Preliminary data on the antimicrobial effect of Cannabis sativa L. variety Futura 75 against food-borne pathogens in vitro as well as against naturally occurring microbial populations on minced meat during storage

Abstract

Introduction

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.

Materials and Methods

Phytocannabinoids detection and quantification

Phytocannabinoids were detected and quantified in the grind as well as in two autoclaved and not autoclaved aliquots of the extract following a previously reported protocol with some minor modifications (Mandrioli et al., 2019). The autoclaved and not-autoclaved aliquots were tested in order to evaluate the exact cannabinoid composition in the in vitro susceptibility and the food experiments respectively. An aliquot of the sample (grind), about 50 mg, was added to 10 mL of methanol-chloroform 9:1 (v/v) extraction solvent. After 10 min on agitation set at 350 oscillations per minute, the sample was ultrasonicated for 10 minutes. After centrifugation for 5 minutes at 1620 xg, the supernatant was collected. The extraction was repeated twice and the two fractions were collected in a 25 mL volumetric flask and brought to volume with methanol/ chloroform (9:1, v/v). The solution was filtered with a 45 μm nylon filter. One mL of this solution was dried under weak nitrogen flow. Dried material was resuspended in 1 mL of acetonitrile, 5.0 μl of which was injected into an RP-HPLCDAD liquid chromatography system. For the extract (autoclaved and not autoclaved),1 mL was transferred and directly dissolved into a 10 mL flask and brought to volume with acetonitrile. For the HPLC analysis, UV detection was used at 230 nm, gradient elution was used at flow rate of 1.5 mL/min according to the following procedure: eluent mixture A water + 0.1% phosphoric acid, B acetonitrile + 0.1% phosphoric acid; gradient elution: 75% of B up to 0.7 min. 85% of B to 2 min. 100% of B to 3.0-3.5 min and 100% of B to 3.6-5.0 min. Data were acquired using Chemstation software for LC3D (Rev.A.08.03 Agilent Technologies, USA). The quantification of analytes was carried out using the external standard method, through the construction of calibration curves prepared with standard reference compounds of chromatographic purity. Standard references of CBDA, CBGA, CBG, CBD, THCV, CBN, Δ9-THC, Δ8-THC, CBC, and THCA were diluted wit acetonitrile in a concentration range between 0.1-100 μg/mL. Eight dilutions of each cannabinoid was used to build the calibration curve. The standard solutions were stored away from light at a temperature of −20 ◦C. The equations of the calibration curves were as follows:

Figure 1.

Minced beef with extract (ETA) and without (CONTR) after 8 days of storage at 4°C.

Table 2.

Susceptibility to CBD and Cannabis sativa of S. Typhimurium (strains ST208, ST63), E. coli (strains EC ATCC 25922, EC135), L. monocytogenes (LM1, LM2) and Staphylococcus spp. (strain S661).

CBD Concentration (mg/mL)	ST208	ST63	EC ATCC	EC135	LM1	LM2	S661
Extract
0.6	+	+	+	+	–	–	–
0.3	+	+	+	+	–	–	–
0.15	+	+	+	+	–	–	+
0.07	+	+	+	+	–	–	+
0.035	+	+	+	+	–	–	+
0.017	+	+	+	+	–	–	+
CBD
0.6	+	+	+	+	–	–	–
0.3	+	+	+	+	–	–	–
0.15	+	+	+	+	–	–	+
0.07	+	+	+	+	–	–	+
0.035	+	+	+	+	–	–	+
0.017	+	+	+	+	–	–	+
Negative control
0	+	+	+	+	+	+	+

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CBD, y=6.5473x-1.6742 (r2=0.9999); CBG, y=5.9001x-0.4766 (r2=0.9964); Δ9-THC, y=6.3627x-0.2229 (r2=0.9962); CBN, y=14.792x-0.6310 (r2=0.9990); CBC, y=15.619x-0.788 (r2=0.9989); CBDA, y=10.018x+1.1843 (r2=0.9981); CBGA, y=9.5259x-1.1675 (r2=0.9959); THCA, y=9.154x-0.9992 (r2=0.9954); Δ8-THC, y=4.8513x+1.0499(r2=0.9979); THCV, y=4.9225x+1.883 (r2=0.9987).

Results

In vitro evaluation of Cannabis sativa susceptibility against foodborne pathogens

The antimicrobial properties of Cannabis sativa L. variety Futura 75 were investigated in vitro in comparison to pure CBD against S. Typhimurium, E. coli, L. monocytogenes and Staphylococcus spp. (Table 2). At the tested concentrations, hemp as well as pure CBD were effective in inhibiting Gram positive bacteria but not Gram negative ones (ST208, ST63, EC ATCC and EC135). In particular, all tested concentrations were effective against L. monocytogenes (LM1 and LM2) and 0.3 mg/mL was effective against Staphylococcus spp (S661). This result confirmed previous published data (Duarte et al., 2016).

Figure 2.

Enumeration of enterobacteriaceae in minced meat with extract (treated) and without (control) during storage at 4°C for 8 days.

Figure 3.

Enumeration of coliforms in minced meat with extract (treated) and without (control) during storage at 4°C for 8 days.

Discussion

Cannabis sativa variety 75 was investigated as antimicrobial against foodborne pathogens as well as against microbial populations of minced beef. Results in vitro confirmed previously reported data on inhibitory properties of this plant against Staphylococcus aureus (Appendino et al., 2008; Duarte et al., 2016). Both L. monocytogenes tested strains (LM1 and LM2) were susceptible to the whole range of CBD and plant concentrations tested. In literature, data are available on oil extract efficacy against this microorganism. Nevertheless controversial results are reported with moderate (Minimum Inhibitory Concentration (MIC) > 2048 μg/mL) to high efficacy (MIC 2-32 μg/mL) (Iseppi et al., 2019; Marini et al., 2018). In contrast, S. Typhimurium and E. coli strains were resistant to both CBD and hemp extract. Efficacy against Gram negative bacteria and in particular E. coli is controversial since both susceptibility and resistance to hemp were described (Ferrante et al., 2019; Tardon et al., 2017). In particular, oil, aqueous and ethanol extracts were described as inactive or slightly active against E. coli, whereas high activity was attributed to methanol and petroleum hemp extracts (Tardon et al., 2017). In the present study, results confirmed the lack of efficacy of ethanol extract against E. coli and they extended the same observation to S. Typhimurium, which was never tested before to the best of author’s knowledge. Moreover, the lack of activity was observed independently from the antibiotic susceptibility profile of selected strains. E. coli and S. Typhimurium, multiresistant as well as antibiotic susceptible strains were equally resistant to Cannabis sativa and CBD at the tested concentrations.

Industrial hemp seeds and seed oil have been recently applied as functional food or functional food ingredient thanks to their significant contribution in polyunsaturated fatty acids, optimum Omega-6 and Omega- 3 ratio, fibres and amino acids (Apostol et al. 2015; Frassinetti et al., 2017; Radočaj et al., 2014). However, to the best of author’s knowledges, the effect of hemp extract as natural food preservative was never investigated before. In particular, in the present study the antimicrobial effect of hemp extract was investigated against microbial indicators of food hygiene of minced meat. After one week of storage at 4°C, a significant reduction was achieved for Enterobacteriaceae and coliforms respectively, along with a delay on the growth of aerobic colony count. Moreover, a striking lighter red colour of the minced meat was observed in comparison to the control, suggesting: i) hemp antioxidant properties as previously described (Bonini et al., 2018); ii) reduction of strict aerobic spoilage bacteria of the Pseudomonas genus.

Figure 4.

Aerobic colony count in minced meat with extract (treated) and without (control) during storage at 4°C for 8 days.

Figure 5.

Escherichia coli enumeration in minced meat with extract (treated) and without (control) during storage at 4°C for 8 days.

Interestingly, the hemp extract showed antimicrobial properties both after heat treatment due to autoclaving in the in vitro experiment and without heat treatment in the food experiment. In particular, in the food experiment a final CBD concentration of 6.45 μg/g was enough to significantly impact on the growth of tested bacterial groups. As demonstrated by chemical results heat treatment allows the decarboxylation of cannabinoids compounds which shifted from an inactive acid form to an active decarboxylated form. In particular 90% of CBDA and 100 % of THCA and CBGA shifted to their decarboxylated forms CBD, THC and CBG respectively. Additionally, heat treatment might be responsible of the loss of other compounds such as volatile terpenes. Further analyses should be performed in order to detect and quantify the exact profile of terpenoid compounds characterizing the hemp extract as well as the antioxidant activity of cannabinoids in minced meat and their inhibitory effect on spoilage bacteria such as Pseudomonas.

Conclusions

The ethanol extract of industrial hemp (Cannabis sativa L. variety Futura 75) showed in vitro antimicrobial properties against foodborne pathogens such as L. monocytogenes and Staphylococcus spp. but not against S. Typhimurium and E. coli. These results along with the antimicrobial properties observed for hemp extract when mixed to minced beef, suggest that hemp extract might have promising applications as natural food preservative which deserves further investigations especially on the specific molecules as well as their mechanisms of action linked to antimicrobial activity.

References