In Europe, Cannabis sativa L. varieties can be legally cultivated if it is registered in the EU Plant variety database of agricultural plant species and its tetrahydrocannabinol (THC) content does not exceed 0.2 % (w/w) hereafter addressed as industrial hemp (EC, 2019a). These plants find different applications in pharmacotherapy (Bonini et al., 2018), agronomy (Das et al., 2017), food industry (Radočaj et al., 2014; Zając et al., 2018), cosmetic (Nuutinen, 2018), sustainable building (Arizzi et al., 2019), animal production (Khan et al., 2010; Neijat et al., 2016; Vispute et al., 2019), broiler meat and chicken egg production (Goldberg et al., 2012; Jing et al., 2017). In Italy the cultivation of industrial hemp is specifically regulated by law N° 242 of 2016, which specifically promotes the use of this plant in research, cosmetics, food industry, biomaterials, sustainable building.

As food or food ingredient, industrial hemp is regulated at European level as novel food and needs pre-market authorisation before commercialisation (EC, 2019b; Regulation EU 2015/2283). However, some products such as hemp seed, seed flour and oil have a long history of consumption and cannot be considered as novel. For them, the use is regulated at national level. With the exception of Romania, where hemp seed and oil can be used without any limit, in other countries such as The Netherlands, Germany, Danmark and Italy, the maximum limits of THC in hemp seed, oil, flour and other foods and drinks is specifically regulated. In particular for Italy, the 30^th of October 2018, the Ministry of Health submitted to the European Commission a draft regulation, not yet approved, including the following maximum limits of THC: hemp oil – 5 ppm; hemp seeds and seed flour – 2 ppm; dietary supplement including hemp derivatives: 2 ppm (Italian Ministry of Health, 2018). According to this draft regulation, for all the other hemp derivatives, for example hemp inflorescence, food business operators shall provide evidences on the THC concentration in foodstuffs to competent authorities of official control taking into account specific concentration or dilution factors linked to the food processing (Commission Regulation (EC) No 1881/2006).

Industrial hemp inflorescence, especially resin secreted from the trichomes of female plants, are rich in phytocannabinoids. In C. sativa, cannabinoids are biosynthesized and accumulated as cannabinoid acids, and subsequently decarboxylated into their neutral forms (Bonini et al., 2018). At present around 100 molecules have been characterised with Cannabidiol (CBD) as the one of the most represented in industrial hemp. Properties such as anti-inflammatory, immunomodulation, anti-arthritic have been attributed to CBD (Bonini et al., 2018). Along with these properties, CBD has been described as an antimicrobial agent since the 70ies when CBD was shown to be effective against Gram + bacteria, although not against Gram - (Van Klingeren and Ham, 1976). More recently, MICs of 0.5-1 μg/mL were registered for CBD against different strains of multiresistant and methicillin resistant Staphylococcus aureus (MRSA) (Appendino et al., 2008). Antimicrobial activity of CBD against S. aureus was observed also by other authors (Burstein et al., 2015). Along phytocannabinoids, also terpenoids are found in C. sativa. More than 200 terpenoids have been characterised with limonene, β-myrcene, α- and β-pinene as the most common. Terpenoids act synergistically with phytocannabinoids in the defense strategy against predators (Bonini et al., 2018; Russo et al., 2011). Antimicrobial properties were specifically observed for α- and β-pinene against both Gram + and Gram – bacteria as well as fungi namely, Pseudomonas aeruginosa, Escherichia coli, MRSA and Candida albicans, (da Silva et al., 2012; Dai et al., 2013; Leite et al., 2007).

Due to the presence of different antimicrobial compounds acting synergistically, the antimicrobial activity of Cannabis sativa L. might be potentially higher than those of the single molecules (Russo et al., 2011; Blasco-Benito et al., 2018; Andre et al., 2016). Different types of extracts of seeds or female inflorescence, and resin of industrial hemp have been tested against bacteria, fungi and moulds. For a comprehensive and detailed review please refer to Tandon et al. (2017). With particular reference to bacteria, although with broad diverse results related to plant varieties, extraction methods and parts of the plant tested, industrial hemp extracts showed to be effective against microbial pathogens such as Escherichia coli, Pseudomonas aeruginosa and Staphyloccocus aureus. Among all varieties tested, Futura 75 might be addressed as a promising antimicrobial. Futura 75 variety showed superior antimicrobial properties in comparison to other varieties. In particular, higher MIC values were registered against Enterococcus faecium and Clostridium botulinum (Futura MIC values of 1.55 and 1.76 %v/v respectively) (Nissen et al., 2010). More recently, the water flower extract of Cannabis sativa L. variety Futura 75 was described as effective also against E. coli, P. aeruginosa and S. aureus (Ferrante et al., 2019). Unfortunately, all information on antimicrobial activity of Cannabis sativa were collected from experiments performed in vitro against food-unrelated microbial pathogens. Since the great application of industrial hemp as food ingredient for nutraceutical purposes, knowledges are missing on food safety and hygiene and in particular on the antimicrobial properties of this plant against foodborne-pathogens in vitro as well as against bacteria related to the hygiene of food processing (Commission Regulation (EC) No 2073/2005).

The research question of the present study was to investigate the antimicrobial activity of an extract of the female fluorescence of Cannabis sativa L. variety Futura 75 in vitro against E. coli, Salmonella Typhimurium, Listeria monocytogenes and Staphylococcus spp. and against microbial populations occurring in minced beef stored for 8 days at 4°C.