Shotgun Proteomics Analysis, Functional Networks, and Peptide Biomarkers for Seafood-Originating Biogenic-Amine-Producing Bacteria.

Biogenic amine-producing bacteria are responsible for the production of basic nitrogenous compounds (histamine, cadaverine, tyramine, and putrescine) following the spoilage of food due to microorganisms. In this study, we adopted a shotgun proteomics strategy to characterize 15 foodborne strains of biogenic-amine-producing bacteria. A total of 10,673 peptide spectrum matches belonging to 4081 peptides and corresponding to 1811 proteins were identified. Relevant functional pathways were determined, and strains were differentiated into hierarchical clusters. An expected protein-protein interaction network was created (260 nodes/1973 interactions). Most of the determined proteins were associated with networks/pathways of energy, putrescine metabolism, and host-virus interaction. Additionally, 556 peptides were identified as virulence factors. Moreover, 77 species-specific peptide biomarkers corresponding to 64 different proteins were proposed to identify 10 bacterial species. This represents a major proteomic dataset of biogenic-amine-producing strains. These results may also be suitable for new treatments for food intoxication and for tracking microbial sources in foodstuffs.

Proteomic Characterization of Virulence Factors and Related Proteins in Enterococcus Strains from Dairy and Fermented Food Products.

Enterococcus species are Gram-positive bacteria that are normal gastrointestinal tract inhabitants that play a beneficial role in the dairy and meat industry. However, Enterococcus species are also the causative agents of health care-associated infections that can be found in dairy and fermented food products. Enterococcal infections are led by strains of Enterococcus faecalis and Enterococcus faecium, which are often resistant to antibiotics and biofilm formation. Enterococci virulence factors attach to host cells and are also involved in immune evasion. LC-MS/MS-based methods offer several advantages compared with other approaches because one can directly identify microbial peptides without the necessity of inferring conclusions based on other approaches such as genomics tools. The present study describes the use of liquid chromatography−electrospray ionization tandem mass spectrometry (LC−ESI−MS/MS) to perform a global shotgun proteomics characterization for opportunistic pathogenic Enterococcus from different dairy and fermented food products. This method allowed the identification of a total of 1403 nonredundant peptides, representing 1327 proteins. Furthermore, 310 of those peptides corresponded to proteins playing a direct role as virulence factors for Enterococcus pathogenicity. Virulence factors, antibiotic sensitivity, and proper identification of the enterococcal strain are required to propose an effective therapy. Data are available via ProteomeXchange with identifier PXD036435. Label-free quantification (LFQ) demonstrated that the majority of the high-abundance proteins corresponded to E. faecalis species. Therefore, the global proteomic repository obtained here can be the basis for further research into pathogenic Enterococcus species, thus facilitating the development of novel therapeutics.

Rapid Proteomic Characterization of Bacteriocin-Producing Enterococcus faecium Strains from Foodstuffs.

Enterococcus belongs to a group of microorganisms known as lactic acid bacteria (LAB), which constitute a broad heterogeneous group of generally food-grade microorganisms historically used in food preservation. Enterococci live as commensals of the gastrointestinal tract of warm-blooded animals, although they also are present in food of animal origin (milk, cheese, fermented sausages), vegetables, and plant materials because of their ability to survive heat treatments and adverse environmental conditions. The biotechnological traits of enterococci can be applied in the food industry; however, the emergence of enterococci as a cause of nosocomial infections makes their food status uncertain. Recent advances in high-throughput sequencing allow the subtyping of bacterial pathogens, but it cannot reflect the temporal dynamics and functional activities of microbiomes or bacterial isolates. Moreover, genetic analysis is based on sequence homologies, inferring functions from databases. Here, we used an end-to-end proteomic workflow to rapidly characterize two bacteriocin-producing Enterococcus faecium (Efm) strains. The proteome analysis was performed with liquid chromatography coupled to a trapped ion mobility spectrometry-time-of-flight mass spectrometry instrument (TimsTOF) for high-throughput and high-resolution characterization of bacterial proteins. Thus, we identified almost half of the proteins predicted in the bacterial genomes (>1100 unique proteins per isolate), including quantifying proteins conferring resistance to antibiotics, heavy metals, virulence factors, and bacteriocins. The obtained proteomes were annotated according to function, resulting in 22 complete KEGG metabolic pathway modules for both strains. The workflow used here successfully characterized these bacterial isolates and showed great promise for determining and optimizing the bioengineering and biotechnology properties of other LAB strains in the food industry.

Proteomic Characterization of Antibiotic Resistance in Listeria and Production of Antimicrobial and Virulence Factors.

Some Listeria species are important human and animal pathogens that can be found in contaminated food and produce a variety of virulence factors involved in their pathogenicity. Listeria strains exhibiting multidrug resistance are known to be progressively increasing and that is why continuous monitoring is needed. Effective therapy against pathogenic Listeria requires identification of the bacterial strain involved, as well as determining its virulence factors, such as antibiotic resistance and sensitivity. The present study describes the use of liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to do a global shotgun proteomics characterization for pathogenic Listeria species. This method allowed the identification of a total of 2990 non-redundant peptides, representing 2727 proteins. Furthermore, 395 of the peptides correspond to proteins that play a direct role in Listeria pathogenicity; they were identified as virulence factors, toxins and anti-toxins, or associated with either antibiotics (involved in antibiotic-related compounds production or resistance) or resistance to toxic substances. The proteomic repository obtained here can be the base for further research into pathogenic Listeria species and facilitate the development of novel therapeutics for these pathogens.

Preservative Effect of Aqueous and Ethanolic Extracts of the Macroalga Bifurcaria bifurcata on the Quality of Chilled Hake (Merluccius merluccius).

This work addressed the preservative behaviour of different icing media containing extracts from the alga Bifurcaria bifurcata. A comparative study of the antimicrobial and antioxidant effects of aqueous and ethanolic extracts of this macroalga was carried out. Whole hake (Merluccius merluccius) pieces were stored in ice containing either kind of extract and analysed for quality changes throughout a 13-day storage period. A progressive loss of microbial and biochemical quality was detected in all batches as chilling time increased. A significant inhibitory effect (p < 0.05) on microbial activity could be observed as a result of including the aqueous (lowering of psychrotrophic and lipolytic counts and pH value) and ethanolic (lowering of psychrotrophic and lipolytic counts) extracts. Additionally, both kinds of extract led to a substantial inhibition (p < 0.05) in the lipid hydrolysis rate (formation of free fatty acids), greater in the case of the batch containing ethanolic extract. Concerning lipid oxidation, a similar inhibitory effect (p < 0.05) on the formation of secondary compounds (thiobarbituric acid substances) was noticed in fish specimens corresponding to both alga extracts; however, more (p < 0.05) peroxide formation was detected in fish corresponding to the ethanolic extract batch. A preservative effect can be concluded for both kinds of extract; this effect agrees with previous studies reporting the presence of hydrophilic and lipophilic bioactive compounds in B. bifurcata.

The Effect of Gelatine Packaging Film Containing a Spirulina platensis Protein Concentrate on Atlantic Mackerel Shelf Life.

The use of packaging films containing natural preservative compounds attracts great attention for the quality improvement of seafood. Microalga spirulina (Spirulina platensis) represents a potential source of high added-value and preservative biocompounds. The goal of this study was to enhance the quality of refrigerated Atlantic mackerel (Scomber scombrus) by including a protein concentrate (PC) of spirulina in a gelatine-based film. Quality changes in fish muscle were monitored by microbial and chemical analyses throughout an 11-day refrigerated storage (4 °C). As a result of the presence of spirulina PC in the film, an antimicrobial effect (p < 0.05) was concluded as determined by comparative evolution of aerobes and psychrotrophs, while no effect (p > 0.05) was concluded for Enterobacteriaceae, proteolytics and lipolytics counts. Furthermore, a lower (p < 0.05) formation of trimethylamine and free fatty acids was detected. Lipid oxidation, measured by fluorescent compounds formation, also exhibited lower average values in fish corresponding to the batch containing spirulina concentrate. The preservative effects observed can be explained on the basis of the presence of antimicrobial and antioxidant compounds in the microalga concentrate. It is proposed that the current packaging system may constitute a novel and promising strategy to enhance the quality of commercial refrigerated fatty fish.

New icing media for quality enhancement of chilled hake (Merluccius merluccius) using a jumbo squid (Dosidicus gigas) skin extract.

BACKGROUND: An advanced strategy for chilled fish preservation, based on the inclusion in ice of an extract of jumbo squid (Dosidicus gigas) skin (JSS), is proposed. Aqueous solutions including acetic acid-ethanol extracts of JSS were tested at two different concentrations as icing media, with the effects on the quality evolution of chilled hake (Merluccius merluccius) being monitored. RESULTS: A significant inhibition (P < 0.05) of microbial activity (aerobes, psychrotrophs, Enterobacteriaceae, proteolytic bacteria; pH, trimethylamine) was obtained in hake corresponding to the icing batch including the highest JSS concentration. Additionally, fish specimens from such icing conditions showed an inhibitory effect (P < 0.05) on lipid hydrolysis development, while no effect (P > 0.05) was depicted for lipid oxidation. Sensory analysis (skin and mucus development; eyes; gills; texture; external odour; raw and cooked flesh odour; flesh taste) indicated a shelf life extension of chilled hake stored in ice including the highest JSS concentration. CONCLUSION: A profitable use of JSS, an industrial by-product during jumbo squid commercialisation, has been developed in the present work, which leads to a remarkable microbial inhibition and a significant shelf life extension of chilled hake. In agreement with previous research, ommochrome pigments (i.e. lipophilic-type compounds) would be considered responsible for this preservative effect. © 2016 Society of Chemical Industry.

In vitro probiotic profiling of novel Enterococcus faecium and Leuconostoc mesenteroides from Tunisian freshwater fishes.

Novel lactic acid bacteria isolated from different organs of freshwater fish were examined for their potential application as probiotics in raw and processed foods. Four isolates of Enterococcus faecium and Leuconostoc mesenteroides were identified at the molecular level by 16S rRNA sequencing and random amplification of polymorphic DNA - polymerase chain reaction, and their antimicrobial activity against a panel of pathogens and food-poisoning bacteria was investigated. The whole bacteriocins of the 4 isolates were characterized by enterobacterial repetitive intergenic consensus sequences in PCR. The isolates exhibited high inhibitory activities against food-borne pathogens and spoilage microbial species and have significant probiotic profiles, since they survived at pH 3.0 and in the presence of bile salts, pancreatin, and pepsin, without any detectable hemolytic activity. Further, moderate heat resistance, adhesion ability to steel surfaces, and sensitivity to clinically relevant antimicrobial agents were revealed for all the isolates. These results highlight the specific probiotic properties of the strains and give evidence for potential application in minimally processed foods subjected to moderate heat processing.

Metaproteomic Analysis of Microbial Communities Affecting Fishery Products.

Fishery products are one of the main human nutritional sources, and due to the consumption increase, the quality of the derived products may be modified, during catching, technological processing, and storage. Detection and identification of pathogenic and spoilage microorganisms in fishery products is needed because the first may be involved in human diseases, while the second is responsible of significant economic losses. In this sense, liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method and computational analysis of MS data are useful tools for characterizing and identifying different microorganisms and to develop promising strategies for food science investigations. Moreover, in the past decade, metaproteomic methodologies have progressed for the study of microorganisms isolated from their natural samples and independently of the culture restrictions. Metaproteomics enables assessment of proteins and pathways from individual members of the consortium. Metaproteomics can provide a detailed understanding of which organisms occupy specific metabolic niches, how they interact, and how they utilize nutrients, and these insights can be obtained directly from environmental samples.According to that, the sample preparation of the fishery product, the LC-ESI-MS/MS dedicated method, and the MS data analysis were described in the present chapter to obtain the metaproteomic analysis of the respective microbiomes or microbial communities.

High-Resolution Comparative and Quantitative Proteomics of Biogenic-Amine-Producing Bacteria and Virulence Factors Present in Seafood.

The presence of biogenic amines (histamine, tyramine, putrescine, and cadaverine) in seafood is a significant concern for food safety. This review describes for the first time a shotgun quantitative proteomics strategy to evaluate and compare foodborne strains of bacteria that produce biogenic amines in seafoods. This approach recognized 35,621 peptide spectrum matches, belonging to 20,792 peptides, and 4621 proteins. It allowed the determination of functional pathways and the classification of the strains into hierarchical clusters. The study identified a protein-protein interaction network involving 1160 nodes/10,318 edges. Proteins were related to energy pathways, spermidine biosynthesis, and putrescine metabolism. Label-free quantitative proteomics allowed the identification of differentially regulated proteins in specific strains such as putrescine aminotransferase, arginine decarboxylase, and l-histidine-binding protein. Additionally, 123 peptides were characterized as virulence factors and 299 peptide biomarkers were selected to identify bacterial species in fish products. This study presents the most extensive proteomic repository and progress in the science of food biogenic bacteria and could be applied in the food industry for the detection of bacterial contamination that produces histamine and other biogenic amines during food processing/storage.

Comprehensive shotgun proteomic characterization and virulence factors of seafood spoilage bacteria.

This article summarizes the characterization, by shotgun proteomics, of 11 bacterial strains identified as responsible for seafood spoilage. A total of 4455 peptide spectrum matches, corresponding to 4299 peptides and 3817 proteins were identified. Analyses of data determined the functional pathways they are involved in. The proteins identified were integrated into a protein-protein network that involves 371 nodes and 3016 edges. Those proteins are implicated in energy pathways, peptidoglycan biosynthesis, spermidine/putrescine metabolism. An additional 773 peptides were characterized as virulence factors, that participates in bacterial pathogenesis; while 14 peptides were defined as biomarkers, as they can be used to differentiate the bacterial species present. This report represents the most extensive proteomic repository available in the field of seafood spoilage bacteria; the data substantially advances the understanding of seafood decay, as well as provides fundamental bases for the recognition of the bacteria existent in seafood that cause spoilage during food processing/storage.

Shotgun proteomic analyses of Pseudomonas species isolated from fish products.

Numerous Pseudomonas species can infect aquatic animals, such as farmed rainbow trout, sea trout, sea bass, and sea bream, by causing disease or stress reactions. In aquaculture facilities, a number of Pseudomonas species have been isolated and identified as the main pathogens. The present study describes the characterization of 18 Pseudomonas strains, isolated from fish products using shotgun proteomics. The bacterial proteomes obtained were further analyzed to identify the main functional pathway proteins involved. In addition, this study revealed the presence of 1015 non-redundant peptides related to virulence factors. An additional 25 species-specific peptides were identified as putative Pseudomonas spp. biomarkers. The results constitute the largest dataset, described thus far for the rapid identification and characterization of Pseudomonas species present in edible fish; furthermore, these data can provide the basis for further research into the development of new therapies against these harmful pathogens.

Characterization of different food-isolated Enterococcus strains by MALDI-TOF mass fingerprinting.

Enterococcus is a controversial genus due to its great variability; this genus includes pathogenic strains, spoilage strains, and apparently safe strains including some probiotic strains. Previous studies focused on the characterization of strains of Enterococcus spp. involved in nosocomial infections. However, little research has been conducted on Enterococcus strains in foodstuffs. In the present work, 36 strains of different species of Enterococcus have been characterized by means of MALDI-TOF MS, resulting in highly specific mass spectral fingerprints. Characteristic peak masses common to certain bacterial species of Enterococcus have been identified. Thus, a peak at m/z 4426 ± 1 was assigned as a genus-specific biomarker. In addition, phyloproteomic relationships based on the mass spectral data were compared to the results of a phylogenetic analysis based on the 16S rRNA gene sequence. A better grouping at the species level was observed in the phyloproteomic tree, especially for the Enterococcus faecium group. Presumably, the assortment of some strains or ecotypes could be related to their ecological niche specialization. The approach described in this study leads the way toward the rapid and specific identification of different strains and species of Enterococcus in food based on molecular protein markers, aiming at the early detection of pathogenic strains and strains implicated in food poisoning or food spoilage.

Identification and classification of seafood-borne pathogenic and spoilage bacteria: 16S rRNA sequencing versus MALDI-TOF MS fingerprinting.

The present study aims to compare two molecular technologies, 16S rRNA sequencing and MALDI-TOF MS, for bacterial species identification in seafood. With this aim, 70 reference strains from culture collections, including important seafood-borne pathogenic and spoilage bacterial species, and 50 strains isolated from commercial seafood products, were analysed by both techniques. Genomic analysis only identified the species of 50% of the isolated strains, proving to be particularly poor at identifying members of the Pseudomonas and Bacillus genera. In contrast, MALDI-TOF MS fingerprinting identified 76% of the strains at the species level. The mass spectral data were submitted to the SpectraBank database (http://www.spectrabank.org), making this information available to other researchers. Furthermore, cluster analysis of the peak mass lists was carried out with the web application SPECLUST and the calculated groupings were consistent with results determined by a phylogenetic approach that is based on the 16S rRNA sequences. However, the MALDI-TOF MS analysis demonstrated more discriminating potential that allowed for better classification, especially for the Pseudomonas and Bacillus genera. This is of importance with respect to the varying pathogenic and spoilage character at the intragenus and intraspecies level. In this sense, MALDI-TOF MS demonstrated to be a competent bacterial typing tool that extends phenotypic and genotypic approaches, allowing a more ample classification of bacterial strains.

16S rRNA gene sequencing and MALDI TOF mass spectroscopy identification of Leuconostoc mesenteroides isolated from Algerian raw camel milk.

BACKGROUND: Eighty-three strains of Leuconostoc mesenteroides were isolated from Algerian raw camel milk. Based on morphological, biochemical, and physiological characters tests, strains were identified as Ln. mesenteroides subsp. mesenteroides. Seven strains had a remarkable antagonistic and probiotic characterization. The present study aims at identifying these strains by means of 16 s rRNA gene sequencing and Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), extending phenotypic and genotypic studies done previously. RESULTS: The phyloproteomic dendrograms of the studied strains based on MALDI-TOF MS provided the same identification with more intraspecific information from the 16S rRNA gene sequencing based on phylogenetic analysis. The latter were in agreement with the previous biochemical/physiological identification, the seven isolated strains were Ln. mesenteroides subsp. mesenteroides. CONCLUSIONS: Remarkably, MALDI-TOF MS fingerprinting was found to be effective enough as 16S rRNA gene sequencing identification, allowing faster and more reliable analysis than biochemical/physiological methods.

Antioxidant and Antimicrobial Effect of Biodegradable Films Containing Pitaya (Stenocereus thurberi) Extracts during the Refrigerated Storage of Fish.

This study focused on the quality loss inhibition of fish muscle during refrigerated storage. Two parallel experiments were carried out that were focused on the employment of pitaya (Stenocereus thurberi) extracts in biodegradable packing films. On the one hand, a pitaya-gelatin film was employed for hake (Merluccius merluccius) muscle storage. On the other hand, a pitaya-polylactic acid (PLA) film was used for Atlantic mackerel (Scomber scombrus) muscle storage. In both experiments, fish-packing systems were stored at 4 °C for 8 days. Quality loss was determined by lipid damage and microbial activity development. The presence of the pitaya extract led to an inhibitory effect (p < 0.05) on peroxide, fluorescent compound, and free fatty acid (FFA) values in the gelatin-hake system and to a lower (p < 0.05) formation of thiobarbituric acid reactive substances, fluorescent compounds, and FFAs in the PLA-mackerel system. Additionally, the inclusion of pitaya extracts in the packing films slowed down (p < 0.05) the growth of aerobes, anaerobes, psychrotrophs, and proteolytic bacteria in the case of the pitaya-gelatin films and of aerobes, anaerobes, and proteolytic bacteria in the case of pitaya-PLA films. The current preservative effects are explained on the basis of the preservative compound presence (betalains and phenolic compounds) in the pitaya extracts.

Sequential Biorefining of Bioactive Compounds of High Functional Value from Calafate Pomace (Berberis microphylla) Using Supercritical CO2 and Pressurized Liquids.

A biorefinery process was developed for a freeze-dried pomace of calafate berries (Berberis microphylla). The process consisted of extraction of lipophilic components with supercritical CO2 (scCO2) and subsequent extraction of the residue with a pressurized mixture of ethanol/water (1:1 v/v). scCO2 extracted oil from the pomace, while pressurized liquid extraction generated a crude extract rich in phenols and a residue rich in fiber, proteins and minerals. Response surface analysis of scCO2 extraction suggested optimal conditions of 60 °C, 358.5 bar and 144.6 min to obtain a lipid extract yield of 11.15% (d.w.). The dark yellow oil extract contained a good ratio of ω6/ω3 fatty acids (1:1.2), provitamin E tocopherols (406.6 mg/kg), and a peroxide index of 8.6 meq O2/kg. Pressurized liquid extraction generated a polar extract with good phenolic content (33 mg gallic acid equivalents /g d.w.), anthocyanins (8 mg/g) and antioxidant capacity (2,2-diphenyl-1-picrylhydrazyl test = 25 µg/mL and antioxidant activity = 63 µM Te/g). The extraction kinetics of oil by scCO2 and phenolic compounds were optimally adjusted to the spline model (R2 = 0.989 and R2 = 0.999, respectively). The solid extracted residue presented a fiber content close to cereals (56.4% d.w.) and acceptable values of proteins (29.6% d.w.) and minerals (14.1% d.w.). These eco-friendly processes valorize calafate pomace as a source of ingredients for formulation of healthy foods, nutraceuticals and nutritional supplements.

Food authentication of commercially-relevant shrimp and prawn species: from classical methods to Foodomics.

Although seafood species identification has traditionally relied on morphological analysis, sometimes this is difficult to apply for the differentiation among penaeid shrimps owing to their phenotypic similarities and to the frequent removal of external carapace during processing. The objective of this review is to provide an updated and extensive overview on the molecular methods for shrimp and prawn species authentication, in which several omics approaches based on protein and DNA analysis are described. DNA-based methods include the amplification by PCR of different genes, commonly the mitochondrial 16S ribosomal RNA and cytochrome oxidase I genes. A recently described method based on RFLP coupled to PCR turned out to be particularly interesting for species differentiation and origin identification. Protein analysis methods for the characterization and detection of species-specific peptides are also summarized, emphasizing some novel proteomics-based approaches, such as phyloproteomics, peptide fragmentation, and species-specific peptide detection by HPLC coupled to multiple reaction monitoring (MRM) MS, the latter representing the fastest method described to date for species authentication in food.

SpectraBank: an open access tool for rapid microbial identification by MALDI-TOF MS fingerprinting.

MALDI-TOF MS has proved to be an accurate, rapid, and cost-effective technique for microbial identification in which the spectral fingerprint of an unknown strain can be compared to a database of spectra from reference strains. Most of the existing databases are private and often costly to access, and little spectral information is shared among researchers. The objective of the present communication is to introduce the SpectraBank database (http://www.spectrabank.org), which provides open access MALDI-TOF mass spectra from a variety of microorganisms. This work aims to familiarize readers with the SpectraBank database, from the sample preparation, data collection, and data analysis to how the spectral reference data can be used for microbial species identification. The database currently includes more than 200 MALDI-TOF MS spectra from more than 70 bacterial species and links to the freely available web-based application SPECLUST (http://bioinfo.thep.lu.se/speclust.html) to allow comparisons of the obtained peak mass lists and evaluate phyloproteomic relationships. The SpectraBank database is intended to be expanded by the addition of new spectra from microbial strains, obtained in our laboratory and by other researchers.

Characterization of Staphylococcus aureus strains isolated from Italian dairy products by MALDI-TOF mass fingerprinting.

Staphylococcus aureus is a known pathogen, causing serious food-borne intoxications due to the production of enterotoxins, being otherwise a major cause of mastitis. In this sense, the detection of S. aureus is an important issue for the food industry to avoid health hazards and economic losses. The present work applied MALDI-TOF MS for the classification of 40 S. aureus strains, 36 isolated from Italian dairy products and four from human samples. All isolated strains were clearly identified as S. aureus by their spectral fingerprints. The peak masses m/z 3444, 5031, and 6887 were determined to be specific biomarkers for S. aureus. Furthermore, clustering of the peak mass lists was successfully applied as a typing method, resulting in eight groups of strains. This is the first time that a detailed spectral comparison was carried out and characteristic peak masses were determined for every spectral group. Three strains exhibited a peak at m/z 6917 instead of m/z 6887, which was related to four polymorphisms in their 16S rRNA sequences. However, the grouping obtained by MALDI-TOF MS fingerprinting could not be related to toxin production or to the origin of the strains.