A three-years survey of microbial contaminants in industrial hemp inflorescences from two Italian cultivation sites.

BACKGROUND: The use of industrial Cannabis sativa L. for recreational, cosmeceutical, nutraceutical, and medicinal purposes has gained momentum due to its rich content of valuable phytochemicals, such as cannabidiol (CBD) and cannabigerol (CBG). However, there are concerns regarding the risk of microbial contamination in plants grown outside controlled environments. Microbes associated with hemp can be either epiphytes or endophytes and may pose a risk of infectious illness for humans. METHODS: Seven Italian hemp genotypes, including Bernabeo, Carmagnola, Carmaleonte, Codimono, CS, Eletta Campana, and Fibranova, were cultivated in two distinct geographic locations, Catania and Rovigo, for three consecutive years from 2019 to 2021. Total aerobic microbes (TAMC), total combined yeasts/moulds (TYMC), the presence of bile-tolerant Gram-negative bacteria, and the absence of Escherichia coli and Salmonella spp. were evaluated and compared. The main phytocannabinoid content was measured and correlated with microbial contamination. RESULTS: Most samples analyzed in this study did not meet the European Pharmacopoeia microbiological limits. The detection of potential pathogens, such as E. coli and Salmonella spp., in the samples indicates that the use of inflorescences may represent a possible source of infection. Microbial contamination varied among harvesting seasons and production sites, with agroclimatic conditions influencing microbial load and composition. The presence of potentially pathogenic bacteria was less associated with seasonal climate variability and more likely affected by sporadic contamination from external sources. CBD concentration exhibited a negative correlation with bile-tolerant Gram-negative bacteria and total yeasts/moulds levels. Samples with lower CBD content were more contaminated than those with higher CBD levels, suggesting a potential protective effect of this phytochemical on the plant. CONCLUSIONS: The threshing residues (inflorescences, floral bracts, and leaves) of industrial hemp varieties represent a valuable product and a source of beneficial phytochemicals that warrants further exploration. While post-harvest sterilization methods may reduce microbiological risks, they may also degrade heat- and light-sensitive bioactive phytochemicals. The most promising strategy involves implementing best agronomic practices to maintain healthy and uncontaminated cultures. Rigorous monitoring and quality certification protocols are essential to mitigate the microbiological risk associated with the consumption of hemp-derived products.

Microbiota Survey of Sliced Cooked Ham During the Secondary Shelf Life.

Sliced cooked ham packaged in a modified atmosphere is a popular ready-to-eat product, subjected to abundant microbial contamination throughout its shelf life that can lead to deterioration of both sensorial properties and safety. In this study, the microbial load and the chemical-physical features of cooked ham of five producers were monitored for a period of 12 days after the opening of the packages (i.e., the secondary shelf life), during which the products were stored in a domestic refrigerator at 5.2 ± 0.6°C. The sensorial properties presented a perceivable decay after 8 days and became unacceptable after 12 days. High-performance liquid chromatography analysis and solid-phase microextraction coupled with gas chromatography profiling of volatile metabolites indicated that lactic acid, ethanol, acetic acid, acetoin, 3-methyl-1-butanol, and 2-3 butanediol were the main metabolites that characterized the evolution of the analyzed cooked ham. The microbiota was monitored by 16S ribosomal RNA gene profiling and culture-dependent techniques. Already at the opening of packages, all the products presented high microbial load, generally dominated by lactic acid bacteria, with evident differences among the products. The increase of lactic acid bacteria somehow protected samples from abundant contamination by other bacteria, concurring with the evolution of more safe products. This role was exerted by numerous Latilactobacillus, Leuconostoc, and Carnobacterium species, among which the most frequently detected were Latilactobacillus sakei, Latilactobacillus sakei carnosum, Leuconostoc mesenteroides, and Carnobacterium divergens. Some products presented more complex communities that encompassed Proteobacteria such as Moellerella wisconsensis, Proteus hauseri, Brochothrix thermosphacta, and less frequently Pseudomonas, Erwinia, and Massilia. Opportunistic pathogenic bacteria such as Escherichia coli and Vibrio sp. were found in small quantities. The yeasts Kazachstania servazzii and Debaryomyces hansenii occurred already at 0 days, whereas various species of Candida (Candida zeylanoides, Candida sake, Candida norvegica, and Candida glaebosa) were abundant only after 12 days. These results indicated that the microbiological contaminants overgrowing during the secondary shelf life did not derive from environmental cross-contamination at the opening of the tray but were already present when the packages were opened, highlighting the phases of production up to the packaging as those crucial in managing the safety risk associated to this product.

Phylogenomic analysis of the genus Leuconostoc.

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

Phenotypic Traits and Immunomodulatory Properties of Leuconostoc carnosum Isolated From Meat Products.

Twelve strains of Leuconostoc carnosum from meat products were investigated in terms of biochemical, physiological, and functional properties. The spectrum of sugars fermented by L. carnosum strains was limited to few mono- and disaccharides, consistently with the natural habitats of the species, including meat and fermented vegetables. The strains were able to grow from 4 to 37°C with an optimum of approximately 32.5°C. The ability to grow at temperatures compatible with refrigeration and in presence of up to 60 g/L NaCl explains the high loads of L. carnosum frequently described in many meat-based products. Six strains produced exopolysaccharides, causing a ropy phenotype of colonies, according to the potential involvement on L. carnosum in the appearance of slime in packed meat products. On the other side, the study provides evidence of a potential protective role of L. carnosum WC0321 and L. carnosum WC0323 against Listeria monocytogenes, consistently with the presence in these strains of the genes encoding leucocin B. Some meat-based products intended to be consumed without cooking may harbor up to 108 CFU/g of L. carnosum; therefore, we investigated the potential impact of this load on health. No strains survived the treatment with simulated gastric juice. Three selected strains were challenged for the capability to colonize a mouse model and their immunomodulatory properties were investigated. The strains did not colonize the intestine of mice during 10 days of daily dietary administration. Intriguingly, despite the loss of viability during the gastrointestinal transit, the strains exhibited different immunomodulatory effect on the maturation of dendritic cells in vivo, the extent of which correlated to the production of exopolysaccharides. The ability to stimulate the mucosal associated immune system in such probiotic-like manner, the general absence of antibiotic resistance genes, and the lack of the biosynthetic pathways for biogenic amines should reassure on the safety of this species, with potential for exploitation of selected starters.

Comparative Genomics of Leuconostoc carnosum.

Leuconostoc carnosum is a known colonizer of meat-related food matrices. It reaches remarkably high loads during the shelf life in packaged meat products and plays a role in spoilage, although preservative effects have been proposed for some strains. In this study, the draft genomes of 17 strains of L. carnosum (i.e., all the strains that have been sequenced so far) were compared to decipher their metabolic and functional potential and to determine their role in food transformations. Genome comparison and pathway reconstruction indicated that L. carnosum is a compact group of closely related heterofermentative bacteria sharing most of the metabolic features. Adaptation to a nitrogen-rich environment, such as meat, is evidenced by 23 peptidase genes identified in the core genome and by the autotrophy for nitrogen compounds including several amino acids, vitamins, and cofactors. Genes encoding the decarboxylases yielding biogenic amines were not present. All the strains harbored 1-4 of 32 different plasmids, bearing functions associated to proteins hydrolysis, transport of amino acids and oligopeptides, exopolysaccharides, and various resistances (e.g., to environmental stresses, bacteriophages, and heavy metals). Functions associated to bacteriocin synthesis, secretion, and immunity were also found in plasmids. While genes for lactococcin were found in most plasmids, only three harbored the genes for leucocin B, a class IIa antilisterial bacteriocin. Determinants of antibiotic resistances were absent in both plasmids and chromosomes.

Draft Genome Sequences of 12 Leuconostoc carnosum Strains Isolated from Cooked Ham Packaged in a Modified Atmosphere and from Fresh Sausages.

Leuconostoc carnosum is a lactic acid bacterium that preferentially colonizes meat. In this work, we present the draft genome sequences of 12 Leuconostoc carnosum strains isolated from modified-atmosphere-packaged cooked ham and fresh sausages. Three strains harbor bacteriocin genes.

Effect of Rearing Temperature on Growth and Microbiota Composition of Hermetia illucens.

The potential utilization of black soldier fly (Hermetia illucens) as food or feed is interesting due to the nutritive value and the sustainability of the rearing process. In the present study, larvae and prepupae of H. illucens were reared at 20, 27, and 33 °C, to determine whether temperature affects the whole insect microbiota, described using microbiological risk assessment techniques and 16S rRNA gene survey. The larvae efficiently grew across the tested temperatures. Higher temperatures promoted faster larval development and greater final biomass but also higher mortality. Viable Enterobacteriaceae, Bacillus cereus, Campylobacter, Clostridium perfringens, coagulase-positive staphylococci, Listeriaceae, and Salmonella were detected in prepupae. Campylobacter and Listeriaceae counts got higher with the increasing temperature. Based on 16S rRNA gene analysis, the microbiota of larvae was dominated by Providencia (>60%) and other Proteobateria (mainly Klebsiella) and evolved to a more complex composition in prepupae, with a bloom of Actinobacteria, Bacteroidetes, and Bacilli, while Providencia was still present as the main component. Prepupae largely shared the microbiota with the frass where it was reared, except for few lowly represented taxa. The rearing temperature was negatively associated with the amount of Providencia, and positively associated with a variety of other genera, such as Alcaligenes, Pseudogracilibacillus, Bacillus, Proteus, Enterococcus, Pediococcus, Bordetella, Pseudomonas, and Kerstersia. With respect to the microbiological risk assessment, attention should be paid to abundant genera, such as Bacillus, Myroides, Proteus, Providencia, and Morganella, which encompass species described as opportunistic pathogens, bearing drug resistances or causing severe morbidity.