Jerushalmi et al.

Journal of Cannabis Research

https://doi.org/10.1186/s42238-020-00020-6
(2020) 2:12
Journal of Cannabis
Research

ORIGINAL RESEARCH Open Access

Effects of cold plasma, gamma and e-beam

irradiations on reduction of fungal colony
forming unit levels in medical cannabis
inflorescences
Shachar Jerushalmi1,2, Marcel Maymon1, Aviv Dombrovsky1 and Stanley Freeman1*

Abstract
Background: The use of medical cannabis (MC) in the medical field has been expanding over the last decade, as
more therapeutic beneficial properties of MC are discovered, ranging from general analgesics to anti-inflammatory
and anti-bacterial treatments. Together with the intensified utilization of MC, concerns regarding the safety of
usage, especially in immunocompromised patients, have arisen. Similar to other plants, MC may be infected by
fungal plant pathogens (molds) that sporulate in the tissues while other fungal spores (nonpathogenic) may be
present at high concentrations in MC inflorescences, causing a health hazard when inhaled. Since MC is not grown
under sterile conditions, it is crucial to evaluate current available methods for reduction of molds in inflorescences
that will not damage the active compounds. Three different sterilization methods of inflorescences were examined
in this research; gamma irradiation, beta irradiation (e-beam) and cold plasma to determine their efficacy in
reduction of fungal colony forming units (CFUs) in vivo.
Methods: The examined methods were evaluated for decontamination of both uninoculated and artificially
inoculated Botrytis cinerea MC inflorescences, by assessing total yeast and mold (TYM) CFU levels per g plant tissue.
In addition, e-beam treatment was also tested on naturally infected commercial MC inflorescences.
Results: All tested methods significantly reduced TYM CFUs at the tested dosages. Gamma irradiation reduced CFU
levels by approximately 6- and 4.5-log fold, in uninoculated and artificially inoculated B. cinerea MC inflorescences,
respectively. The effective dosage for elimination of 50% (ED50)TYM CFU of uninoculated MC inflorescence treated
with e-beam was calculated as 3.6 KGy. In naturally infected commercial MC inflorescences, e-beam treatments
reduced TYM CFU levels by approximately 5-log-fold. A 10 min exposure to cold plasma treatment resulted in 5-
log-fold reduction in TYM CFU levels in both uninoculated and artificially inoculated B. cinerea MC inflorescences.
Conclusions: Although gamma irradiation was very effective in reducing TYM CFU levels, it is the most expensive
and complicated method for MC sterilization. Both e-beam and cold plasma treatments have greater potential since
they are cheaper and simpler to apply, and are equally effective for MC sterilization.
Keywords: Botrytis cinerea, CFU, Cold plasma, E-beam, Gamma irradiation, Medical Cannabis, Sterilization

* Correspondence: [email protected]
1
Department of Plant Pathology and Weed Research, The Volcani Center,
Agriculture Research Organization, 7505101 Rishon Lezion, Israel
Full list of author information is available at the end of the article

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Jerushalmi et al. Journal of Cannabis Research (2020) 2:12 Page 2 of 12

Background extreme heat or U.V. irradiation which may damage the

In recent years there is a growing trend of research re- active compounds in MC (Hazekamp 2016). Studies re-
garding the beneficial effects of medical cannabis (MC) lated to the effects of gamma and beta irradiation on de-
for treating various diseases and ailments (Ben Amar contamination of the final product of MC is limited,
2006; Ruchlemer et al. 2015). The use of MC is growing however, these treatments do not appear to have a detri-
exponentially, especially in patients suffering from differ- mental effect on the quality of food and spice products
ent types of cancer and HIV, following FDA approval (Arvanitoyannis et al. 2009; Guerreiro et al. 2016; Jeong
(Ruchlemer et al. 2015), MC is also used widely as a gen- et al. 2015; Sádecká 2007).
eral analgesic. Since MC is used by patients with a weak- Gamma irradiation is commonly based on the use of
ened immune system, there are potential risks to their cobalt 60 isotope (60Co) which is reported as safe for de-
health when exposed to microbial-infected cannabis contamination of both MC and various food products
(fungal spores, bacteria, etc.), as shown by a growing (Arvanitoyannis et al. 2009; Jeong et al. 2015; Sádecká
number of reports (Cescon et al. 2008; Gargani et al. 2007). Moreover, long term mammalian studies have
2011; Hazekamp 2016; Ruchlemer et al. 2015). There- shown that irradiated foods are both safe and nutritious
fore, it is critical to supply MC-treated patients with a for human consumption (Thayer et al. 1996). While
“clean”, mold-free and healthy product. gamma irradiation is more commonly used, e-beam is a
In order to achieve a high level of quality control, many newer method showing greater promise. This technique
countries, including Israel, the Netherlands, and the Euro- does not require a radioactive source as the radiation is
pean pharmacopoeia have imposed strict regulations dictat- created using an electron accelerator making it environ-
ing the permitted number of microbial contaminations mentally friendly. Moreover, it was reported that a simi-
present in commercial MC supplied to patients as, 2000, lar efficacy of decontamination was observed when
100 and 50,000 colony forming units (CFUs) of total yeasts Botrytis cinerea (a major MC inflorescence fungal patho-
and molds (TYM) per g inflorescence, respectively (https:// gen) was exposed to either gamma or beta irradiation
www.health.gov.il/hozer/mmk151_2016.pdf, EP 8.0, 5.1.8.C) (McPartland et al. 2017). Furthermore, another fungal
(Hazekamp 2016). These CFU limitations are very low and pathogen Penicillium expansum, was more sensitive to
to date, effective cultivation of MC under sterile conditions e-beam than gamma irradiation (Jeong et al. 2015).
does not exist. Therefore, the need for post-harvest decon- While there was no direct mention of P. expansum as a
tamination of inhaled MC products is essential. Even though specific phytopathogen of MC, Penicillium spp. spores
sterilization methods such as autoclaving or ultraviolet are ubiquitous and common in dry MC products, sug-
(U.V.) irradiation may be the first to come to mind, the gesting that this fungus may be a potential pathogen of
most important therapeutic compounds in MC (cannabi- concern (McPartland et al. 2017; Punja et al. 2019).
noids and terpenes) are heat and light sensitive and undergo While gamma and e-beam irradiation possess a similar
decarboxylation, causing their early decay when exposed to mode of action, cold plasma treatment is a different
the above decontamination methods (Hazekamp 2016; method for sanitation and sterilization. The general defin-
Russo 2011; Small 2016). This highlights the necessity for ition of plasma is a state of ionized gas, with limited net
novel techniques to disinfect MC without exposing the charge. Natural examples of plasma are the sun and the
product to high temperatures or U.V. irradiation. aurora (Misra et al. 2019; Turner 2016). Cold plasma is
Both gamma and beta radiation (e-beam) fall under usually achieved by deploying electrical discharges in gases
the category of ionizing radiation, containing an amount at atmospheric or subatmospheric pressure. When a high
of energy that causes excitation or ionization of atoms enough voltage is reached a breakdown of the gas occurs,
and molecules, leading to the creation of free radicals leading to the formation of a mix of antimicrobial ele-
(Jeong et al. 2015). These free radicals in turn sever cer- ments. The mechanisms that take place during this phase
tain chemical bonds that lead to damage of molecules of cold plasma reaction are numerous and include vibra-
and especially cell DNA. Damage in these cases can be tion and excitation of gas atoms, ion-ion neutralization,
either direct, caused by Reactive Oxygen Species (ROS) quenching and many more (Misra et al. 2019; Sahu et al.
created from the radiolysis of H2O2, or indirect, caused 2017). Addition of H2O2 to the plasma, augments the
by other free radicals (Sádecká 2007). In living organ- sterilization mechanism; e.g. it was shown that at a high
isms, these damaged molecules cause a disruption in the concentration, ROS inhibit cell proliferation and cause
chemical and metabolic functions of living cells thus apoptosis (Thannickal and Fanburg 2000). Many reports
leading to cell death (Hazekamp 2016; Jeong et al. 2015; have reported the efficacy of cold plasma treatment in in-
Sádecká 2007). The advantages of using gamma and beta activating a wide spectrum of bacteria (gram positive and
irradiation for MC decontamination are numerous. Ion- negative) and in many of these studies the method was
izing radiation leaves no residues after application (un- shown to be even more effective in the reduction of fungal
like fungicides for example) and does not involve viability and spore CFU counts (Hertwig et al. 2015a,
Jerushalmi et al. Journal of Cannabis Research (2020) 2:12 Page 3 of 12

2015b; Kim et al. 2014; Misra et al. 2019; Zahoranová plastic planting container (80 × 40 × 50 cm) in a humid en-
et al. 2016). In recent years cold plasma sterilization has vironment, and an additional week without the top cover.
become more popular in various medical applications The rooted shoots were replanted in 0.2 L pots and trans-
such as surface sterilization and sterilization of damaged ferred for vegetative propagation under photoperiodic
tissues (Heinlin et al. 2010; Kolb et al. 2008; Xinpei et al. conditions of 18 h light and 6 h dark of 3000 LUX, for 2
2009). The direct mechanism of inactivation of fungi using months. Plants were retransferred into 0.5 to 2 L pots and
cold plasma is still not entirely clear. Scanning electron placed in a flowering induction chamber 4 × 3 m, for 80–
microscopy examination of plasma post-treated Cordyceps 90 days. The flowering chamber contained six 600 W high
bassiana spores revealed dried, cracked and flattened pressure sodium lamps (SunMaster, Twinsburg, Ohio,
propagules, indicating that cold plasma treatment may USA) with dual red and blue spectrum light, under photo-
cause cell wall leakage and destruction, resulting in re- periodic conditions of 11 h light (50,000 LUX) and 13 h
duced cell viability (Lee et al. 2015). Similar results were dark, until flowers were produced. Mature inflorescences,
achieved with cold plasma treatments of Aspergillus spp. that were produced 80–90 days after floral induction, were
(Dasan et al. 2017), which is of paramount importance used for sterilization experiments.
since Aspergillus spp. are very common in MC floral parts, Two types of plant parts were used: (i) uninoculated (that
and can cause serious health complications in immuno- included asymptomatic natural infections) mature inflores-
compromised patients when mycotoxin-contaminated cences, (ii) artificially inoculated mature inflorescences with
products are inhaled in large quantities (Gargani et al. a culture of Botrytis cinerea originating from naturally in-
2011; Hamadeh et al. 1988; Ruchlemer et al. 2015). Even fected cannabis flowers, isolated and characterized by mor-
more intriguing is the reported ability of cold plasma phological and molecular methods. It should be noted that
treatment to reduce the presence of these toxins as well as “uninoculated” inflorescences from the ARO facility con-
pesticide residues (Misra et al. 2015; Sarangapani et al. tained asymptomatic microbial infections comprised of a
2016). While gamma, e-beam irradiation and cold plasma wide variety of different fungal species. B. cinerae was cul-
treatments appear promising for MC sterilization, there is tured for 2 weeks at 22 °C on 9 cm Petri plates containing
a lack of evidence and knowledge regarding the efficacy of potato dextrose agar (Difco, Franklin Lakes, New Jersey,
each of these methods, specifically in the treatment of har- USA) supplemented with 0.25 g/l chloramphenicol (PDAC)
vested MC inflorescences, and their effect on the desired (Acros Organics, Geel, Belgium). After 14 days, spores were
active chemical compounds. harvested from the plates with a sterile rod by adding a sus-
In this research, we examined the efficacy of three pension of 10 ml sterile saline solution (NaCl 0.85 g/l,
sterilization methods: (i) gamma irradiation, (ii) beta ir- Tween 20, 100 μl/l). The conidia were filtered through four
radiation (e-beam), and (iii) cold plasma sterilization, for layers of sterile gauze and centrifuged (Heraeus, Franklin
reduction and elimination of fungal colony forming units Lakes, New Jersey, USA) at 9000 RPM for 10 min at 4 °C.
(CFUs) in uninoculated and artificially inoculated B. The pellet was resuspended in 20 ml fresh saline solution
cinerea inflorescences, naturally infected commercial and adjusted to a concentration of 106 spores/ml. The in-
trimmed floral parts and inflorescences. oculum was sprayed till run-off on healthy mature cannabis
plants that were subsequently covered by a plastic bag.
Methods After 5 days, the bags were removed and harvested flowers
Plant and fungal samples were dried in an Excalibur 3548/3948 digital oven (Sacra-
Cannabis sativa cultivar (BB 734) was grown in the Agri- mento, California, USA) at 35 °C for 12 h, then stored in a
culture Research Organization (ARO) Volcani Center re- STATUS innovations vacuum pack (Metlika, Slovenia) at
search facility (authorized by the Israeli Medical Cannabis room temperature before experimentation. CFU’s of the
Agency, IMCA, Ministry of Health, State of Israel) for this microbial cultures from affected floral parts, before and
research. Uncharacterized cannabis seedlings were kindly after each sterilization treatment, were determined (see
provided by Dr. Moshe Flaishman, ARO that established below).
the genetic source. Plants were propagated and five male
flowers were used for pollination of 30 female flower Quantification of fungal colonies
plants. Seeds were collected from the harvested female One g of each floral sample was inserted into a 10 ml
flowers and sown for continuous breeding and selection sterile saline solution in 50 ml Falcon tubes, vortexed for
for a range of parameters. The strain that was used in this 30 s and kept at room temperature for 10 min. There-
study (BB 734) was derived from shoots of third gener- after, serial dilutions were conducted and spread on
ation mother plants. This strain is a “drug-type” cannabis PDAC plates that were maintained at room temperature
with Cannabis indica characteristics. (22°-25 °C) for 3–5 days, and developing CFU’s of total
Shoots were rooted under continuous 24 h light photo- yeasts and mold (TYM) species were enumerated and
periodic conditions of 880 LUX, for 1 week in a closed characterized.
Jerushalmi et al. Journal of Cannabis Research (2020) 2:12 Page 4 of 12

Survey of CFU levels from commercial MC farms Irradiation treatments of naturally infected commercial
In order to evaluate common CFU levels under commer- plant material including (i) dried and packed floral parts,
cial conditions, samples of MC inflorescences were col- and (ii) dried and packed trimmed leaves were assessed
lected from 4 different farms located in Israel. CFU levels for efficacy of treatments by determining CFU counts.
were evaluated as described. Plating of each sample was Inflorescences were naturally infected indicating that
conducted 3 times to achieve higher reproducibility. Vari- CFUs from these inflorescences were comprised of a
ability in sampled inflorescences existed, as certain sam- wide variety of fungal species. Each product contained
ples exhibited disease symptoms while others remained two 500 g vacuum-sealed bags that were treated with e-
asymptomatic, and certain inflorescences were dry. beam irradiation at Sorvan nuclear facility. A 5 g sample
that was removed from each bag before the irradiation
Gamma and beta irradiation treatments served as a control. To determine efficacy of
Commercial cannabis samples were received from a num- e-beam irradiation treatments at different locations in
ber of commercial farms in Israel for irradiation treatments. the bag, six samples of 5 g each were removed after
Treatments comprising of e-beam (beta irradiation) and treatments from different locations of each bag: from the
gamma irradiation were conducted at Sorvan Radiation upper right corner, upper left corner, lower right corner,
Ltd., Soreq Nuclear Research Center, Israel. Gamma radi- lower left corner, upper middle area and lower middle
ation was based on a 60Co isotope, doubly encapsulated in area, and CFU’s were determined as described.
stainless steel source pencils type C-188, with radiation dos- Cold plasma treatment was conducted on noncom-
ages of 7.5 and 8.37 KGy (KiloGray) in two consecutive ex- mercial floral material. The experimental design was
periments, respectively. E-beam radiation was created using identical to that described for the noncommercial irradi-
an electron accelerator, with 15 kW (KWs) and an energy ation experiments. Floral parts were placed on the elec-
capacity of 5.25 megaelectronvolt (MeV). The radiation trode and H2O2 was injected around the perimeter. Each
dosages were 4.18, 8.2 and 10.26 KGy, and 4.06, 8.5, and treatment comprised of 8 min of vacuum and different
10.26 KGy in two consecutive experiments, respectively. plasma exposure periods described. An untreated sample
served as control.
Cold plasma irradiation
Cold plasma treatment was conducted using a prototype ED50 calculation
created by NovaGreen company, (Kibbutz Megiddo, Israel). In order to determine the effective radiation dosage for
The gas in this treatment was low pressure air with the eliminating 50% (ED50) CFUs, a response curve with
addition of H2O2 liquid at a concentration of 35% (Chen R2 > 0.95 was produced for each treatment. CFU levels
Shmuel Chemicals Ltd., Haifa, Israel). A vacuum chamber in the controls of each treatment were calculated as the
was generated using an Edwards i10 dry pump to eliminate 100%. This value divided by two was used as the Y value
possible oil contamination that may have occurred during in the response curve formula of each treatment, and
wet pump usage. Although the H2O2 liquid had no direct served as the radiation dosage required for reducing
contact with the MC, it affects the gaseous environment CFU levels by 50% (ED50). All other ED values were cal-
and generates a highly reactive plasma with elevated con- culated using the same method.
centrations of oxygen species. An RF generator at a voltage
of 6 kV generated the plasma and exposure periods lasted Results
for 2.5, 5.0 and 10 min for each experiment. CFU survey of inflorescences from commercial farms
The initial CFU survey that was conducted on 21 sam-
Sampling procedures and experimental design ples indicated that in all four tested commercial farms
Two sample types [uninoculated (that included asymp- levels of TYM fungal contamination exceeded the max-
tomatic infections) and artificially inoculated Botrytis imum CFU values of 2000 yeasts and molds per g inflor-
cinerea] of noncommercial plant material were ob- escence permitted by the IMCA (Fig. 1); some samples
tained from the ARO Volcani Center facilities and di- exceeded this level by as much as 3.08 log-fold CFU/g
vided into bags containing 5 g MC floral parts each inflorescences. Morphological identification of the CFUs
(total of 20 g per sample type). Artificially inoculated indicated an abundance of the following fungal species;
Botrytis cinerea and uninoculated samples were treated Alternaria spp., Aspergillus spp., Botrytis cinerea, Fusar-
with beta and gamma radiation at Sorvan facility. A 5 g ium spp., and Penicillium spp.
non-irradiated sample of each floral MC type served as
a control. After irradiation treatments, four and three Irradiation treatments
biological repeats (from two consecutive experiments, Gamma irradiation of noncommercial MC inflorescences
respectively) were removed from each bag and CFU’s Gamma irradiation treatments caused a considerable re-
were determined, as described. duction in TYM CFU levels in noncommercial MC
Jerushalmi et al. Journal of Cannabis Research (2020) 2:12 Page 5 of 12

Fig. 1 CFU levels [log10(CFU/g inflorescence)] detected from four farms designated A, B, C and D. Consecutive numbers after each individual
farm indicate different samples of inflorescences taken from that farm. Samples D1 and D2 represent dried inflorescences. Asterisks indicate MC
inflorescence that were asymptomatic, all other samples exhibited varying degrees of disease symptoms. Bars represent SE of the mean of 3
replicates per sample. The gray line represents maximum levels of total yeasts and molds permitted according to protocols of the IMCA for
commercial MC inflorescences (2000 CFU/g)

inflorescences. CFU levels were reduced in the uninocu- The effective dosages calculated for reduction of per-
lated MC inflorescence samples from 6.16 ± 0.2 and from cent population of CFUs for e-beam treatments in artifi-
6.04 ± 0.08 to 0 log CFU/g inflorescence in the first (7.5 cially inoculated Botrytis cinerea and uninoculated MC
KGy irradiation dosage) and second (8.37 KGy) experi- inflorescences are shown in Table 1.
ments, respectively. While the ED values in the uninoculated MC inflores-
In artificially inoculated Botrytis cinerea MC inflores- cences were considerably lower than those for the artifi-
cences, gamma irradiation treatments resulted in a re- cially inoculated Botrytis cinerea inflorescences, it is
duction of CFU levels from 8.05 ± 0.12 and 7.7 ± 0.11 to worth mentioning that in the inoculated inflorescences
1.88 ± 0.96 and 3.02 ± 0.11 log CFU/g inflorescence, in (Fig. 3) initial CFU levels were 100-fold higher than
consequent experiments respectively, a respective reduc- those of the uninoculated inflorescences (Fig. 2).
tion of 6- and 4.5 log-fold. Peak temperatures measured
during irradiation treatments reached 32.5 and 30.0 °C
in the first and second experiments, respectively. E-beam treatments of commercial material Beta-ir-
radiation of commercial material significantly reduced
and eliminated CFUs in MC inflorescences at all loca-
E-beam (beta irradiation) tions in the vacuum-sealed packages. In the first experi-
ment, e-beam reduced CFU levels from 4.9±0.25 to 0 log
E-beam treatments of noncommercial material E- CFU/g inflorescence, compared to the untreated control.
beam treatments at 10.26 KGy of noncommercial MC (Fig. 5). Likewise, in the second experiment, CFUs were
floral parts were very effective in completely eliminating reduced to undetected levels at all sampled locations in
contamination of uninoculated inflorescences from the package (Fig. 5).
6.16 ± 0.26 and 6.04 ± 0.08 to 0 log CFU/g inflorescence The results of irradiation of commercial MC trimmed
in two consequent experiments, respectively (Fig. 2). leaves were more varied (Fig. 6). In the first experiment,
A similar pattern was found when noncommercial, arti- no viable CFUs were detected from three of the sam-
ficially inoculated Botrytis cinerea MC inflorescences were pled locations, although CFU levels were significantly
treated under the same conditions, (Figs. 3 and 4a). E- reduced in three of the other locations, compared to
beam irradiation reduced CFU levels in two consecutive the untreated controls (Fig. 6). In the second experi-
experiments to 0 and 1.75 ± 0.5 log CFU/g inflorescence, ment, even though initial CFU levels were higher, no
respectively (Fig. 3). In both experiments, peak tempera- CFUs were detected from four sampled locations after
tures during radiation were less than 27 °C. the treatment (Fig. 6).
Jerushalmi et al. Journal of Cannabis Research (2020) 2:12 Page 6 of 12

Fig. 2 CFU levels [log10(CFU/g inflorescence)] of uninoculated MC inflorescences, exposed to different e-beam irradiation dosages in two
experiments. Bars represent SE of the mean of 9 replicates per sample. A value of 3.55 KGy was calculated, according to the polynominal formula
(dotted line), to reduce CFUs by 50% (ED50), represented by the dashed line

Cold plasma treatments of noncommercial material heavily infected uninoculated inflorescences, a reduction
Cold plasma treatments resulted in a reduction in CFU of approximately 6 logs was recorded, to below 10 CFU/
levels of uninoculated inflorescences, according to ex- g inflorescence after 12 min of plasma exposure (data
posure times (Fig. 7). After 2.5 min of plasma exposure, not shown).
CFU levels were reduced from 3.01 ± 0.08 and 2.81 ± A similar pattern was observed in artificially inoculated
0.99 log CFU/g inflorescence to 0 and 0.79 ± 0.39 log Botrytis cinerea MC inflorescences treated with cold
CFU/g inflorescence, in the first and second experi- plasma (Figs. 4b and 8). After 2.5 min of plasma expos-
ments, respectively (Fig. 7). However, after 5 min expos- ure, CFU levels were reduced by approximately 3-log-
ure the detected CFU levels were 1.83 ± 0.47 and 0.92 ± fold, by 2-log-fold after 5 min exposure, and 4-log-fold
0.46 log CFU/g inflorescence, and after 10 min of expos- after 10 min exposure (Fig. 8).
ure, CFU levels were reduced to 0 and 0.25 ± 0.25 log
CFU/g inflorescence, in the respective consecutive ex- Discussion
periments, a reduction of approximately 3-log-fold in The use of medical cannabis (MC) has increased tre-
detected CFU levels. In an experiment performed with mendously in the last decade (Ruchlemer et al. 2015).

Fig. 3 CFU levels, [log10(CFU/g inflorescence)] of artificially inoculated Botrytis cinerea MC inflorecences, exposed to different e-beam irradiation
dosages in two experiments. Bars represent SE of the mean of 9 replicates per sample. A value of 5.18 KGy was calculated, according to the
polynominal formula (dotted line) to reduce CFUs by 50% (ED50), represented by the dashed line
Jerushalmi et al. Journal of Cannabis Research (2020) 2:12 Page 7 of 12

Fig. 4 Fungal CFUs from artificially inoculated Botrytis cinerea inflorescences after: a e-beam irradiation (4, 8, 10 KGy and untreated control), and b
cold plasma (2.5, 5, 10 min exposure and untreated control) treatments

One of the main reasons for the rising popularity and (Guerreiro et al. 2016; Jeong et al. 2015; Sádecká 2007). Al-
interest in MC are the therapeutic qualities of this plant ternatively, e-beam (beta irradiation) and cold plasma, are
(Ben Amar 2006; Cascio et al. 2017; Russo 2011; Sirikan- effective for food sterilization and are safe for human con-
taramas and Taura 2017). With the increase in use, both sumption (Jeong et al. 2015; Misra et al. 2019; Moreno
for recreational and therapeutic means, reports are start- et al. 2007; Van Impe et al. 2018). Even though these
ing to accumulate concerning the threat of microbial methods have been applied or suggested for decontaminat-
presence in MC inflorescences and the harmful potential ing MC inflorescences to safeguard its use by immunocom-
to cannabis consumers, especially in immunocomprom- promised patients, there is a lack of knowledge in regards
ised patients (Gargani et al. 2011; Hamadeh et al. 1988; to their efficacy in eliminating deleterious microorganisms.
McPartland and McKernan 2017; Ruchlemer et al. In this research, we examined the effect of gamma ir-
2015). The extent of MC inflorescence infections by fun- radiation, e-beam and cold plasma treatments, on the re-
gal CFUs was determined in our initial survey of com- duction of CFU contamination in artificially inoculated
mercial farms, indicating that without sterilization and naturally infected MC inflorescences and trimmed
treatments, levels of CFUs were extremely high, above leaves (Table 2). Gamma irradiation was very effective in
that permitted by the IMCA in all tested sites, disregard- reducing the CFU levels by approximately 6-log-fold
ing the sample condition; dried or un-dried, and with or CFU/g inflorescences, at a minimal dosage of 7.5 KGy.
without visible disease symptoms (Fig. 1). Since it is not Similarly, in the Netherlands, MC is sterilized using 60Co
feasible to cultivate commercial MC under a sterile en- gamma irradiation at a dosage of 10 KGy, which is well
vironment there is an acute need for postharvest MC in- below the authorized dosage of 30 KGy, permitted by the
florescence sterilization before usage (Hazekamp 2016). FDA for irradiation of aromatic herbs and spices (Sádecká
There have been many reports regarding the efficacy of 2007). Likewise, in a recent report, 60Co gamma irradi-
different non-thermal treatments for food and herb ation was used to sterilize cherry tomatoes with a radi-
sterilization, especially the utilization of gamma irradiation ation dosage of 5.7 KGy that reduced CFU levels from 2.2
log CFU/g to nearly zero, an inactivation efficacy of 99.8%
(Guerreiro et al. 2016). In spite of its effectiveness, gamma
Table 1 Effective dosage of e-beam treatments for reducing
percent CFU populations in both infected and artificially irradiation remains an expensive sterilization method re-
inoculated Botrytis cinerea MC inflorescences quiring the usage of radioactive isotopes, specialized
Effective dosage (ED)(%) Irradiation dosage Irradiation dosage
equipment and facilities. In contrast, e-beam does not
(KGy) of uninoculated (KGy) of B. cinerea require the use of radioactive isotopes and as such, is con-
inflorescencesa inoculated siderably more environmentally friendly (Leonhardt 1990).
inflorescencesb
Moreover, in this research we found that e-beam treat-
90 8.1 12.4 ments were very effective in eliminating CFUs from
70 5.5 8 infected MC inflorescences applied at low temperatures,
50 3.6 5.2 below 27 °C, that do not detrimentally affect the active in-
10 0.6 1 gredients of MC. In fact, this method is so effective that at
a
Calculated using the polynomial formula in Fig. 2
a radiation dosage of 10.26 KGy, CFU levels were reduced
b
Calculated using the polynomial formula in Fig. 3 from 6 log CFU/g inflorescence to 0 (Fig. 2). A similar
Jerushalmi et al. Journal of Cannabis Research (2020) 2:12 Page 8 of 12

Fig. 5 CFU levels [log10(CFU/g inflorescence)] in naturally infected commercial MC inflorescence before (control) and after e-beam treatments in
two experiments. Post-treatment samples were taken from different locations of vacuum-sealed packages. Bars represent SE of 3 replicates per
sample per location

reduction in CFUs was observed with artificially inocu- beam irradiation (Table 1) were also very promising; e.g.
lated inflorescences (Fig. 3). ED10, the radiation dosage required to reduce 10% CFU
ED values are an important and useful tool as they in- populations of uninoculated and artificially inoculated
dicate the efficacy of treatments and also allow for com- inflorescences were calculated as 0.6 KGy and 1 KGy, re-
parison between different sterilization systems; in spectively. In comparison, the ED10 value for inactivation
general lower ED values represent a higher acute toxicity of enteric viruses e.g. poliovirus Type 1 on cantaloupe
(Lorke 1983). ED values are extremely relevant in this surfaces using e-beam was 4.76 KGy (Shurong et al.
study since they indicate a measurable value for the effi- 2006). Similarly, e-beam treatment of red pepper powder
cacy of the treatments. The ED values calculated for e- at a dosage of 3 KGy, reduced CFU levels of total yeasts

Fig. 6 CFU levels, [log10(CFU/g inflorescence)] in naturally infected commercial MC trimmed leaves before (control) and after e-beam treatments
in two experiments. Post-treatment samples were taken from different locations of vacuum-sealed packages. Bars represent SE of 3 replicates per
sample per location
Jerushalmi et al. Journal of Cannabis Research (2020) 2:12 Page 9 of 12

Fig. 7 CFU levels [log10(CFU/g inflorescence)] of uninoculated MC inflorescences exposed to different cold plasma treatments. Bars represent SE
of the mean of 9 replicates per sample

and molds from 6.62 to 3.71 log CFU/g (Kyung et al. than the maximum limit of 30 KGy, according to that
2019). In our research, we found that a dosage of 12.4 authorized by the FDA for irradiation of dry herbs
KGy resulted in 90% reduction in CFU levels in artifi- (Sádecká 2007).
cially inoculated B. cinerea MC inflorescence (Table 1). Cold plasma treatment of MC inflorescences was also
Although 12.4 KGy is considered a rather high radiation found to be effective for elimination of fungal propagules
dosage, it is important to note that in this experiment in this study. After 10 min of plasma exposure, CFU
the initial CFU levels were very high (more than 7.7 ± levels in uninoculated MC inflorescences were reduced
0.11 log CFU/g). Accordingly, initial CFU levels in com- by approximately 3 log-fold CFU/g. It is important to
mercial MC were constantly much lower, measuring note that initial recorded infection levels in these experi-
below 5 ± 0.25 CFU/g. This indicates that irradiation ments were approximately 3 log CFU/g indicating that
values of 8.1 KGy, resulting in a reduction of 90% of un- given a higher infection level, an exposure to 10 min
inoculated MC inflorescences are very reasonable con- plasma treatment may have resulted in an even greater
sidering that the irradiation dosages are much lower CFU reduction (Fig. 7). In an additional experiment

Fig. 8 CFU levels, log10(CFU/g inflorescence), of Botrytis cinerea-inoculated MC inflorescences exposed to different cold plasma tretments. Bars
represent SE of the mean of 9 replicates per sample
Jerushalmi et al. Journal of Cannabis Research (2020) 2:12 Page 10 of 12

Table 2 Effect of radiation and sterilization methods on fungal contamination of cannabis plant samples
Treatment Experiment Sample type Average CFU before Radiation Plasma Average CFU after
number treatment [log 10 CFU/g dose exposure treatment [log 10 CFU/g
inflorescence] [kGy] time [min] inflorescence]
Gamma irradiation 1 Uninoculated 6.16 ± 0.26 7.5 – 0
inflorescence
1 Botrytis cinerea- 8.05 ± 0.12 7.5 – 1.88 ± 0.96
inoculated
inflorescence
2 Uninoculated 6.04 ± 0.08 8.37 – 0
inflorescence
2 Botrytis cinerea- 7.7 ± 0.11 8.37 – 3.02 ± 0.11
inoculated
inflorescence
E-beam 1 Uninoculated 6.16 ± 0.26 10.26 – 0
inflorescence
1 Botrytis cinerea- 8.05 ± 0.12 10.26 – 0
inoculated
inflorescence
2 Uninoculated 6.04 ± 0.08 10.26 – 0
inflorescence
2 Botrytis cinerea- 7.7 ± 0.11 10.26 – 1.75 ± 0.5
inoculated
inflorescence
1 Naturally infected 5 ± 0.25 11.99 – 0.211 ± 0.1
commercial
inflorescence
2 Naturally infected 2.2 ± 0.47 11.99 – 0
commercial
inflorescence
1 Naturally infected 4.3 ± 0.03 11.99 – 0.314 ± 0.095
commercial trimmed
leaves
2 Naturally infected 5.27 ± 0.05 11.99 – 0.6 ± 0.48
commercial trimmed
leaves
Cold plasma 1 Uninoculated 3 ± 0.08 – 10 0
inflorescence
2 Uninoculated 2.81 ± 0.91 – 10 0.25 ± 0.25
inflorescence
2 Botrytis cinerea- 7.82 ± 0.04 – 10 3.84 ± 0.08
inoculated
inflorescence
Cold plasma with 1 Uninoculated 6.6 ± 0.35 – 12.5 min + 10 0.25 ± 0.25
changing vacuum inflorescence min vacuum
timea
1 Botrytis cinerea- 6.88 ± 0.11 – 10 min + 8 4.05 ± 0.31
inoculated min vacuum
inflorescence
Abbreviations: CFU colony forming units, kGy kiloGray
a
in the cold plasma treatment with changing vacuum time, only one experiment was conducted

(data not shown) a cold plasma treatment of 8 min vac- Similarly, cold plasma treatments conducted in Shaare
uum time followed by 12 min of plasma exposure re- Zedek Medical Center Israel, resulted in complete
sulted in CFU reduction of approximately 6 log CFU/g sterilization of MC inflorescences (Ruchlemer et al.
of uninoculated MC inflorescences. Accordingly, in arti- 2015). In that research, autoclaving and ethylene gas
ficially inoculated B. cinerea MC inflorescences the re- sterilization were found to be as effective for MC inflor-
sults were improved with a reduction of approximately 4 escence sterilization, although the two latter methods re-
log CFU/g, after 10 min of plasma exposure (Fig. 8). sulted in a greater reduction of Δ9-THC, which is one of
Jerushalmi et al. Journal of Cannabis Research (2020) 2:12 Page 11 of 12

the major phytocannabinoids in MC. Cold plasma was Abbreviations

60
also found useful in decontamination of wheat seeds, Co: 60 cobalt isotope; ARO: Agriculture Research Organization; CFU: Colony
forming unit; E-BEAM: Electron beam; ED: Effective dosage; IMCA: Israeli
resulting in complete inactivation of Fusarium nivale-ar- Medical Cannabis Agency; KGy: KiloGray; KWs: Kilowatts; MC: Medical
tificially inoculated seeds after as little as 90 s treatment Cannabis; MeV: Megaelectronvolt; PDAC: Potato dextrose agar and
(Zahoranová et al. 2016). An increase in CFU counts chloramphenicol; ROS: Reactive oxygen species; TYM: Total yeast and mold

after 5 min of plasma exposure compared to that after

Acknowledgements
2.5 min was recorded in our experiments, which is still The authors thank M. Borenstein for technical and greenhouse assistance.
unclear (Figs. 7 and 8) and will require further research. The authors are indebted to Dr. Moshe Flaishman from ARO for kindly
In various studies, cold plasma was reported to degrade providing us with cannabis seedlings and to the various cannabis farms for
providing commercial plant material for the experiments. We thank Sorvan
both mycotoxins and pesticides in in vitro experiments Radiation Ltd., Soreq Nuclear Research Center, Yavne, Israel, and Novagreen
(Ten Bosch et al. 2017; Misra et al. 2011; Sarangapani company, Kibbutz Megiddo, Israel, for cooperation in performing the
et al. 2016). Mycotoxins such as aflatoxins, zearalenone sterilization experiments.
and fumonisins are secondary metabolites produced by
Authors’ contributions
certain fungi such as Aspergillus and Fusarium spp. that
SJ conducted the research, MM assisted with technical and lab experiments,
are ubiquitous in MC inflorescences and are known to AD raised partial funding and SF raised partial funding, conceived and
cause health risks to humans and mammals (McPartland supervised the project. The author(s) read and approved the final
manuscript.
et al. 2017; Misra et al. 2019). Thus, cold plasma treat-
ments may be even more beneficial by not only reducing
Funding
CFU counts but also by reducing levels of mycotoxins The authors thank the Chief Scientist of the Israeli Ministry of Agriculture,
(Ten Bosch et al. 2017; Misra et al. 2019). grant numbers 20-02-0070 and 20-02-0099, for funding this research.
Another important aspect when dealing with MC are
the active compounds, comprised mainly of different Availability of data and materials
All data generated or analyzed during this study are included in this
phytocannabinoids and terpenoids. These compounds, published article.
especially phytocannabinoids, are responsible for MC
therapeutic effects and currently more than 100 different Competing interests
phytocannabinoids have been identified (Cascio et al. The authors declare that they have no competing interests.
2017; Russo 2011). In recent years, there is a growing
Author details
understanding that some of the MC therapeutic affects 1
Department of Plant Pathology and Weed Research, The Volcani Center,
are a result of synergism among the bioactive com- Agriculture Research Organization, 7505101 Rishon Lezion, Israel. 2The Robert
H. Smith Faculty of Agriculture, Food and Environment, The Hebrew
pounds in certain MC lines and cultivars (Russo 2011).
University of Jerusalem, 7610001 Rehovot, Israel.
Thus, it is crucial to decontaminate MC inflorescences
using a method that causes the least damage to the pro- Received: 9 September 2019 Accepted: 18 February 2020
files of these active compounds.
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