borne disease. Lactococcus piscium CNCM I-4031 has the capacity to prevent the growth of L. monocytogenes in contaminated peeled and cooked shrimp. To investigate the inhibititory mechanism, a chemically defined medium (MSMA) based on shrimp composition and reproducing the inhibition observed in shrimp was developed. In co-culture at 26 °C, L. monocytogenes was reduced by 3-4 log CFU g(-1) after 24 h. We have demonstrated that the inhibition was not due to secretion of extracellular antimicrobial compounds as bacteriocins, organic acids and hydrogen peroxide. Global metabolomic fingerprints of these strains in pure culture were assessed by liquid chromatography coupled with high resolution mass spectrometry. Consumption of glucose, amino-acids, vitamins, nitrogen bases, iron and magnesium was measured and competition for some molecules could be hypothesized. However, after 24 h of co-culture, when inhibition of L. monocytogenes occurred, supplementation of the medium with these compounds did not restore its growth. The inhibition was observed in co-culture but not in diffusion chamber when species were separated by a filter membrane. Taken together, these data indicate that the inhibition mechanism of L. monocytogenes by L. piscium is cell-to-cell contact-dependent.

Changes in microbial diversity of brined green asparagus upon treatment with high hydrostatic pressure.

The application of high hydrostatic pressure (HHP, 600MPa, 8 min) on brined green asparagus and the changes in bacterial diversity after treatments and during storage at 4 °C (30 days) or 22 °C (10 days) were studied. HHP treatments reduced viable cell counts by 3.6 log cycles. The residual surviving population did not increase during storage at 4 °C. However, bacterial counts significantly increased at 22 °C by day 3, leading to rapid spoilage. The microbiota of green asparagus was composed mainly by Proteobacteria (mainly Pantoea and Pseudomonas), followed by Firmicutes (mainly Lactococcus and Enterococcus) and to a less extent Bacteroidetes and Actinobacteria. During chill storage of untreated asparagus, the relative abundance of Proteobacteria as well as Enterococcus and Lactococcus decreased while Lactobacillus increased. During storage of untreated asparagus at 22 °C, the abundance of Bacteroidetes decreased while Proteobacteria increased during late storage. The HHP treatment determined a reduction of the Proteobacteria both early after treatment and during chill storage. In the HHP treated samples stored at 22 °C, the relative abundance of Pseudomonas rapidly decreased at day 1, with an increase of Bacteroidetes. This was followed by a marked increase in Enterobacteriaceae (Escherichia) simultaneously with increase in viable counts and spoilage. Results from the study indicate that the effect of HHP treatments on the viability ofmicrobial populations in foods also has an impact on the dynamics of microbial populations during the storage of the treated foods.

Impact of NaCl reduction in Danish semi-hard Samsoe cheeses on proliferation and autolysis of DL-starter cultures.

Reduction of sodium chloride (NaCl) in cheese manufacturing is a challenge for the dairy industry. NaCl has a profound role on microbial development influencing cheese sensory and technological properties. The purpose of this work was to investigate how proliferation, distribution and autolysis of two commercial DL-starter cultures (C1 and C2) used in the production of Danish semi-hard Samsoe cheeses were affected by reduced NaCl levels. Cheeses containing <0.3% (unsalted), 2.3% (reduced-salt) and 3.4% (normal-salted) (w/v) NaCl in moisture were produced and analyzed during 12 weeks of ripening. Lactic acid bacteria (LAB), distribution of bacteria as single cells or microcolonies, their viability in the cheeses and cell autolysis were monitored during ripening, as well as the impact of NaCl content and autolysis on the formation of free amino acids (FAA). Reduction of NaCl resulted in higher LAB counts at the early stages of ripening, with differences between the two DL-starter cultures. The unsalted cheeses produced with C1 had retained a significantly higher number of the initial LAB counts (cfu/g) after 1 and 2 weeks of ripening (i.e. 58% and 71%), compared to the normal-salted cheeses (i.e. 22% and 21%), whereas no significant difference was found between the reduced-salt (i.e. 31% and 35%) and normal-salted cheeses. At the later stages of ripening (i.e. 7 and 11 weeks) NaCl had no significant influence. For cheeses produced with C2, a significant influence of NaCl was only found in cheeses ripened for 7 weeks, where the unsalted and reduced-salt cheeses had retained a significantly higher number of the initial LAB counts (cfu/g) (i.e. 39% and 38%), compared to the normal-salted cheeses (i.e. 21%). In the Samsoe cheeses, bacteria were organized as single cells, in groups of 2-3 cells or in groups of ≥4 cells. During ripening the decrease in the number of viable bacteria was mainly due to a reduction in the number of viable bacteria organized in groups of ≥4 cells. A negative correlation between NaCl content and PepX activity was observed. At the end of ripening the total FAA content was lower in the unsalted cheeses, compared to the reduced- and normal-salted cheeses. In conclusion, NaCl had a significant influence on proliferation of both DL-starter cultures. However, the influence of NaCl on culture development was more pronounced in cheeses produced with DL-starter culture C1. As both texture and taste are parameters known to be affected by the development of the starter culture, the design of starter cultures for reduced NaCl cheeses is recommended.

Genome Sequence and Transcriptome Analysis of Meat-Spoilage-Associated Lactic Acid Bacterium Lactococcus piscium MKFS47.

Lactococcus piscium is a psychrotrophic lactic acid bacterium and is known to be one of the predominant species within spoilage microbial communities in cold-stored packaged foods, particularly in meat products. Its presence in such products has been associated with the formation of buttery and sour off-odors. Nevertheless, the spoilage potential of L. piscium varies dramatically depending on the strain and growth conditions. Additional knowledge about the genome is required to explain such variation, understand its phylogeny, and study gene functions. Here, we present the complete and annotated genomic sequence of L. piscium MKFS47, combined with a time course analysis of the glucose catabolism-based transcriptome. In addition, a comparative analysis of gene contents was done for L. piscium MKFS47 and 29 other lactococci, revealing three distinct clades within the genus. The genome of L. piscium MKFS47 consists of one chromosome, carrying 2,289 genes, and two plasmids. A wide range of carbohydrates was predicted to be fermented, and growth on glycerol was observed. Both carbohydrate and glycerol catabolic pathways were significantly upregulated in the course of time as a result of glucose exhaustion. At the same time, differential expression of the pyruvate utilization pathways, implicated in the formation of spoilage substances, switched the metabolism toward a heterofermentative mode. In agreement with data from previous inoculation studies, L. piscium MKFS47 was identified as an efficient producer of buttery-odor compounds under aerobic conditions. Finally, genes and pathways that may contribute to increased survival in meat environments were considered.

Selection of autochthonous strains as promising starter cultures for Fior di Latte, a traditional cheese of southern Italy.

BACKGROUND: This paper describes the selection of promising strains intended as starter cultures from the autochthonous lactic acid bacteria of Fior di Latte cheese (Apulia region, southern Italy). RESULTS: Ninety-five isolates were randomly selected from whey and Fior di Latte. After preliminary characterization based upon Gram staining, deamination of arginine, hydrolysis of esculine and production of CO2 from glucose and citrate, the isolates were studied for their growth at different temperatures (10, 15 and 45 °C), with salt addition (20, 40 and 65 g L(-1) ) and at pH 4.4 and their acidification score in MRS broth after 6 and 24 h. Data were modeled through the growth index and used as input to run a preliminary cluster analysis and a principal component analysis. In this way, nine promising strains were selected and used for validation at laboratory level, to study the acidification score in milk and propose some possible microbial mixtures. CONCLUSION: This paper reports the first research focusing on the design of a lactic starter for the production of Fior di Latte cheese, using a quantitative approach based on the evaluation of growth index and acidification score as well as on the use of a multivariate approach to select the most promising nine strains.