To develop a method for organic gluten-free (GF) sourdough bread production, a long-term and original wheat sourdough was refreshed with GF flours. The dynamics of the sourdough microbiota during five months of back-slopping were analyzed by classical enumeration and molecular methods, including PCR-temporal temperature gel electrophoresis (PCR-TTGE), multiplex PCR, and pulsed field gel electrophoresis (PFGE). The results showed that the yeast counts remained constant, although Saccharomyces cerevisiae, present in the initial wheat sourdough, was no longer detected in the GF sourdough, while lactic acid bacteria (LAB) counts increased consistently. In the first phase, which was aimed at obtaining a GF sourdough from wheat sourdough, Lactobacillus sanfranciscensis, L. plantarum, and L. spicheri were the main LAB species detected. During the second phase, aimed at maintaining the GF sourdough, the L. plantarum and L. spicheri populations decreased whereas L. sanfranciscensis persisted and L. sakei became the predominant species. Multiplex PCRs also revealed the presence of several L. sakei strains in the GF sourdough. In a search for the origin of the LAB species, PCR-TTGE was performed on the flour samples but only L. sanfranciscensis was detected, suggesting a flour origin for this typical sourdough species. Thus, while replacement of the wheat flour by GF flour influenced the sourdough microbiota, some of the original sourdough LAB and yeast species remained in the GF sourdough.
The microbial spoilage of meat and seafood products with short shelf lives is responsible for a significant amount of food waste. Food spoilage is a very heterogeneous process, involving the growth of various, poorly characterized bacterial communities. In this study, we conducted 16S ribosomal RNA gene pyrosequencing on 160 samples of fresh and spoiled foods to comparatively explore the bacterial communities associated with four meat products and four seafood products that are among the most consumed food items in Europe. We show that fresh products are contaminated in part by a microbiota similar to that found on the skin and in the gut of animals. However, this animal-derived microbiota was less prevalent and less abundant than a core microbiota, psychrotrophic in nature, mainly originated from the environment (water reservoirs). We clearly show that this core community found on meat and seafood products is the main reservoir of spoilage bacteria. We also show that storage conditions exert strong selective pressure on the initial microbiota: alpha diversity in fresh samples was 189±58 operational taxonomic units (OTUs) but dropped to 27±12 OTUs in spoiled samples. The OTU assemblage associated with spoilage was shaped by low storage temperatures, packaging and the nutritional value of the food matrix itself. These factors presumably act in tandem without any hierarchical pattern. Most notably, we were also able to identify putative new clades of dominant, previously undescribed bacteria occurring on spoiled seafood, a finding that emphasizes the importance of using culture-independent methods when studying food microbiota.The ISME Journal advance online publication, 21 October 2014; doi:10.1038/ismej.2014.202.
Within the lactic acid bacterium genus Carnobacterium, Carnobacterium maltaromaticum is one of the most frequently isolated species from natural environments and food. It potentially plays a major role in food product biopreservation. We report here on the 3.649-Mb chromosome sequence of C. maltaromaticum LMA 28, which was isolated from ripened soft cheese.
In silico analysis of the genome sequence of the meat-borne lactic acid bacterium (LAB) Lactobacillus sakei 23K has revealed a repertoire of potential functions related to the adaptation of this bacterium to the meat environment. Among these functions, the ability to use N-acetyl-neuraminic acid (NANA) as a carbon source could provide a competitive advantage for growth on meat in which this amino sugar is present. In this work, we proposed to analyze the functionality of a gene cluster encompassing nanTEAR and nanK (nanTEAR-nanK). We established that this cluster encoded a pathway allowing transport and early steps of the catabolism of NANA in this genome. We also demonstrated that this cluster was absent from the genome of other L. sakei strains that were shown to be unable to grow on NANA. Moreover, L. sakei 23K nanA, nanT, nanK, and nanE genes were able to complement Escherichia coli mutants. Construction of different mutants in L. sakei 23K ?nanR, ?nanT, and ?nanK and the double mutant L. sakei 23K ?(nanA-nanE) made it possible to show that all were impaired for growth on NANA. In addition, two genes located downstream from nanK, lsa1644 and lsa1645, are involved in the catabolism of sialic acid in L. sakei 23K, as a L. sakei 23K ?lsa1645 mutant was no longer able to grow on NANA. All these results demonstrate that the gene cluster nanTEAR-nanK-lsa1644-lsa1645 is indeed involved in the use of NANA as an energy source by L. sakei.
Lactobacillus sakei plays a major role in meat fermentation and in the preservation of fresh meat. The large diversity of L. sakei strains represents a valuable and exploitable asset in the development of a variety of industrial applications; however, an efficient method to identify and classify these strains has yet to be developed. In this study, we used multilocus sequence typing (MLST) to analyze the polymorphism and allelic distribution of eight loci within an L. sakei population of 232 strains collected worldwide. Within this population, we identified 116 unique sequence types with an average pairwise nucleotide diversity per site (?) of 0.13%. Results from Structure, goeBurst, and ClonalFrame software analyses demonstrated that the L. sakei population analyzed here is derived from three ancestral lineages, each of which shows evidence of a unique evolutionary history influenced by independent selection scenarios. However, the signature of selective events in the contemporary population of isolates was somewhat masked by the pervasive phenomenon of homologous recombination. Our results demonstrate that lineage 1 is a completely panmictic subpopulation in which alleles have been continually redistributed through the process of intra-lineage recombination. In contrast, lineage 2 was characterized by a high degree of clonality. Lineage 3, the earliest-diverging branch in the genealogy, showed evidence of both clonality and recombination. These evolutionary histories strongly indicate that the three lineages may correspond to distinct ecotypes, likely linked or specialized to different environmental reservoirs. The MLST scheme developed in this study represents an easy and straightforward tool that can be used to further analyze the population dynamics of L. sakei strains in food products.
Lactobacillus sakei is a meat-borne lactic acid bacterium species exhibiting a wide genomic diversity. We have investigated the diversity of response to various oxidative compounds, between L. sakei strains, among a collection representing the genomic diversity. We observed various responses to the different compounds as well as a diversity of response depending on the aeration conditions used for cell growth. A principal component analysis revealed two main phenotypic groups, partially correlating with previously described genomic clusters. We designed strains mixes composed of three different strains, in order to examine the behavior of each strain, when cultured alone or in the presence of other strains. The strains composing the mixtures were chosen as diverse as possible, i.e. exhibiting diverse responses to oxidative stress and belonging to different genomic clusters. Growth and survival rates of each strain were monitored under various aeration conditions, with or without heme supplementation. The results obtained suggest that some strains may act as "helper" or "burden" strains depending on the oxidative conditions encountered during incubation. This study confirms that resistance to oxidative stress is extremely variable within the L. sakei species and that this property should be considered when investigating starter performance in the complex meat bacterial ecosystems.
We have investigated the population structure of lactic acid bacteria (LAB) for several beef carpaccio available on the market with the purpose of comparing the effect of storage process (modified-atmosphere packaging and vacuum-packaging) and of seasonal changes on this microbial population. Out of 60 samples we have characterised 214 isolates accounting for 10 LAB species and 35 isolates accounting for 11 non-LAB species. Lactobacillus sakei, Leuconostoc carnosum and Leuconostoc mesenteroides were the most prevailing LAB species with a frequency of identification within 66%, 62% and 52% of the samples respectively. These 3 species were also characterised by a phenotypic intra-species diversity of isolates based on colony morphology. We showed that the prevalence was increased 1.5 fold for L. sakei and L. mesenteroides during the summer sampling in comparison to the spring or the fall sampling suggesting an environmental origin of these two species. Seasonal variations were also observed for the prevalence of Lactobacillus fuchuensis and L. carnosum in spring (2- and 1.5-fold increase, respectively) and of Brochothrix thermosphacta in fall (6-fold increase). Finally, we demonstrated that the growth potential after the sell-by-date was favourable of 1.25 log(10) cfu g(-1) to Leuconostoc spp. in modified-atmosphere packaging and of 1.38 log(10) cfu g(-1) to Lactobacillus spp. in vacuum-packaging. In conclusion, we show that important and unsuspected traits in bacterial population dynamics can be unravelled by large sampling strategies. We discuss about the need to take this assessment into account for further studies on bacterial ecosystems of meat.
The entire genome of Lactobacillus casei BL23, a strain with probiotic properties, has been sequenced. The genomes of BL23 and the industrially used probiotic strain Shirota YIT 9029 (Yakult) seem to be very similar.
We recently showed that Lactobacillus sakei, a natural meat-borne lactic acid bacterium, can colonize the gastrointestinal tracts (GIT) of axenic mice but that this colonization in the intestinal environment selects L. sakei mutants showing modified colony morphology (small and rough) and cell shape, most probably resulting from the accumulation of various mutations that confer a selective advantage for persistence in the GIT. In the present study, we analyzed such clones, issued from three different L. sakei strains, in order to determine which functions were modified in the mutants. In the elongated filamentous cells of the rough clones, transmission electron microscopy (TEM) analysis showed a septation defect and dotted and slanted black bands, suggesting the presence of a helical structure around the cells. Comparison of the cytoplasmic and cell wall/membrane proteomes of the meat isolate L. sakei 23K and of one of its rough derivatives revealed a modified expression for 38 spots. The expression of six oxidoreductases, several stress proteins, and four ABC transporters was strongly reduced in the GIT-adapted strain, while the actin-like MreB protein responsible for cell shaping was upregulated. In addition, the expression of several enzymes involved in carbohydrate metabolism was modified, which may correlate with the observation of modified growth of mutants on various carbon sources. These results suggest that the modifications leading to a better adaptation to the GIT are pleiotropic and are characterized in a rough mutant by a different stress status, a cell wall modification, and modified use of energy sources, leading to an improved fitness for the colonization of the GIT.
Lactobacillus sakei is an important food-associated lactic acid bacterium commonly used as starter culture for industrial meat fermentation, and with great potential as a biopreservative in meat and fish products. Understanding the metabolic mechanisms underlying the growth performance of a strain to be used for food fermentations is important for obtaining high-quality and safe products. Proteomic analysis was used to study the primary metabolism in ten food isolates after growth on glucose and ribose, the main sugars available for L. sakei in meat and fish.
Bifidobacteria are natural inhabitants of the human gastrointestinal tract. In full-term newborns, these bacteria are acquired from the mother during delivery and rapidly become the predominant organisms in the intestinal microbiota. Bifidobacteria contribute to the establishment of healthy intestinal ecology and can confer health benefits to their host. Consequently, there is growing interest in bifidobacteria, and various strains are currently used as probiotic components in functional food products. However, the probiotic effects have been reported to be strain-specific. There is thus a need to better understand the determinants of the observed benefits provided by these probiotics. Our objective was to compare three human B. longum isolates with the sequenced model strain B. longum NCC2705 at the chromosome and proteome levels.
Lactobacillus sakei is a lactic acid bacterium mainly found in meat and meat products. In order to understand the factors favoring its adaptation to meat matrix, growth parameters and survival of the strain L. sakei 23K in the presence of sarcoplasmic or myofibrillar extracts were assessed. Cytosolic proteins putatively involved in the response of this strain to meat proteins were determined using 2D electrophoresis and the significantly regulated proteins were identified by Maldi Tof-MS analyses. From the 31 differentially expressed spots, 16 occurred in the presence of myofibrillar extract while 6 proteins were modulated by the sarcoplasmic extract. Two dipeptidases were overexpressed in the presence of sarcoplasmic proteins, in correlation to the protein degradation patterns obtained by SDS-PAGE. In the presence of the myofibrillar extract, L. sakei 23K overexpressed proteins related to energy and pyrimidine metabolism as well as ala- and tyr-tRNA synthetases, involved in translation, while others corresponding to general stress response, pyrimidine, vitamin and cofactor biosynthesis were down-regulated. The supplementary nutrients furnished by meat extracts modulated the overexpression of proteins related to translation, peptide/amino acid metabolism and energy production while the stress proteins were under regulated. The results obtained here suggest that meat proteins would not represent a stress environment per se for L. sakei 23K in contrast to the harsh conditions during meat processing. This study has extended the understanding of the molecular responses and growth mechanisms of L. sakei 23K in the presence of meat proteins. The transference of genomic information into useful biological insight is an important step for the selection of well-adapted strains for the achievement of high-quality fermented products.
Lactobacillus sakei is a lactic acid bacterium naturally found on meat. Although it is generally acknowledged that lactic acid bacteria are rare species in the microbial world which do not have iron requirements, the genome sequence of L. sakei 23K has revealed quite complete genetic equipment dedicated to transport and use of this metal. Here, we aimed to investigate which iron sources could be used by this species as well as their role in the bacteriums physiology. Therefore, we developed a microscopy approach based on electron energy loss spectroscopy (EELS) analysis and nano-scale secondary-ion mass spectrometry (SIMS) in order to analyze the iron content of L. sakei cells. This revealed that L. sakei can use iron sources found in its natural ecosystem, myoglobin, hemoglobin, hematin, and transferrin, to ensure long-term survival during stationary phase. This study reveals that analytical image methods (EELS and SIMS) are powerful complementary tools for investigation of metal utilization by bacteria.
A Lactobacillus sakei strain named FLEC01 was isolated from human feces and characterized genotypically. Comparison of the genetic features of this strain with those of both the meat-borne L. sakei strain 23K and another human isolate, LTH5590, showed that they belong to different but closely related clusters. The three L. sakei strains did not persist and only transited through the gastrointestinal tracts (GITs) of conventional C3H/HeN mice. In contrast, they all colonized the GITs of axenic mice and rapidly reached a population of 10(9) CFU/g of feces, which remained stable until day 51. Five days after mice were fed, a first subpopulation, characterized by small colonies, appeared and reached 50% of the total L. sakei population in mice. Fifteen to 21 days after feeding, a second subpopulation, characterized by rough colonies, appeared. It coexisted with the two other populations until day 51, and its cell shapes were also affected, suggesting a dysfunction of the cell division or cell wall. No clear difference between the behaviors of the meat-borne strain and the two human isolates in both conventional and axenic mice was observed, suggesting that L. sakei is a food-borne bacterium rather than a commensal one and that its presence in human feces originates from diet. Previous observations of Escherichia coli strains suggest that the mouse GIT environment could induce mutations to increase their survival and colonization capacities. Here, we observed similar mutations concerning a food-grade gram-positive bacterium for the first time.
Lactobacillus sakei is a food-borne bacterium naturally found in meat and fish products. A study was performed to examine the intraspecies diversity among 73 isolates sourced from laboratory collections in several different countries. Pulsed-field gel electrophoresis analysis demonstrated a 25% variation in genome size between isolates, ranging from 1,815 kb to 2,310 kb. The relatedness between isolates was then determined using a PCR-based method that detects the possession of 60 chromosomal genes belonging to the flexible gene pool. Ten different strain clusters were identified that had noticeable differences in their average genome size reflecting the natural population structure. The results show that many different genotypes may be isolated from similar types of meat products, suggesting a complex ecological habitat in which intraspecies diversity may be required for successful adaptation. Finally, proteomic analysis revealed a slight difference between the migration patterns of highly abundant GapA isoforms of the two prevailing L. sakei subspecies (sakei and carnosus). This analysis was used to affiliate the genotypic clusters with the corresponding subspecies. These findings reveal for the first time the extent of intraspecies genomic diversity in L. sakei. Consequently, identification of molecular subtypes may in the future prove valuable for a better understanding of microbial ecosystems in food products.
The abilities of five Lactobacillus sakei strains and one Lactococcus lactis strain to retain inhibitory activity against several target organisms in the flora of product during 12 weeks storage of vacuum-packaged lamb and beef was investigated. L. sakei strains were generally found capable of developing dominant populations on both beef and lamb. L. lactis 75 grew poorly on lamb did not inhibit co-inoculated Brochothrix thermosphacta. Lamb inoculated with the Sakacin-A producer L. sakei Lb706 had lower Listeria monocytogenes populations than lamb inoculated with a bacteriocin-negative variant. In beef packs inoculated with Clostridium estertheticum spores and L. sakei strain 27, 44 or 63, the development of blown-pack spoilage was delayed by up to one week. Campylobacter jejuni inoculated onto beef was recovered from fewer packs when it was co-inoculated with 3000 CFU cm(-2) of L. sakei strain 27, 44 or 63. Observed inhibition did not always correlate with inhibition observed in earlier media-based studies, supporting the view that functionality identified using simple media-based screening methods may not be replicated in the complex environment of stored foods, and vice-versa. These findings further define a set of L. sakei strains with potential for the extended bio-preservation of minimally processed fresh beef and lamb.
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