Aproximación a la evaluación de la potencia de la resina de cannabis en Madrid: ¿Un riesgo para la salud? / An approach to the evaluation of the potency of cannabis resin in Madrid: A health hazard?

El presente estudio investiga la concentración de Delta(9)-tetrahidrocannabinol (THC), cannabidiol (CBD) y cannabinol (CBN) en 60 muestras de resina de cannabis adquiridas en las calles de Madrid y su potencial riesgo para la salud del consumidor. Adicionalmente, estudiamos la posible asociación entre la potencia de las muestras y sus características organolépticas. El análisis de cannabinoides se llevó a cabo mediante cromatografía líquida de alta resolución (RP-HPLC-UV). Atendiendo al contenido en THC se estableció una escala de potencia para clasificar las muestras. El 76,7% de las muestras tenía un contenido en THC superior al 15%, esta potencia las cataloga como drogas de Grado I con “riesgo inaceptable” para la salud. El 36,7% de las muestras presentaron un contenido medio en THC del 28,8% (potencia muy alta). El contenido medio en CBD fue del 5% y el de CBN 1,74%; ambas ratios, CBD/THC y CBN/THC, mostraron una correlación negativa con la potencia. Al investigar la posible asociación entra potencia y características organolépticas, se observó que las muestras que presentaban a la vez una textura pegajosa, una elasticidad alta y un color marrón claro, tenían una potencia muy alta, con un contenido medio en THC del 28.7%. Nuestro estudio muestra que el contenido en THC de la mayoría de la resina de cannabis que puede adquirirse en Madrid es superior al 15% y supone un elevado riesgo para la salud. Adicionalmente, demostramos por primera vez que solo aquellas muestras con una potencia muy alta pueden asociarse directamente con ciertas características organolépticas. (AU)

The present study investigates the concentration of Delta (9)-tetrahidrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in 60 samples of cannabis resin acquired on the streets of Madrid region and its potential danger to consumers’ health. Additionally, we study the possible correlation between the potency of samples and their organoleptic characteristics. The analysis of cannabinoids was carried out using a high performance liquid chromatography (RP-HPLC-UV). To classify samples, a strength scale based on THC content was established. THC content in 76.7% of the samples was higher than 15%. This potency allows these samples to be classified as Schedule I or drugs with “unacceptable risk” for human health. THC content in 36.7% of the samples was 28.8% on average, which means very high potency. The mean CBD content was 5%, while the correlation between the CBD/THC ratio and potency was negative. The mean content of CBN was 1.74% and the CBN/THC ratio also showed a negative correlation in respect to potency. When investigating the possible correlation between sample potency and organoleptic characteristics, those samples which simultaneously presented sticky texture, high elasticity and light brown colour had very high potency, with an average THC content of 28.7%. Our study shows that the THC content of most of the cannabis that can be purchased in Madrid region is over 15% and poses a health hazard. Additionally, we demonstrate for the first time that only those samples with very high potency can be directly associated with certain organoleptic characteristics. (AU)

Validation of the 3M™ Molecular Detection Assay 2-STEC Gene Screen (stx) for Detection of Shiga Toxin Gene (stx1 and/or stx2) in Dried Cannabis Flower and Dried Hemp Flower: AOAC Performance Tested MethodSM 071903.

BACKGROUND: The 3M™ Molecular Detection Assay 2-STEC Gene Screen (stx) method is based on gene amplification by the use of real-time loop-mediated isothermal amplification when used with the 3M Molecular Detection System for the rapid and specific detection of Shiga toxin gene (stx1 and/or stx2) from Shiga toxin-producing Escherichia coli (STEC) in enriched products. The 3M Molecular Detection Assay 2-STEC Gene Screen (stx) was approved as AOAC Performance Tested MethodSM Certificate No. 071903. OBJECTIVE: This matrix extension study evaluated the 3M Molecular Detection Assay 2-STEC Gene Screen (stx) method for detection of STECs in dried cannabis flower [>0.3% delta 9-tetrahydrocannabinol (THC)] and dried hemp flower (≤0.3% THC) at a 10 g test portion size. METHOD: Testing followed procedures outlined in 3M Molecular Detection Assay 2-STEC Gene Screen (stx) product instructions and Standard Method Performance Requirement (SMPR®) for Detection of Shiga Toxin-Producing Escherichia coli in Cannabis and Cannabis Products (AOAC SMPR 2020.012). The method was evaluated at low, high, and non-inoculated levels. RESULTS: Results showed no statistically significant difference between the presumptive positive 3M Molecular Detection Assay 2-STEC Gene Screen (stx) results and the SMPR 2020.012 recommended cultural confirmations. CONCLUSIONS: This study provides data that demonstrate the 3M Molecular Detection Assay 2-STEC Gene Screen (stx) is a reliable method for the rapid and specific detection of STECs in dried cannabis flower and dried hemp flower. HIGHLIGHTS: The 3M Molecular Detection Assay 2-STEC Gene Screen (stx) method is suitable for the rapid and specific detection of STECs in dried cannabis flower and dried hemp flower.

Validation of the 3M™ Molecular Detection Assay 2-STEC Gene Screen (stx and eae) for Detection of Shiga Toxin Gene (stx1 and/or stx2) and Intimin Gene (eae) in Dried Cannabis Flower and Dried Hemp Flower: AOAC Performance Tested MethodSM 071902.

BACKGROUND: The 3M™ Molecular Detection Assay 2-STEC Gene Screen (stx and eae) method is based on gene amplification by the use of real time loop-mediated isothermal amplification when used with the 3M Molecular Detection System for the rapid and specific detection of Shiga toxin gene (stx1 and/or stx2) and intimin gene (eae) from Shiga toxin-producing Escherichia coli (STEC) in enriched products. The 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) was approved as AOAC Performance Tested MethodSM Certificate No. 071902. OBJECTIVE: This matrix extension study evaluated the 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) method for detection of STECs in dried cannabis flower [>0.3% delta 9-tetrahydrocannabinol (THC)] and dried hemp flower (≤0.3% THC) at a 10 g test portion size. METHOD: Testing followed procedures outlined in 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) product instructions and Standard Method Performance Requirements (SMPR®) for Detection of Shiga Toxin-Producing Escherichia coli in Cannabis and Cannabis Products (AOAC SMPR 2020.012). The method was evaluated at low, high, and non-inoculated levels. RESULTS: Results showed no statistically significant difference between the presumptive positive 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) results and the SMPR 2020.012 recommended cultural confirmations. CONCLUSIONS: This study provides data that demonstrate the 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) is a reliable method for the rapid and specific detection of STEC organisms in dried cannabis flower and dried hemp flower. HIGHLIGHTS: The 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) method is suitable for the rapid and specific detection of STEC organisms in dried cannabis flower and dried hemp flower.

Core RNA Interference Genes Involved in miRNA and Ta-siRNA Biogenesis in Hops and Their Expression Analysis after Challenging with Verticillium nonalfalfae.

RNA interference is an evolutionary conserved mechanism by which organisms regulate the expression of genes in a sequence-specific manner to modulate defense responses against various abiotic or biotic stresses. Hops are grown for their use in brewing and, in recent years, for the pharmaceutical industry. Hop production is threatened by many phytopathogens, of which Verticillium, the causal agent of Verticillium wilt, is a major contributor to yield losses. In the present study, we performed identification, characterization, phylogenetic, and expression analyses of three Argonaute, two Dicer-like, and two RNA-dependent RNA polymerase genes in the susceptible hop cultivar Celeia and the resistant cultivar Wye Target after infection with Verticillium nonalfalfae. Phylogeny results showed clustering of hop RNAi proteins with their orthologues from the closely related species Cannabis sativa, Morus notabilis and Ziziphus jujuba which form a common cluster with species of the Rosaceae family. Expression analysis revealed downregulation of argonaute 2 in both cultivars on the third day post-inoculation, which may result in reduced AGO2-siRNA-mediated posttranscriptional gene silencing. Both cultivars may also repress ta-siRNA biogenesis at different dpi, as we observed downregulation of argonaute 7 in the susceptible cultivar on day 1 and downregulation of RDR6 in the resistant cultivar on day 3 after inoculation.

Ultra-High-Performance Liquid Chromatography Coupled with Quadrupole Orbitrap High-Resolution Mass Spectrometry for Multi-Residue Analysis of Mycotoxins and Pesticides in Botanical Nutraceuticals.

Cannabidiol (CBD) food supplements made of Cannabis sativa L. extracts have quickly become popular products due to their health-promoting effects. However, potential contaminants, such as mycotoxins and pesticides, can be coextracted during the manufacturing process and placed into the final product. Accordingly, a novel methodology using ultra-high-performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was developed to quantify 16 mycotoxins produced by major C. sativa fungi, followed by a post-target screening of 283 pesticides based on a comprehensive spectral library. The validated procedure was applied to ten CBD-based products. Up to six different Fusarium mycotoxins were found in seven samples, the most prevalent being zearalenone (60%) and enniatin B1 (30%), both found at a maximum level of 11.6 ng/g. Co-occurrence was observed in four samples, including one with enniatin B1, enniatin A and enniatin A1. On the other hand, 46 different pesticides were detected after retrospective analysis. Ethoxyquin (50%), piperonyl butoxide (40%), simazine (30%) and cyanazine (30%) were the major residues found. These results highlight the necessity of monitoring contaminants in food supplements in order to ensure a safe consumption, even more considering the increase trend in their use. Furthermore, the developed procedure is proposed as a powerful analytical tool to evaluate the potential mycotoxin profile of these particular products.

Pararcticibacter amylolyticus gen. nov., sp. nov., Isolated from a Rotten Hemp Rope, and Reclassification of Pedobacter tournemirensis as Pararcticibacter tournemirensis comb. nov.

A Gram-stain-negative, rod-shaped, non-motile, facultatively anaerobic bacterium, designated FJ4-8T, was isolated from a rotten hemp rope in Chongqing City, PR China. Phylogenetic analysis of 16S rRNA gene sequences indicated that the isolate was closely related to members of the family Sphingobacteriaceae, with the highest similarity to Pedobacter tournemirensis TF5-37.2-LB10T (95.3%) and low similarities to all other species of the genus Pedobacter (90.4-93.9%). Phylogenetic analyses demonstrated that strain FJ4-8T formed a stable subclade with Pedobacter tournemirensis TF5-37.2-LB10T. The clade with these two strains branched adjacent to a clade containing three species of the genus Arcticibacter. MK-7 was detected as the only respiratory quinone. The major fatty acids composed iso-C15:0, iso-C17:0 3-OH and summed feature three. Phosphatidylethanolamine, three aminophospholipids and one unidentified lipid were found as the major polar lipids. The major polyamine was identified as sym-homospermidine. The DNA-DNA hybridization value between strain FJ4-8T and Pedobacter tournemirensis TF5-37.2-LB10T was 42.0 ± 2.5%. Based on its phylogenetic, chemotaxonomic and phenotypic characteristics, the novel strain and TF5-37.2-LB10T were found to be different from members of genera Pedobacter and Arcticibacter. FJ4-8T and TF5-37.2-LB10T represented different species. Therefore, FJ4-8T should be classified as a novel species of a novel genus in the family Sphingobacteriaceae, for which the name Pararcticibacter amylolyticus gen. nov., sp. nov. is proposed. The type strain is FJ4-8T (= KCTC 62640T = CCTCC AB 2018052T). The draft genome sequence is 6290, 449 bp in length, the genomic DNA G+C content was 44.4 mol%. Pedobacter tournemirensis TF5-37.2-LB10T should be transferred to the novel genus as Pararcticibacter tournemirensis comb. nov. (The type strain is TF5-37.2-LB10T (= DSM 23085T = CIP 110085T = MOLA 820T).

Automated image analysis with ImageJ of yeast colony forming units from cannabis flowers.

Currently, in the state of Colorado and all other states within the United States of America with legalized marijuana programs, testing is required for bacteria, yeast, and mold on marijuana products. The Code of Colorado Regulations, 1 CCR 212-1, considers a passing result when a 1 g sample contains <104 colony forming units (CFU) for the total yeast and mold count (TYMC). These measurements are usually obtained by manually counting colonies on petri-dishes or 3 M™ Petrifilms™, which is a time consuming and user subjective process. Therefore, an automated counting method utilizing ImageJ has been developed for CFU analysis of TYMC on Petrifilms. The performance of this colony counting method was demonstrated by comparing manual and automated counts from marijuana flower samples containing spikes of Candida albicans as well as samples that tested positive for the presence of yeast and mold. Fifteen images of Petrifilms showing various concentrations of colonies were studied by fifteen users at two institutions using both the automated and manual counting methods. All counts from the automated ImageJ procedure were within 12% of those obtained manually. In twelve out of fifteen Petrifilms, the average count of the automated method was statistically similar to the manual counts. The statistical differences of the other three samples were observed to be random and caused by user errors. The automated counting method could be used to quickly count numbers that are as high as 400 CFUs, reducing time of analysis with improved documentation because the images and the electronic colony counts can be saved on a computer or cloud for long term storage and data access.

The Role of Mass Spectrometry in the Cannabis Industry.

The focus of this critical insight article is a brief overview of analytical challenges the cannabis industry faces and how analytical chemists have new opportunities to demonstrate the merits of employing mass spectrometry for the chemical analysis of cannabis and its products. The current range of cannabis products extends from recreational use to medicines, edibles, beverages, and beyond. The standards employed to assure product quality, integrity, and safety are lacking compared to those currently used by the pharmaceutical, food, and beverage industries. This manuscript overviews some of the important analytical issues that exist for the growth and harvest of the cannabis plant to the production of a wide variety of its products. Currently, the topics of interest for safety in cannabis testing where mass spectrometry can play an important role include what are currently referred to as potency, pesticides, terpenes, heavy metals, and mycotoxins from bacteria. Since each state in the USA as well as several countries has their own regulations, the analytical opportunities and challenges vary depending upon which jurisdiction a laboratory is supporting. This Critical Insight report will suggest where mass spectrometry can play an important role and provide valuable input on these topics. Graphical Abstract.

Mapping the Secretome and Its N-Linked Glycosylation of Pleurotus eryngii and Pleurotus ostreatus Grown on Hemp Stalks.

Our previous research showed that Pleurotus eryngii and Pleurotus ostreatus were effective fungi for pretreatment of industrial hemp stalks to improve enzymatic saccharification. The secretomes of these two fungi were analyzed to search for the effective enzyme cocktails degrading hemp lignin during the pretreatment process. In total, 169 and 155 proteins were identified in Pleurotus eryngii and Pleurotus ostreatus, respectively, and 50% of the proteins involved in lignocellulose degradation were CAZymes. Because most of the extracellular proteins secreted by fungi are glycosylated proteins, the N-linked glycosylation of enzymes could be mapped. In total, 27 and 24 N-glycosylated peptides were detected in Pleurotus eryngii and Pleurotus ostreatus secretomes, respectively. N-Glycosylated peptides of laccase, GH92, exoglucanase, phenol oxidase, α-galactosidase, carboxylic ester hydrolase, and pectin lyase were identified. Deglycosylation could decrease enzymatic saccharification of hemp stalks. The activities of laccase, α-galactosidase, and phenol oxidase and the thermal stability of laccase were reduced after deglycosylation.

Endophytes of industrial hemp (Cannabis sativa L.) cultivars: identification of culturable bacteria and fungi in leaves, petioles, and seeds.

Plant endophytes are a group of microorganisms that reside asymptomatically within the healthy living tissue. The diversity and molecular and biochemical characterization of industrial hemp-associated endophytes have not been previously studied. This study explored the abundance and diversity of culturable endophytes residing in petioles, leaves, and seeds of three industrial hemp cultivars, and examined their biochemical attributes and antifungal potential. A total of 134 bacterial and 53 fungal strains were isolated from cultivars Anka, CRS-1, and Yvonne. The number of bacterial isolates was similarly distributed among the cultivars, with the majority recovered from petiole tissue. Most fungal strains originated from leaf tissue of cultivar Anka. Molecular and phylogenetic analyses grouped the endophytes into 18 bacterial and 13 fungal taxa, respectively. The most abundant bacterial genera were Pseudomonas, Pantoea, and Bacillus, and the fungal genera were Aureobasidium, Alternaria, and Cochliobolus. The presence of siderophores, cellulase production, and phosphorus solubilization were the main biochemical traits. In proof-of-concept experiments, re-inoculation of tomato roots with some endophytes confirmed their migration to aerial tissues of the plant. Taken together, this study demonstrates that industrial hemp harbours a diversity of microbial endophytes, some of which could be used in growth promotion and (or) in biological control designed experiments.

Genome of Serratia nematodiphila MB307 offers unique insights into its diverse traits.

A pigment-producing species of Serratia was isolated from the rhizosphere of a heavy metal resistant Cannabis sativa plant growing in effluent-affected soil of Hattar Industrial Estate, Haripur, Pakistan. Here, we report the genome sequence of this bacterium, which has been identified as Serratia nematodiphila on the basis of whole genome comparison using the OrthoANI classification scheme. The bacterium exhibited diverse traits, including plant growth promotion, antimicrobial, bioremediation, and pollutant tolerance capabilities including metal tolerance, azo dye degradation, ibuprofen degradation, etc. Plant growth-promoting exoenzyme production as well as phosphate solubilisation properties were observed. Genes for phosphate solubilisation, siderophore production, and chitin destruction were identified in addition to other industrially important enzymes like nitrilase and lipase. Secondary metabolite producing apparatus for high value chemicals in the whole genome was also analysed. The number of antibiotic resistance genes was then profiled in silico, through a match with Antibiotic Resistant Gene and CAR database. This is the first report of a S. nematodiphila genome from a polluted environment. This could significantly contribute to the understanding of pollution tolerance, antibiotic resistance, association with nematodes, production of bio-pesticide, and their role in plant growth promotion.

Pro-technological and functional characterization of lactic acid bacteria to be used as starters for hemp (Cannabis sativa L.) sourdough fermentation and wheat bread fortification.

Lactic acid bacteria were isolated from hemp (Cannabis sativa L.) flour, spontaneously fermented dough, and type I sourdough. Isolates were identified and further selected based on pro-technological, nutritional and functional properties. Lactobacillus plantarum/s5, Pediococcus acidilactici/s5, and Leuconostoc mesenteroides/s1 were used as mixed starter to produce hemp sourdough. Significant decreases of the concentration of phytic acid, condensed tannins, and total saponins were observed during fermentation. The in vitro protein digestibility increased up to 90%. Experimental wheat breads were made adding 5% to 15% (w/w) hemp sourdough to the formula, characterized, and compared to baker's yeast wheat bread manufactured without hemp sourdough. The use of hemp sourdough improved the textural features of wheat bread, without adversely affect the sensory profile. Proportionally to the fortification with hemp sourdough, protein digestibility of the breads increased, while the predicted glycemic index significantly decreased (87 vs 100%). This work demonstrated that the fermentation with selected starters improved nutritional functionality of hemp flour, allowing its large-scale use in different food applications, meeting the consumers and producers request for novel fermented baked goods with a well-balanced nutritional profile.

High pressure homogenization combined with pH shift treatment: A process to produce physically and oxidatively stable hemp milk.

Hemp milk, an emerging beverage with high nutritional value and low allergenicity, is an attractive alternative to dairy, soy, and nut milks. To obtain a non-thermally processed, physically and oxidatively stable hemp milk, high pressure homogenization (HPH) combined with pH shift treatment was investigated. For hemp milk (4% protein, 5% fat) without pH shift, increasing the homogenization pressure (up to 60 MPa) resulted in a more uniform distribution of emulsion droplets (2.2-2.7 µm). When pH shift was applied prior to HPH, large clusters and aggregates of oil droplets (3.5-8.2 µm) were formed. Interestingly, hemp milk with such interactive structures was remarkably stable, showing negligible phase separation within 3-day storage at 4 °C. Moreover, hemp milk made by combined pH shift and HPH exhibited delayed hydroperoxides (expressed as peroxide value, PV) and malondialdehyde (expressed as thiobarbituric acid-reactive substances, TBARS) production, suggesting the resistance of such emulsion cluster structures to radicals. On the other hand, a significant reduction of microbial population was observed in hemp milk prepared by pH shift combined with HPH. The results indicate that the pH shift + HPH combination treatment may potentially be employed for the production of non-thermally processed hemp milk.

Microbial hazards during harvesting and processing at an outdoor United States cannabis farm.

Cannabis cultivation is an emerging industry within the United States. Organic dust derived in part from naturally occurring microorganisms is known to cause byssinosis in the hemp industry. In this pilot study, bacteria and fungi encountered by workers at an outdoor cannabis farm that utilized organic practices were elucidated by 16 S ribosomal RNA (rRNA) and Internal Transcribed Spacer (ITS) region sequencing, respectively. Area (n = 14) and personal air samples (n = 12) were collected during harvesting and processing activities. 16 S rRNA and ITS regions of extracted bacterial and fungal genomic DNA were amplified and sequenced using Sanger sequencing. Bacterial sequencing resolved 1,077 sequences that were clustered into 639 operational taxonomic units (OTUs) and predominantly placed in the phylum, Actinobacteria (46%). Personal air samples revealed higher bacterial and Actinobacteria diversity compared to outdoor area samples collected within the facility (p < 0.05). A high degree of dissimilarity between bacteria was identified within and between samples. Fungal sequences (n = 985) were identified and predominantly clustered in the phylum Ascomycota (53%). Of the 216 fungal OTUs elucidated, the cannabis plant pathogenic species, Botrytis cinerea, was the most prevalent and accounted for 34% of all fungal sequences. The relative abundance of B. cinerea was highest in personal air samples (59%) compared to area samples collected in the drying room (19%), greenhouse (18%), and outdoor environment (6%). There was 49% sample similarity between fungi identified within personal air samples, but higher dissimilarity coefficients were observed within and between greenhouse, drying room, and outdoor area air samples. The results of this pilot study suggest that the cannabis farm workers are potentially exposed to Actinobacteria as well as the cannabis plant pathogen, B. cinerea during harvesting, bud-stripping, and hand-trimming processes.

Evaluación de contaminantes microbianos en muestras de cannabis incautadas en Costa Rica / Evaluation of microbial contamination in samples of cannabis seized in Costa Rica

Diferentes literaturas sugieren que el consumo de cannabis es un importante problema para la salud pública. El cannabis, por su naturaleza ilegal, llega a sus usuarios sin cumplimiento de normas adecuadas de cultivo, almacenamiento, procesamiento y tráfico; lo que puede generar diversos contaminantes, que pueden ser químicos, físicos o microbianos. La presencia de contaminantes microbianos es un riesgo para la salud de los usuarios, sobre todo en pacientes con el sistema inmune comprometido. Costa Rica no cuenta con estudios sobre la presencia de contaminantes microbianos en el cannabis comercializado, por lo que este estudio pretendía establecer si los hay en las muestras incautadas en seis regiones del territorio costarricense y relacionar este hecho con riesgos para la salud de sus consumidores. En dichas muestras se realizaron pruebas de conteo total de bacterias aeróbicas, levaduras y hongos según los procedimientos establecidos por la Farmacopea de los Estados Unidos de América (USP). Para la identificación de microorganismos potencialmente patógenos, se realizaron los procedimientos descritos por la USP, el uso del sistema API y metodología de identificación morfológica de hongos. Los conteos totales de bacterias, de levaduras y hongos superan los límites establecidos por USP en la mayoría de las muestras. Se hallaron cuatro tipos de hongos: Aspergillus sp., Scopulariopsis sp, Fusarium sp y Penicillum sp y cinco especies bacterianas: Staphylococcus aureus, S. xylosus, S. lentus, Enterobacter cloacae y E. sakazakki. La contaminación hallada requiere de un debate para el establecimiento de una verdadera política de regulación, tanto para fin terapéutico como para fines recreativos

Different literature suggests that cannabis use is a major problem for public health. Cannabis, through its illegal nature, reaches its users without complying with any suitable standards of growth, storage, processing and transport, which may generate diverse pollutants, which can be chemical, physical or microbial. The presence of microbial pollutants is a risk for the user’s health, especially in patients whose immune system is compromised. In Costa Rica there are no studies on the presence of microbial pollutants in commercialized cannabis, which is why the aim of this study was to establish the presence of the aforementioned pollutants in cannabis samples seized in six regions of Costa Rican territory and to relate this fact to risks for the health of its consumers. These samples were tested for their total count in aerobic bacteria, yeast and fungi according to procedures established by the Pharmacopoeia of the United States (USP). Identification of potentially pathogenic microorganisms was performed using methods described by USP; using the API system and methodology for morphological identification of fungi. The total counts of bacteria, yeast and fungus exceed limits set by USP on most of the seized samples. Four types of fungus were found: Aspergillus sp, Scopulariopsis sp, Fusarium sp and Penicillium sp, and five bacterial species: Staphylococcus aureus, S. xylosus, S. lentus, Enterobacter cloacae and E. sakazakki. The contamination found requires a debate for establishing a genuine policy of cannabis regulation, both for therapeutic or recreational purposes

Microbial diversity observed during hemp retting.

Historically used in textile and paper industry, hemp fibres have started to find new applications in composite materials with important economic and ecological advantages. However, their applications are limited since manufacturers have some difficulties to standardise fabrication processes. This study is a first step before selection and isolation of strains that could later be used to optimise microbial retting efficiency and hence fibre quality. We studied six samples harvested on different ground types, at different dates and with different retting durations on field to obtain an exhaustive representation of the process. After DNA extraction, total bacteria and fungi associated with stems during retting were specifically quantified using real-time PCR. Then, using sequence analysis of randomly cloned 16S and 18S ribosomal RNA (rRNA) genes, a phylogenetic characterisation of the dominant microorganisms was carried out. Quantitatively, we showed that there were 8.1-9.5 log10 16S rRNA gene copies per gram of hemp straw for bacteria and 8.6-9.6 log10 18S rRNA gene copies per gram for fungi. Qualitatively, we noticed a higher bacterial diversity in comparison to fungi. This work showed that in the different samples, the same species were present but in significantly different proportions according to ground type, harvest dates and retting durations on field. The most frequent bacterial sequences were affiliated to species Escherichia coli, Pantoea agglomerans, Pseudomonas rhizosphaerae, Rhodobacter sp., Pseudomonas fulva, Rhizobium huautlense and Massilia timonae, whereas fungal sequences were principally related to the genera Cladosporium and Cryptococcus.