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Article
In Pursuit of Optimal Quality: Cultivar-Specific Drying
Approaches for Medicinal Cannabis
Matan Birenboim 1,2 , Nimrod Brikenstein 1,2 , Danielle Duanis-Assaf 1 , Dalia Maurer 3 , Daniel Chalupowicz 3 ,
David Kenigsbuch 3, * and Jakob A. Shimshoni 1, *

1 Department of Food Science, Institute for Postharvest and Food Sciences, Agricultural Research Organization,
Volcani Center, P.O. Box 15159, Rishon LeZion 7505101, Israel
2 Department of Plant Science, The Robert H Smith Faculty of Agriculture, Food and Environment, The Hebrew
University, Rehovot 7610001, Israel
3 Department of Postharvest Science, Institute for Postharvest and Food Sciences, Agricultural Research
Organization, Volcani Center, Rishon LeZion 7505101, Israel
* Correspondence: [email protected] (D.K.); [email protected] (J.A.S.)

Abstract: A limited number of studies have examined how drying conditions affect the cannabinoid
and terpene content in cannabis inflorescences. In the present study, we evaluated the potential of
controlled atmosphere drying chambers for drying medicinal cannabis inflorescence. Controlled
atmosphere drying chambers were found to reduce the drying and curing time by at least 60%
compared to traditional drying methods, while preserving the volatile terpene content. On the other
hand, inflorescences subjected to traditional drying were highly infested by Alternaria alternata and
also revealed low infestation of Botrytis cinerea. In the high-THC chemovar (“240”), controlled N2
and atm drying conditions preserved THCA concentration as compared to the initial time point
(t0 ). On the other hand, in the hybrid chemovar (“Gen12”) all of the employed drying conditions
preserved THCA and CBDA content. The optimal drying conditions for preserving monoterpenes
and sesquiterpenes in both chemovars were C5O5 (5% CO2 , 5% O2 , and 90% N2 ) and pure N2 ,
respectively. The results of this study suggest that each chemovar may require tailored drying
conditions in order to preserve specific terpenes and cannabinoids. Controlled atmosphere drying
chambers could offer a cost-effective, fast, and efficient drying method for preserving cannabinoids
Citation: Birenboim, M.; Brikenstein,
N.; Duanis-Assaf, D.; Maurer, D.;
and terpenes during the drying process while reducing the risk of mold growth.
Chalupowicz, D.; Kenigsbuch, D.;
Shimshoni, J.A. In Pursuit of Optimal Keywords: Cannabis sativa L.; controlled drying; atmospheric drying; cannabinoids; terpenes
Quality: Cultivar-Specific Drying
Approaches for Medicinal Cannabis.
Plants 2024, 13, 1049. https://doi.org/
10.3390/plants13071049 1. Introduction
Academic Editor: Alison Ung Cannabis sativa L., the sole species in the Cannabaceae family, is an annual herb with
proven therapeutic benefits for conditions like pain, epilepsy, and cancer, among oth-
Received: 3 March 2024 ers [1–4]. Despite various available cannabis products, dried inflorescences are among
Revised: 4 April 2024
the prevalent medicinal cannabis products accessible to patients. The plant’s therapeutic
Accepted: 7 April 2024
effects are attributed mainly to two major secondary metabolite classes, the cannabinoids
Published: 8 April 2024
and terpenes [5,6]. The cannabinoids interact with the body’s endocannabinoid system,
influencing various physiological processes [6,7]. The most studied cannabinoids are
(−)-∆9-trans-tetrahydrocannabinol (THC) and cannabidiol (CBD) [8]. THC is known for its
Copyright: © 2024 by the authors.
psychoactive effects but also provides medicinal benefits such as reducing chronic pain,
Licensee MDPI, Basel, Switzerland. stimulating appetite, and proving beneficial in conditions like Alzheimer’s disease and
This article is an open access article cancer [2,9]. CBD, a non-psychoactive cannabinoid, is recognized for its anti-inflammatory,
distributed under the terms and anxiolytic, and antiepileptic properties [8–10]. Terpenes, the volatile aromatic compounds
conditions of the Creative Commons found in the inflorescence of medicinal cannabis, play a pivotal role in enhancing cannabis
Attribution (CC BY) license (https:// therapeutic efficacy and consumer experience [9,11]. This aspect of consumer experience
creativecommons.org/licenses/by/ is crucial in medicinal cannabis formulations, as it aids in consistent patient adherence
4.0/). to treatment regimens, ultimately impacting the effectiveness of the therapy and patient

Plants 2024, 13, 1049. https://doi.org/10.3390/plants13071049 https://www.mdpi.com/journal/plants

Plants 2024, 13, 1049 2 of 22

compliance [11,12]. Scientific research has highlighted the importance of terpenes in the “en-
tourage effect”, a synergistic interaction where terpenes, in conjunction with cannabinoids
like THC and CBD, enhance the overall therapeutic potential of cannabis [9,13]. This inter-
action potentially amplifies the analgesic, anti-inflammatory, and anxiolytic properties of
medicinal cannabis, thus contributing to its clinical effectiveness [9]. Furthermore, terpenes
have been individually noted for their medicinal properties. For example, β-myrcene pos-
sesses anti-inflammatory and analgesic qualities, while d-limonene and linalool are known
for their anxiety-reducing and antidepressant effects [9]. The complexity and variability of
terpene profiles in different cannabis cultivars underscore the importance of employing
optimal drying, curing, and storage techniques to preserve consistent terpene composition
during post-harvest processes, which is paramount for ensuring patient satisfaction and
therapeutic consistency.
Cannabis cultivars are categorized into three main classes based on the ratio of the
major cannabinoids (−)-∆9-trans-tetrahydrocannabinolic acid (THCA) and cannabidiolic
acid (CBDA) and their neutral homologs THC and CBD: high-THCA (total THC/total CBD
ratio ≥ 10), high-CBDA (total CBD/total THC ratio ≥ 10), and hybrid (10 > total THC/total
CBD ratio > 0.1) [14–18]. These acidic cannabinoids convert to their neutral forms by a
decarboxylation process under specific post-harvest conditions such as light intensity and
temperature [19–21]. Given the chemical variability within and between cultivars, the
term “chemovar”, encompassing the full cannabinoid and terpene profile, is preferred for
classification [7,15,19].
Medicinal cannabis inflorescences undergo rigorous post-harvest processes to ensure
optimal quality [1,22]. Initial stages include trimming and a 2–3 week drying period,
influenced by factors like temperature and humidity [1,19,21,23]. This processing stage
results in up to 75% weight loss, with slight variations due to environmental conditions
and different chemovars [19,24]. Drying can be conducted by hanging buds or using trays,
though the latter has drawbacks like increased mold susceptibility [19,23,25]. Final drying,
or curing, often involves sealing the inflorescences in containers and periodically airing
them to remove residual moisture before final packaging [1,25].
A limited number of studies have examined how drying conditions affect the cannabi-
noid and terpene content in cannabis inflorescences [19,21,23–29]. Recent literature suggests
that industrial drying techniques have seen little innovation and are often more art than sci-
ence, emphasizing the need for empirically grounded, efficient methods [21,23,25]. Recent
reports have explored various alternative drying methods, including microwave, convec-
tion, freeze-drying, infrared, non-isothermal, and vacuum drying techniques [19,21,23–29].
However, these methods have shown limitations in effectively preserving the volatile ter-
penes, necessitating additional research. Future investigations are also required to establish
consistent results across diverse cannabis chemovars, and to ensure the retention of a
broader range of cannabinoids and terpenes.
Controlled atmosphere drying uses controlled atmosphere chambers which allow
for controlling the temperature, humidity, and gas composition during the drying pro-
cess [30–32]. The drying or storing of fruits and vegetables in controlled atmosphere cham-
bers under a controlled gas environment has been shown to increase the produce’s shelf-life,
increase produce quality over time, and preserve the volatile composition [30–33]. Hence,
controlled atmosphere drying could potentially offer a superior solution for cannabis inflo-
rescence drying in terms of both speed and the preservation of terpene and cannabinoid
content [30,31].
This research aimed to determine the optimal atmospheric composition within con-
trolled atmosphere chambers to facilitate the swift drying of two specific commercially
available cannabis inflorescence chemovars, with a focus on preserving their distinct ter-
pene and cannabinoid content.
Plants 2024, 13, 1049 3 of 22

2. Results and Discussion

2.1. 240 and Gen12 Initial Chemical Composition Comparison
The chemovar with high THCA content, 240, exhibited initial THCA levels approx-
imately two times greater than those found in the Gen12 hybrid chemovar (Table 1). In
contrast, the Gen12 chemovar demonstrated initial CBDA levels that were two orders of
magnitude higher than in the 240 chemovar (Table 1). For minor cannabinoids (below
1 DW%), the Gen12 chemovar revealed significantly higher levels of CBGA, CBG, and
CBCA than the 240 chemovar, yet both chemovars revealed similar levels of THC (Table 1).
Furthermore, only the 240 chemovar had detectable levels of THCVA and (−)-∆9-trans-
tetrahydrocannabiorcolic-C4 acid (THCA-C4), while CBD and CBDVA were unique to the
Gen12 chemovar (Table 1).

Table 1. The 240 and Gen12 cannabinoid composition at harvest day (t0 , absolute concentration, nor-
malized to DW%) and absolute cannabinoid concentrations (in DW%) after drying under four drying
conditions. For each drying procedure and t0 n = 5.

Absolute
Absolute Concentration ± SE
Absolute Concentration ± SE after 6 Days of Controlled Atmospheric
Cannabinoid Concentration ± SE after 15 Days of
Drying
at t0 Drying and Curing
(Traditional Drying)
240
Atm N2 C5O5 Open-air
CBDVA <LOD
CBDA 0.066 ± 0.007 0.066 ± 0.003 (ns) a 0.067 ± 0.005 (ns) 0.058 ± 0.004 (ns) 0.050 ± 0.008 (**) b
CBGA 0.36 ± 0.03 0.34 ± 0.02 (ns) 0.38 ± 0.02 (ns) 0.28 ± 0.02 (***) 0.20 ± 0.02 (****)
CBG 0.102 ± 0.007 0.09 ± 0.01 (ns) 0.12 ± 0.01 (ns) 0.094 ± 0.005 (ns) 0.07 ± 0.02 (**)
CBD <LOD
THCVA 0.037 ± 0.004 0.035 ± 0.002 (ns) 0.040 ± 0.004 (ns) 0.033 ± 0.002 (ns) 0.033 ± 0.004 (ns)
THCA-C4 0.020 ± 0.006 0.016 ± 0.004 (ns) 0.021 ± 0.003 (ns) 0.018 ± 0.004 (ns) 0.017 ± 0.006 (ns)
THC 0.07 ± 0.01 0.09 ± 0.02 (ns) 0.12 ± 0.01 (**) 0.10 ± 0.02 (*) 0.24 ± 0.03 (****)
THCA 9.5 ± 0.7 8.8 ± 0.4 (ns) 9.3 ± 0.7 (ns) 8.4 ± 0.6 (*) 7.6 ± 0.5 (***)
CBCA 0.12 ± 0.01 0.11 ± 0.02 (ns) 0.13 ± 0.02 (ns) 0.10 ± 0.02 (ns) 0.11 ± 0.02 (ns)
Total cannabinoids 10.3 ± 0.6 9.5 ± 0.47 (ns) 10.2 ± 0.78 (ns) 9.1 ± 0.67 (ns) 8.3 ± 0.6 (***)
Total minor
0.80 ± 0.08 0.74 ± 0.07 (ns) 0.90 ± 0.08 (ns) 0.70 ± 0.07 (ns) 0.7 ± 0.1 (ns)
cannabinoids
total THC 8.4 ± 0.5 7.8 ± 0.3 (ns) 8.3 ± 0.6 (ns) 7.5 ± 0.5 (ns) 6.9 ± 0.5 (***)
Gen12
Atm N2 C5O5 Open-air
CBDVA 0.015 ± 0.004 0.020 ± 0.003 (ns) 0.018 ± 0.004 (ns) 0.016 ± 0.003 (ns) 0.016 ± 0.003 (ns)
CBDA 10.0 ± 1.0 10.5 ± 0.7 (ns) 10.0 ± 0.6 (ns) 10.3 ± 0.5 (ns) 10.9 ± 0.4 (ns)
CBGA 0.73 ± 0.08 0.65 ± 0.04 (ns) 0.61 ± 0.03 (**) 0.63 ± 0.03 (*) 0.46 ± 0.04 (****)
CBG 0.17 ± 0.02 0.19 ± 0.03 (ns) 0.17 ± 0.02 (ns) 0.20 ± 0.02 (ns) 0.16 ± 0.01 (ns)
CBD 0.15 ± 0.02 0.17 ± 0.04 (ns) 0.15 ± 0.02 (ns) 0.18 ± 0.01 (ns) 0.45 ± 0.03 (****)
THCVA <LOD
THCA-C4 <LOD
THC 0.05 ± 0.02 0.08 ± 0.02 (ns) 0.07 ± 0.01 (ns) 0.09 ± 0.01 (ns) 0.40 ± 0.04 (****)
THCA 4.5 ± 0.5 4.8 ± 0.3 (ns) 4.3 ± 0.2 (ns) 4.7 ± 0.3 (ns) 4.6 ± 0.2 (ns)
CBCA 0.54 ± 0.11 0.52 ± 0.04 (ns) 0.49 ± 0.02 (ns) 0.53 ± 0.04 (ns) 0.56 ± 0.05 (ns)
Total cannabinoids 16.1 ± 1.7 16.9 ± 1.2 (ns) 15.8 ± 0.9 (ns) 16.6 ± 0.9 (ns) 17.6 ± 0.8 (ns)
Plants 2024, 13, 1049 4 of 22

Table 1. Cont.

Absolute
Absolute Concentration ± SE
Absolute Concentration ± SE after 6 Days of Controlled Atmospheric
Cannabinoid Concentration ± SE after 15 Days of
Drying
at t0 Drying and Curing
(Traditional Drying)
Total minor
1.6 ± 0.2 1.6 ± 0.2 (ns) 1.5 ± 0.1 (ns) 1.6 ± 0.1 (ns) 2.1 ± 0.2 (**)
cannabinoids
Total THC 4.0 ± 0.4 4.2 ± 0.3 (ns) 3.9 ± 0. 2 (ns) 4.2 ± 0.2 (ns) 4.4 ± 0.2 (ns)
Total CBD 8.9 ± 0.9 9.4 ± 0.7 (ns) 8.9 ± 0.5 (ns) 9.2 ± 0.5 (ns) 10.0 ± 0.4 (ns)
a ns, not significant; b One-way ANOVA results of absolute cannabinoid concentrations between the different
drying conditions and t0 at an adjusted significance value of p < 0.05. p value < 0.0332 (*), p < 0.0021 (**), p < 0.0002
(***), p < 0.0001 (****).

β-myrcene and d-limonene were the primary monoterpenes in the Gen12 and
240 chemovars, respectively (Table 2). However, the monoterpene content in the 240 chemovar
was significantly lower compared to the Gen12 chemovar (Table 2). The Gen12 chemovar
had one order of magnitude higher β-myrcene and (−)-β-pinene levels and two orders of
magnitude higher α-pinene levels as compared to the 240 chemovar at t0 (Table 2). Further-
more, the Gen12 chemovar had 2.5-fold higher initial sesquiterpene content compared to
the 240 chemovar (Table 2). Though both chemovars had β-caryophyllene as the dominant
sesquiterpene, Gen12’s concentration was two times higher than that of the 240 chemovar
at t0 (Table 2). Nerolidol, α-guaiene, α-bulnesene, α-gurjunene, and elemol were the only
sesquiterpenes found in higher concentrations in the 240 chemovar (<0.1 DW%, Table 2).

Table 2. The 240 and Gen12 terpene composition at harvest day (t0 , absolute concentration, normal-
ized to DW%) and absolute terpene concentrations (in DW%) after drying under the four drying
conditions. For each drying procedure and t0 n = 5.

Absolute
Absolute Concentration ± SE
Absolute Concentration ± SE after 6 Days of Controlled Atmospheric
Terpene Concentration ± SE after 15 Days of
Drying
at t0 Drying and Curing
(Traditional Drying)
240
Atm N2 C5O5 Open-air
α-pinene 0.0047 ± 0.0003 0.0046 ± 0.0006 a 0.0047 ± 0.0003 0.0052 ± 0.0003 0.003 ± 0.002
Camphene 0.0020 ± 0.0001 0.001 ± 0.001 0.0018 ± 0.0009 0.0024 ± 0.0001 0.0004 ± 0.0004
(−)-β-pinene 0.0116 ± 0.0007 0.012 ± 0.001 0.0124 ± 0.0006 0.0137 ± 0.0005 0.0110 ± 0.0008
0.054 ± 0.004 b 0.047 ± 0.003 (ns) c 0.052 ± 0.003 (ns) 0.043 ± 0.004 (***)
β-myrcene 0.044 ± 0.005 (**)
δ-3-carene <LOD
d-limonene 0.074 ± 0.004 0.075 ± 0.007 (ns) 0.075 ± 0.004 (ns) 0.083 ± 0.004 (ns) 0.069 ± 0.005 (ns)
Linalool <LOD
Fenchol 0.003 ± 0.002 0.004 ± 0.002 0.0048 ± 0.0007 0.0057 ± 0.0005 0.0050 ± 0.0006
Pinalol <LOD
β-caryophyllene 0.31 ± 0.01 0.41 ± 0.04 (***) 0.40 ± 0.03 (***) 0.408 ± 0.006 (***) 0.40 ± 0.04 (***)
α-humulene 0.132 ± 0.006 0.18 ± 0.03 (*) 0.18 ± 0.03 (*) 0.173 ± 0.001 (ns) 0.18 ± 0.03 (*)
(−)-guaiol 0.056 ± 0.002 0.074 ± 0.009 (*) 0.08 ± 0.01 (**) 0.0747 ± 0.0009 (**) 0.071 ± 0.008 (*)
(−)-α-bisabolol <LOD
Nerolidol 0.063 ± 0.002 0.09 ± 0.02 (*) 0.09 ± 0.02 (*) 0.0804 ± 0.0008 (ns) 0.08 ± 0.01 (ns)
γ-elemene 0.039 ± 0.002 0.055 ± 0.009 (**) 0.055 ± 0.009 (**) 0.053 ± 0.001 (*) 0.048 ± 0.008 (ns)
α-bergomotene 0.0152 ± 0.0006 0.020 ± 0.006 0.022 ± 0.006 0.0183 ± 0.0002 0.021 ± 0.005
α-guaiene 0.028 ± 0.001 0.04 ± 0.01 0.04 ± 0.01 0.0350 ± 0.0005 0.04 ± 0.01
Plants 2024, 13, 1049 5 of 22

Table 2. Cont.

Absolute
Absolute Concentration ± SE
Absolute Concentration ± SE after 6 Days of Controlled Atmospheric
Terpene Concentration ± SE after 15 Days of
Drying
at t0 Drying and Curing
(Traditional Drying)
β-farensene 0.023 ± 0.001 0.03 ± 0.02 0.03 ± 0.01 0.024 ± 0.001 0.029 ± 0.009
β-eudesmene 0.0124 ± 0.0006 0.015 ± 0.001 0.0166 ± 0.0005 0.0162 ± 0.0006 0.016 ± 0.001
α-selinene 0.0164 ± 0.0008 0.024 ± 0.007 0.026 ± 0.007 0.0220 ± 0.0005 0.024 ± 0.0006
α-bulnesene 0.054 ± 0.003 0.07 ± 0.01 (ns) 0.08 ± 0.01 (*) 0.070 ± 0.001 (ns) 0.07 ± 0.01 (ns)
β-bisabolene <LOD
cis-α-bisabolene <LOD
Eudesma-3,7(11)-
0.042 ± 0.002 0.06 ± 0.01 (ns) 0.06 ± 0.01 (*) 0.057 ± 0.001 (ns) 0.06 ± 0.01 (*)
diene
γ-eudesmol 0.055 ± 0.002 0.073 ± 0.009 (***) 0.073 ± 0.005 (***) 0.0734 ± 0.0009 (***) 0.068 ± 0.006 (**)
β-eudesmol 0.053 ± 0.002 0.072 ± 0.007 (****) 0.075 ± 0.006 (****) 0.075 ± 0.001 (****) 0.072 ± 0.007 (****)
Bulnesol 0.051 ± 0.003 0.071 ± 0.007 (***) 0.074 ± 0.007 (****) 0.071 ± 0.001 (***) 0.067 ± 0.007 (**)
α-gurjunene 0.0136 ± 0.0005 0.02 ± 0.01 0.022 ± 0.009 0.0175 ± 0.0002 0.021 ± 0.007
γ-gurjunene 0.031 ± 0.001 0.04 ± 0.01 0.04 ± 0.01 0.0403 ± 0.0005 0.039 ± 0.008
Elemol 0.0162 ± 0.0006 0.020 ± 0.006 0.020 ± 0.006 0.0172 ± 0.0001 0.012 ± 0.003
Total terpenes 1.17 ± 0.05 1.55 ± 0.22 (*) 1.55 ± 0.21 (*) 1.49 ± 0.03 (ns) 1.43 ± 0.21 (ns)
Total monoterpenes 0.15 ± 0.01 0.15 ± 0.02 (ns) 0.146 ± 0.009 (ns) 0.162 ± 0.008 (ns) 0.13 ± 0.01 (ns)
Total sesquiterpenes 1.02 ± 0.04 1.4 ± 0.2 (*) 1.4 ± 0.2 (*) 1.33 ± 0.02 (ns) 1.3 ± 0.2 (ns)
Gen12
Atm N2 C5O5 Open-air
α-pinene 0.26 ± 0.02 0.30 ± 0.02 (ns) 0.28 ± 0.03 (ns) 0.36 ± 0.03 (****) 0.29 ± 0.01 (ns)
Camphene 0.005 ± 0.001 0.0057 ± 0.0005 0.0054 ± 0.0006 0.0069 ± 0.0004 0.0053 ± 0.0003
(−)-β-pinene 0.14 ± 0.01 0.16 ± 0.01 (ns) 0.15 ± 0.02 (ns) 0.20 ± 0.03 (****) 0.151 ± 0.008 (ns)
β-myrcene 0.41 ± 0.03 0.35 ± 0.03 (ns) 0.33 ± 0.04 (**) 0.48 ± 0.06 (ns) 0.29 ± 0.02 (***)
δ-3-carene <LOD
d-limonene 0.093 ± 0.005 0.06 ± 0.01 (****) 0.06 ± 0.01 (***) 0.079 ± 0.007 (ns) 0.039 ± 0.002 (****)
Linalool 0.025 ± 0.005 0.032 ± 0.002 (ns) 0.028 ± 0.005 (ns) 0.04 ± 0.01 (**) 0.023 ± 0.002 (ns)
Fenchol 0.011 ± 0.001 0.012 ± 0.001 0.013 ± 0.002 0.016 ± 0.004 0.010 ± 0.002
Pinalol 0.018 ± 0.001 0.0192 ± 0.0008 0.020 ± 0.002 0.024 ± 0.006 0.017 ± 0.001
β-caryophyllene 0.60 ± 0.03 0.64 ± 0.04 (ns) 0.69 ± 0.07 (ns) 0.70 ± 0.04 (*) 0.73 ± 0.05 (**)
α-humulene 0.22 ± 0.01 0.24 ± 0.01 (ns) 0.25 ± 0.03 (*) 0.25 ± 0.02 (*) 0.26 ± 0.02 (**)
(−)-guaiol 0.092 ± 0.005 0.116 ± 0.005 (*) 0.12 ± 0.01 (**) 0.12 ± 0.01 (**) 0.13 ± 0.01 (****)
(−)-α-bisabolol 0.24 ± 0.01 0.29 ± 0.01 (*) 0.30 ± 0.03 (**) 0.30 ± 0.03 (**) 0.34 ± 0.03 (****)
Nerolidol <LOD
γ-elemene 0.094 ± 0.006 0.085 ± 0.003 (ns) 0.086 ± 0.006 (ns) 0.084 ± 0.003 (ns) 0.08 ± 0.01 (*)
α-bergomotene 0.032 ± 0.002 0.035 ± 0.002 0.036 ± 0.005 0.038 ± 0.002 0.036 ± 0.002
α-guaiene <LOD
β-farensene 0.026 ± 0.002 0.030 ± 0.003 0.032 ± 0.004 0.031 ± 0.002 0.028 ± 0.003
β-eudesmene 0.088 ± 0.006 0.092 ± 0.006 (ns) 0.11 ± 0.01 (*) 0.100 ± 0.008 (ns) 0.105 ± 0.009 (*)
α-selinene 0.091 ± 0.006 0.102 ± 0.006 (ns) 0.11 ± 0.01 (*) 0.108 ± 0.007 (ns) 0.107 ± 0.009 (ns)
α-bulnesene <LOD
β-bisabolene 0.070 ± 0.004 0.081 ± 0.005 (ns) 0.08 ± 0.01 (*) 0.082 ± 0.006 (*) 0.074 ± 0.006 (ns)
cis-α-bisabolene 0.120 ± 0.009 0.15 ± 0.01 (*) 0.16 ± 0.02 (**) 0.15 ± 0.01 (*) 0.14 ± 0.01 (ns)
Plants 2024, 13, 1049 6 of 22

Table 2. Cont.

Absolute
Absolute Concentration ± SE
Absolute Concentration ± SE after 6 Days of Controlled Atmospheric
Terpene Concentration ± SE after 15 Days of
Drying
at t0 Drying and Curing
(Traditional Drying)
Eudesma-3,7(11)-
0.24 ± 0.01 0.31 ± 0.02 (**) 0.32 ± 0.04 (***) 0.32 ± 0.02 (***) 0.34 ± 0.02 (****)
diene
γ-eudesmol 0.087 ± 0.005 0.109 ± 0.005 (*) 0.11 ± 0.01 (**) 0.11 ± 0.01 (**) 0.13 ± 0.01 (****)
β-eudesmol 0.110 ± 0.007 0.138 ± 0.006 (*) 0.14 ± 0.01 (**) 0.14 ± 0.02 (**) 0.17 ± 0.02 (****)
Bulnesol 0.081 ± 0.004 0.110 ± 0.006 (****) 0.11 ± 0.01 (****) 0.104 ± 0.009 (***) 0.079 ± 0.007 (ns)
α-gurjunene <LOD
γ-gurjunene 0.26 ± 0.01 0.29 ± 0.02 (ns) 0.31 ± 0.04 (*) 0.31 ± 0.02 (ns) 0.25 ± 0.02 (ns)
Elemol <LOD
Total terpenes 3.41 ± 0.22 3.75 ± 0.23 (ns) 3.85 ± 0.4 (ns) 4.15 ± 0.35 (**) 3.8 ± 0.24 (ns)
Total monoterpenes 0.96 ± 0.08 0.94 ± 0.08 (ns) 0.85 ± 0.10 (ns) 1.2 ± 0.15 (**) 0.80 ± 0.04 (ns)
Total sesquiterpenes 2.45 ± 0.14 2.81 ± 0.15 (ns) 3.0 ± 0.3 (*) 3.0 ± 0.2 (*) 3.0 ± 0.2 (**)
a Statistical analysis conducted only for terpene with initial concentrations higher than 0.04 DW%. b One-way

ANOVA results of absolute terpene concentrations between the different drying conditions and t0 at a significance
value of p < 0.05. p value < 0.0332 (*), p < 0.0021 (**), p < 0.0002 (***), p < 0.0001 (****). c ns, not significant.

2.2. Drying Process Efficiency

After six days, all the cannabis inflorescences subjected to the three distinct controlled
atmosphere drying conditions were found to be completely dried. This conclusion was
drawn from the absence of weight change on the sixth day, indicating that no further curing
was necessary. On the other hand, the cannabis inflorescences in the reference group from
both chemovars failed to reach a final stable dry weight value after 14 days of open-air
drying, indicating that excessive water content was still present (Table 3). Consequently, an
additional day of curing was necessary, employing silica gel pearls humidity absorbers, to
achieve completely dried cannabis inflorescences (Table 3). After 14 days of drying in the
open air, the 240 and Gen12 inflorescences contained 45% and 16% of water, respectively,
as compared to the 240 and Gen12 inflorescences dried under CA drying conditions. This
study’s findings reveal that drying cannabis inflorescences under controlled atmosphere
drying conditions results in a process that is 2.5 times faster than open-air traditional drying.
It is important to highlight that the traditional method of cannabis curing involves large,
manually-ventilated containers where the complete removal of the remaining water can
span two to three weeks [1]. Hence, controlled atmosphere drying chambers substantially
speed up the drying process compared to the methods typically used today.

Table 3. Percentage weight loss during the drying of the 240 and Gen12 chemovars under controlled
atmosphere and open-air drying conditions.

Drying Conditions 240 Gen12

Weight loss in controlled atmosphere drying chambers
78.5 ± 1.6% 77.4 ± 2.5%
after 6 days
Weight loss in open-air drying conditions after 14 days
60.5 ± 2.4% (**) a 73 ± 1.5% (*) a
(before curing)
Weight loss in open-air drying conditions after 15 days
75.8 ± 1.5% (ns) a 80 ± 4% (ns) a
(including curing)
a t-test statistical results comparing between controlled atmosphere and open-air drying conditions

(p value < 0.0332 (*); p < 0.0021 (**); ns, not significant).

Prolonged drying can elevate mold risks due to slow moisture removal which allows
mold to promote growth, crucial for medicinal cannabis [19]. In this study, no visible
 Plants 2024, 13, 1049 7 of 22

molds were detected after drying in all inflorescences from both chemovars subjected
to CA drying chambers. On the other hand, approximately 80% of the 240 chemovar
inflorescences subjected to traditional drying were highly infested by Alternaria alternata
and also revealed low infestation of Botrytis cinerea on day 12 of the drying process (Figure 1
and Table 4). In order to assess the infestation level in all samples, both CFU and real-time
PCR assays were conducted (Table 4). The extent of infestation with Alternaria alternata after
CA drying was higher but not statistically significant as compared to the infestation level of
fresh inflorescence (Table 4). Moreover, the infestation with Botrytis cinerea at t0 and after CA
drying was below the limit of detection and therefore reported as undetermined (Table 4).
On the other hand, Alternaria alternata infestation levels and CFU levels of inflorescences
subjected to traditional drying were higher by one to two orders of magnitude as compared
to samples subjected to CA drying conditions or at t0 (Table 4). Therefore, CA drying
hastened the drying process and delayed mold growth in cannabis inflorescences, due to
the lower duration of available water content conducive to mold. Regarding Gen12, the
Plants 2024, 13, x FOR PEER REVIEW presence of Botrytis cinerea and Alternaria alternata, both at the initial time point (t 7 of 23
0 ) and
post-drying, was found to be below the limit of detection.

Figure 1. Mycelium development in the 240 inflorescence: (a) at day 12 of drying, (b) at day 14 and
Figure 1. Mycelium development in the 240 inflorescence: (a) at day 12 of drying, (b) at day 14 and
(c) at day 15 after one day of curing in the presence of dried silica gel pearls.
(c) at day 15 after one day of curing in the presence of dried silica gel pearls.

2.3. Impact of the Drying Process on the Cannabinoid Content Preservation

Table 4. Mold determination of the 240 chemovar inflorescences at t0 , under controlled atmosphere
2.3.1.
dryingHigh-THCA Chemovar—240
conditions and open-air drying conditions.
The cannabinoid content within the 240 chemovar was notably influenced by the dif-
ferent drying conditions (Figure 2 and Table Alternaria alternata
1). Specifically, Botrytis cinerea
the predominant canna-
Drying Conditions CFU/g Dry × 106 Extent of Infestation Extent of Infestation
binoid—THCA—exhibited a concentration reduction (by 2–20%, see detailed explanation
[2−∆∆Ct ] [2−∆∆Ct ]
for relative concentration calculation in Section 3.7) throughout the different drying pro-
Fresh inflorescences
cedures implementedat(Figure
t0 2a2.8 ± Table
and 0.8 400 ± 200
1). The smallest reduction in THCAUndetermined
levels was
Inflorescences
observed underfrom N2 drying conditions followed by atm (both are not statistically significant
controlled atmosphere ± 0.06
2.0 On a ± 1200 (ns) a
from t0, Figure 2a and Table 1). the(ns)
other hand, 2100
open-air Undetermined
drying conditions led to the
chambers at day 6
lowest THCA concentration (Figure 2a and Table 1). Although the atmospheric composi-
tionOpen-air
of the inflorescences at
controlled atmosphere 31 ±chamber
19 (*) a and15, open-air conditions
000 ± 2800 (****) a were 290identical,
± 110 they
day 12
produced significantly different cannabinoid concentrations (Table 1). This discrepancy
a One-way ANOVA followed by Tukey’s post hoc test compared to t results (p value < 0.0332 (*), p < 0.0001 (****);
may be attributed to factors such as the duration of the 0 drying process, the inclusion of
ns, not significant).
silica gel pearls humidity absorbers, the use of dry gases within the controlled atmosphere
chamber,
2.3. Impact and/or to the high
of the Drying mold
Process infestation
on the Cannabinoid observed
Content in open-air samples (Table 4).
Preservation
CBGA demonstrated
2.3.1. High-THCA a similar degradation pattern to THCA (Figure 2b). CBGA concen-
Chemovar—240
tration remained stable during drying under N2 and atm drying conditions (not statisti-
The cannabinoid content within the 240 chemovar was notably influenced by the dif-
cally significant from t0), while the most pronounced decrease was observed under open-
ferent drying conditions (Figure 2 and Table 1). Specifically, the predominant cannabinoid—
air drying conditions (by 42%, Figure 2b and Table 1). CBGA, being the first acidic canna-
THCA—exhibited a concentration reduction (by 2–20%, see detailed explanation for relative
binoid produced by the cannabis plant, serves as the primary cannabinoid transformed
concentration calculation in Section 3.7) throughout the different drying procedures im-
by three individual FAD-dependent dehydrogenases into THCA, CBDA, and CBCA [6,9].
plemented (Figure 2a and Table 1). The smallest reduction in THCA levels was observed
This enzymatic activity may persist during the initial drying process at stages where the
under N2 drying conditions followed by atm (both are not statistically significant from t0 ,
inflorescences remain sufficiently moist [34]. The decline in CBGA concentration did not
Figure 2a and Table 1). On the other hand, open-air drying conditions led to the lowest
yield increased levels of these three downstream acidic cannabinoids, nor an increase in
CBG concentration due to ongoing CBGA decarboxylation (Figure 2a,c,d,h).
Plants 2024, 13, 1049 8 of 22

THCA concentration (Figure 2a and Table 1). Although the atmospheric composition of
the controlled atmosphere chamber and open-air conditions were identical, they produced
significantly different cannabinoid concentrations (Table 1). This discrepancy may be at-
tributed to factors such as the duration of the drying process, the inclusion of silica gel
pearls humidity absorbers, the use of dry gases within the controlled atmosphere chamber,
and/or to the high mold infestation observed in open-air samples (Table 4). CBGA demon-
strated a similar degradation pattern to THCA (Figure 2b). CBGA concentration remained
stable during drying under N2 and atm drying conditions (not statistically significant from
t0 ), while the most pronounced decrease was observed under open-air drying conditions
(by 42%, Figure 2b and Table 1). CBGA, being the first acidic cannabinoid produced by
the cannabis plant, serves as the primary cannabinoid transformed by three individual
FAD-dependent dehydrogenases into THCA, CBDA, and CBCA [6,9]. This enzymatic
activity may persist during the initial drying process at stages where the inflorescences
remain sufficiently moist [34]. The decline in CBGA concentration did not yield increased
Plants 2024, 13, x FOR PEER REVIEW 8 of 23
levels of these three downstream acidic cannabinoids, nor an increase in CBG concentration
due to ongoing CBGA decarboxylation (Figure 2a,c,d,h).

Figure2.2.Mean
Figure Meancannabinoid
cannabinoid concentrations
concentrations (in(in DW%,
DW%, y-axis,
y-axis, for
for each
each drying
dryingprocedure
procedureand andt0t;0n; n= = 5)
5) of the 240 chemovar determined under four different drying conditions by HPLC–PDA: con-
of the 240 chemovar determined under four different drying conditions by HPLC–PDA: controlled
trolled atmospheric conditions (atm), controlled N2 ≥ 99% conditions (N2), controlled CO2 5%/O2
5%/N2 90% conditions (C5O5), and open-air drying process used as a reference system (air). (a)
THCA, (b) CBGA, (c) CBDA, (d) CBCA, (e) THCVA, (f) THCA-C4, (g) THC, (h) CBG, (i) total minor
cannabinoids, (j) total cannabinoids and (k) total THC. Statistical significance between the different
drying conditions and t0 for each compound was calculated using one-way ANOVA followed by
Tukey’s post hoc test (p value < 0.0332 (*), p < 0.0021 (**), p < 0.0002 (***), p < 0.0001 (****)).
Plants 2024, 13, 1049 9 of 22

atmospheric conditions (atm), controlled N2 ≥ 99% conditions (N2 ), controlled CO2 5%/O2 5%/N2
90% conditions (C5O5), and open-air drying process used as a reference system (air). (a) THCA,
(b) CBGA, (c) CBDA, (d) CBCA, (e) THCVA, (f) THCA-C4, (g) THC, (h) CBG, (i) total minor cannabi-
noids, (j) total cannabinoids and (k) total THC. Statistical significance between the different drying
conditions and t0 for each compound was calculated using one-way ANOVA followed by Tukey’s
post hoc test (p value < 0.0332 (*), p < 0.0021 (**), p < 0.0002 (***), p < 0.0001 (****)).

Under open-air drying conditions, CBDA concentration dropped by 24%, whereas

under all other drying conditions, CBDA concentration remained stable (Figure 2c and
Table 1). Varying decarboxylation patterns of THCA to THC were observed between the
different drying conditions (Figure 2a,g). THC concentration under the controlled atmo-
sphere drying conditions increased by 40–70% (Figure 2g and Table 1). In contrast, open-air
drying conditions, which yielded the lowest post-drying THCA concentration, resulted
in approximately 3.5-fold higher THC levels as compared to t0 (Figure 2a,g and Table 1).
Notwithstanding, this increase in THC concentration during drying is still one order of
magnitude lower as compared to the amount of THCA degraded during drying under
open-air drying conditions (Figure 2a,g and Table 1). This observation may imply that
controlled atmosphere drying conditions could either slow down the decarboxylation of
THCA to THC or that the extended drying duration under open-air drying conditions
allowed THCA decarboxylation to occur over a longer period, resulting in elevated THC
levels. Total minor cannabinoid content after drying remained similar to t0 , with the highest
content observed for N2 drying conditions, especially in cannabinoids like CBGA, CBDA,
THCVA, and CBG (Figure 2b,c,e,h,i). Consequently, total cannabinoid content was highest
under N2 drying conditions, and lowest under open-air drying conditions (Figure 2i).

2.3.2. Hybrid Chemovar—Gen12

The cannabinoids in the Gen12 chemovar displayed different drying patterns as
compared to the 240 chemovar (Figure 3). Gen12 is a hybrid chemovar containing relatively
high concentrations of both THCA and CBDA (Figure 3a,c). Unlike the degradation pattern
of the most predominant cannabinoids observed in the 240 chemovar, in Gen12, stable levels
were observed for the most predominant cannabinoids—THCA and CBDA under all drying
conditions (Figure 3a,c). The relative changes compared to t0 for both THCA and CBDA
under all drying conditions were smaller than 10% and not statistically significant from t0
(Figure 3a,c and Table 1). The stability of primary cannabinoids can differ notably between
chemovars under identical drying conditions. This variation might stem from distinct
genetic compositions, leading to significant disparities in enzyme activities, especially those
associated with cannabinoid metabolism during drying [9].
CBGA concentration declined significantly under the different drying conditions, with
the largest decrease observed under open-air drying conditions (by 37%, Figure 3b and
Table 1) and the lowest concentration reduction observed under atm drying conditions
(by 11%, not statistically significant from t0 , Figure 3b and Table 1). Despite significant
CBGA degradation occurring under all drying conditions, stable THCA, CBDA, CBCA,
and CBG concentrations were observed (Figure 3a,c,d,f). Based on the known CBGA
metabolic pathway, we anticipated seeing a corresponding increase in at least one of
the CBGA metabolites following CBGA degradation. However, such a mass balance
was not observed. Therefore, we speculate that an additional, unidentified degradation
product may have been formed. Furthermore, the three controlled atmosphere drying
conditions maintained stable THC and CBD concentrations, while under open-air drying
conditions, approximately eight- and three-fold higher THC and CBD levels, respectively,
were observed as compared to t0 (Figure 3e,g and Table 1). These results strengthen the
hypothesis that controlled atmosphere drying conditions slow down the decarboxylation
of the major cannabinoids or that the observed increase in neutral cannabinoids (THC and
CBD) became possible due to the longer duration of the drying process under open-air
drying conditions. Interestingly, in the Gen12 chemovar, the concentrations of THC and
Plants 2024, 13, 1049 10 of 22

CBD increased under open-air drying conditions without any corresponding decrease in
Plants 2024, 13, x FOR PEER REVIEW 10 of 23
THCA and CBDA concentrations (Figure 3a,c,e,g). This suggests that the formation and
degradation of cannabinoids continue to occur during the drying process.

Figure 3. Mean
Figure 3. cannabinoid concentrations
Mean cannabinoid concentrations(in
(inDW%,
DW%,y-axis,
y-axis,for
foreach
eachdrying
drying procedure
procedure and
and t0 ;tn0; =n 5)
=
5) of the Gen12 chemovar determined under four different drying conditions by HPLC–PDA:
of the Gen12 chemovar determined under four different drying conditions by HPLC–PDA: controlled con-
trolled atmospheric
atmospheric conditionsconditions (atm), controlled
(atm), controlled N2 ≥ 99% ≥ 99% conditions
N2conditions (N2), controlled
(N2 ), controlled CO2 5%/O CO25%/N5%/O2
2 2
5%/N2 90% conditions (C5O5), and open-air drying process used as a reference system (air). (a)
90% conditions (C5O5), and open-air drying process used as a reference system (air). (a) THCA,
THCA, (b) CBGA, (c) CBDA, (d) CBCA, (e) THC, (f) CBG, (g) CBD, (h) total minor cannabinoids, (i)
(b) CBGA, (c) CBDA, (d) CBCA, (e) THC, (f) CBG, (g) CBD, (h) total minor cannabinoids, (i) total
total cannabinoids, (j) total THC and (k) total CBD. Statistical significance between the different
cannabinoids,
drying (j) total
conditions and THC
t0 forand
each(k) total CBD.was
compound Statistical significance
calculated between
using one-way the different
ANOVA drying
followed by
conditions and t for each compound was calculated using one-way ANOVA followed
Tukey’s post hoc0test (p value < 0.0332 (*), p < 0.0021 (**), p < 0.0002 (***), p < 0.0001 (****)). by Tukey’s
post hoc test (p value < 0.0332 (*), p < 0.0021 (**), p < 0.0002 (***), p < 0.0001 (****)).
CBGA concentration declined significantly under the different drying conditions,
with The controlled
the largest atmosphere
decrease observeddrying conditions
under open-air maintained stable total
drying conditions minorFigure
(by 37%, cannabi-
3b
noids concentration (not statistically significant from
and Table 1) and the lowest concentration reduction observed t 0 , Figure 3h). The highest
under atm drying condi- total
minor(by
tions cannabinoids concentration
11%, not statistically was observed
significant from t0, under
Figure open-air drying
3b and Table 1). conditions (25%
Despite signifi-
relative increase compared to t0 ), due to the increase in THC and CBD concentrations
cant CBGA degradation occurring under all drying conditions, stable THCA, CBDA,
(Figure 3h). Moreover, none of the drying conditions yielded statistically significant dif-
CBCA, and CBG concentrations were observed (Figure 3a,c,d,f). Based on the known
ferences in total cannabinoid content as compared to t0 (Figure 3i). In both chemovars, an
CBGA metabolic pathway, we anticipated seeing a corresponding increase in at least one
of the CBGA metabolites following CBGA degradation. However, such a mass balance
was not observed. Therefore, we speculate that an additional, unidentified degradation
product may have been formed. Furthermore, the three controlled atmosphere drying
conditions maintained stable THC and CBD concentrations, while under open-air drying
conditions, approximately eight- and three-fold higher THC and CBD levels, respectively,
Plants 2024, 13, 1049 11 of 22

increase in isomerization or oxidation degradation products such as ∆8-THC or CBN was

not observed.
Uziel et al. recently found that both traditional and microwave oven drying of a high-
THCA chemovar led to the decarboxylation of THCA to THC during the drying process
(p < 0.001) [19]. In contrast, our study revealed that only traditional drying conditions
resulted in significant decarboxylation (>3-fold and p < 0.0001) of major cannabinoids.
Given that microwave drying is quicker than controlled atmosphere drying [19], this find-
ing reinforces our conclusion that controlled atmosphere drying conditions are ideal for
slowing the decarboxylation of THCA in high-THCA chemovars and the decarboxylation
of THCA and CBDA in hybrid chemovars. Additionally, certain cannabinoids, such as
THCA, CBGA, and THC, were found to have higher concentrations under various drying
conditions [19], paralleling the varied cannabinoid concentration patterns observed in our
study across different drying conditions used. Oduola et al. recently demonstrated that hot
air, infrared, and microwave drying significantly reduce the drying time; however, they led
to a substantial reduction of more than 45% in CBDA concentration, CBGA concentration,
total CBD content, and total cannabinoid content compared to fresh inflorescence in all
methods examined [26]. In a separate study, Chen et al. noted that hot air drying signifi-
cantly shortened the drying time by one to two orders of magnitude in three high-CBDA
chemovars [24]. However, it led to a reduction in total CBD content by 1.5–16%, depending
on the drying temperature, compared to traditional drying and freeze-drying, both of
which produced similar total CBD content [24]. The recent literature together with the
results obtained from our study highlight the need for a drying procedure that will shorten
the drying time without reducing the active compound content.
In summary, considering both the current findings and previous research, different
chemovars, characterized by distinct secondary metabolite compositions and thus unique
genotypic characteristics, can respond differently to various drying conditions, therefore
necessitating chemovar-tailored drying conditions. These differences may be attributed to
the varied kinetic behavior of enzymes involved in the cannabinoid biosynthetic pathways.

2.4. Impact of the Drying Process on the Terpene Content Preservation

2.4.1. High-THCA Chemovar—240
Throughout the drying process, the formation or degradation patterns of monoter-
penes varied among the different drying conditions. The highest concentrations of various
monoterpenes in the 240 chemovar inflorescence were observed under C5O5 drying condi-
tions (Figure 4). Specifically, (−)-β-pinene concentration remained stable under all drying
conditions, except for C5O5, which led to a 18% relative concentration increase (Figure 4b
and Table 2). Conversely, β-myrcene was highly sensitive to the drying process, losing
approximately 3–20% of its initial concentration during drying, with the smallest reduction
observed under C5O5 drying conditions (not statistically significant from t0 ) and the largest
reduction under atm and open-air drying conditions (Figure 4c and Table 2). On the other
hand, α-pinene and d-limonene concentrations remained stable during drying (Figure 4a,d
and Table 2).
During the drying process, the formation or degradation patterns of sesquiterpenes
were similar among the different drying conditions. No particular drying conditions
were found to be preferable for the various sesquiterpenes, as all conditions resulted
in a 20–50% relative increase in their concentrations (Figure 5). β-caryophyllene and
α-humulene revealed similar drying patterns under all drying conditions (Figure 5a,b and
Table 2), a finding consistent with our prior study that demonstrated a high correlation in
concentrations of these two sesquiterpenes across 14 different chemovars [18].
 approximately 3–20% of its initial concentration during drying, with the smallest reduc-
tion observed under C5O5 drying conditions (not statistically significant from t0) and the
largest reduction under atm and open-air drying conditions (Figure 4c and Table 2). On
Plants 2024, 13, 1049 the other hand, α-pinene and d-limonene concentrations remained stable during drying12 of 22
(Figure 4a,d and Table 2).

Figure
Figure 4.4.Mean
Meanmonoterpene
monoterpeneconcentrations
concentrations (in(in
DW%,
DW%, y-axis, forfor
y-axis, each drying
each procedure
drying andand
procedure t0; nt=0 ;
5)
n =of5)the 240240
of the chemovar
chemovardetermined
determined under four
under fourdifferent
differentdrying
dryingconditions
conditions byby GC/MS:
GC/MS: controlled
controlled
atmospheric
atmospheric conditions
conditions (atm),
(atm),controlled
controlledN N22≥≥99%
99% conditions
conditions(N(N2), controlled CO2 5%/O2 5%/N2 90%
2 ), controlled CO2 5%/O2 5%/N2
conditions (C5O5), and open-air drying process used as a reference system (air). (a) α-pinene, (b)
90% conditions (C5O5), and open-air drying process used as a reference system (air). (a) α-pinene,
(−)-β-pinene, (c) β-myrcene and (d) d-limonene. Statistical significance between the different drying
(b) (−)-β-pinene, (c) β-myrcene and (d) d-limonene. Statistical significance between the different
conditions and t0 for each compound was calculated using one-way ANOVA followed by Tukey’s
Plants 2024, 13, x FOR PEER REVIEW
drying 13 of 23 by
post hocconditions and
test (p value t0 for each
< 0.0332 (*), pcompound was
< 0.0021 (**), p <calculated using one-way ANOVA followed
0.0002 (***)).
Tukey’s post hoc test (p value < 0.0332 (*), p < 0.0021 (**), p < 0.0002 (***)).
During the drying process, the formation or degradation patterns of sesquiterpenes
were similar among the different drying conditions. No particular drying conditions were
found to be preferable for the various sesquiterpenes, as all conditions resulted in a 20–
50% relative increase in their concentrations (Figure 5). β-caryophyllene and α-humulene
revealed similar drying patterns under all drying conditions (Figure 5a,b and Table 2), a
finding consistent with our prior study that demonstrated a high correlation in concentra-
tions of these two sesquiterpenes across 14 different chemovars [18].
Taken altogether, the monoterpene content remained stable during drying with the
highest monoterpene content observed under C5O5 drying conditions (not statistically
significant from t0, Figure 6a and Table 2). Overall, sesquiterpene content significantly in-
creased by 30–40%, with the highest increase observed under atm and N2 drying condi-
tions, as compared to t0 (Figure 6b and Table 2). Although open-air samples were highly
infested by Alternaria alternata and also revealed low infestation of Botrytis cinerea, their
sesquiterpene composition was similar to controlled atmosphere samples. The total ter-
pene content, encompassing both monoterpenes and sesquiterpenes, exhibited an in-
crease of approximately 25–30% during the drying process (Figure 6c). Notably, atm and
N2 drying conditions displayed a statistically significant enhancement in total terpene
content compared to t0 (Figure 6c and Table 2).

Figure5.5. Mean
Figure Mean sesquiterpene
sesquiterpeneconcentrations (in (in
concentrations DW%,
DW%,y-axis, for each
y-axis, drying
for each procedure
drying and t0; and
procedure n= t ;
0
5) of the 240 chemovar determined under four different drying conditions by GC/MS: controlled
n = 5) of the 240 chemovar determined under four different drying conditions by GC/MS: controlled
atmospheric conditions (atm), controlled N2 ≥ 99% conditions (N2), controlled CO2 5%/O2 5%/N2 90%
conditions (C5O5), and open-air drying process used as a reference system (air). (a) β-caryo-
phyllene, (b) α-humulene, (c) (−)-guaiol, (d) nerolidol, (e) eudesma-3,7(11)-diene, (f) γ-eudesmol,
(g) β-eudesmol, (h) bulnesol, (i) γ-elemene and (j) α-bulnesene. Statistical significance between the
different drying conditions and t0 for each compound was calculated using one-way ANOVA fol-
lowed by Tukey’s post hoc test (p value < 0.0332 (*), p < 0.0021 (**), p < 0.0002 (***), p < 0.0001 (****)).
Plants 2024, 13, 1049 13 of 22

atmospheric conditions (atm), controlled N2 ≥ 99% conditions (N2 ), controlled CO2 5%/O2
5%/N2 90% conditions (C5O5), and open-air drying process used as a reference system (air).
(a) β-caryophyllene, (b) α-humulene, (c) (−)-guaiol, (d) nerolidol, (e) eudesma-3,7(11)-diene,
(f) γ-eudesmol, (g) β-eudesmol, (h) bulnesol, (i) γ-elemene and (j) α-bulnesene. Statistical sig-
nificance between the different drying conditions and t0 for each compound was calculated using
one-way ANOVA followed by Tukey’s post hoc test (p value < 0.0332 (*), p < 0.0021 (**), p < 0.0002 (***),
p < 0.0001 (****)).

Taken altogether, the monoterpene content remained stable during drying with the
highest monoterpene content observed under C5O5 drying conditions (not statistically
significant from t0 , Figure 6a and Table 2). Overall, sesquiterpene content significantly
increased by 30–40%, with the highest increase observed under atm and N2 drying condi-
tions, as compared to t0 (Figure 6b and Table 2). Although open-air samples were highly
infested by Alternaria alternata and also revealed low infestation of Botrytis cinerea, their
sesquiterpene composition was similar to controlled atmosphere samples. The total terpene
content, encompassing both monoterpenes and sesquiterpenes, exhibited an increase of
approximately 25–30% during the drying process (Figure 6c). Notably, atm and N2 dry-
Plants 2024, 13, x FOR PEER REVIEW 14 of 23
ing conditions displayed a statistically significant enhancement in total terpene content
compared to t0 (Figure 6c and Table 2).

Figure 6.
Figure Mean total
6. Mean total terpene content (in DW%, y-axis, for each drying procedure
terpene content procedure and and tt00; n == 5)
5) of
of the
the
240 chemovar determined
240 chemovar determinedunder
underfour
four different
different drying
drying conditions
conditions by GC/MS:
by GC/MS: controlled
controlled atmos-
atmospheric
pheric conditions
conditions (atm), controlled
(atm), controlled N2 ≥ 99% N2 conditions
≥ 99% conditions (N2), controlled
(N2 ), controlled CO2 5%/OCO225%/O
5%/N 2 5%/N 90% con-
2 90% 2conditions
ditions
(C5O5),(C5O5), and open-air
and open-air drying process
drying process used as used as a reference
a reference system
system (air). (a)(air).
total (a) total monoterpenes,
monoterpenes, (b) total
(b) total sesquiterpenes and (c) total terpenes. Statistical significance between the
sesquiterpenes and (c) total terpenes. Statistical significance between the different drying different drying
conditions
conditions and t0 for each compound was calculated using one-way ANOVA followed by Tukey’s
and t0 for each compound was calculated using one-way ANOVA followed by Tukey’s post hoc test
post hoc test (p value < 0.0332 (*), p < 0.0021 (**)).
(p value < 0.0332 (*), p < 0.0021 (**)).

Recent studies have

Recent studies haverevealed
revealedthat
thatcertain
certain terpenes,
terpenes, such
such as β-myrcene
as β-myrcene andand d-limo-
d-limonene,
nene,
are susceptible to degradation through freeze-drying in some chemovars [28,29].[28,29].
are susceptible to degradation through freeze-drying in some chemovars These
These
studiesstudies havereported
have also also reported that
that the the concentrations
concentrations of certain
of certain terpenes,
terpenes, such assuch as α-
α-pinene,
pinene, (−)-β-pinene, β-caryophyllene, and α-humulene, can increase under
(−)-β-pinene, β-caryophyllene, and α-humulene, can increase under specific drying condi- specific dry-
ing conditions [28,29]. Moreover, hot air drying was found to reduce various
tions [28,29]. Moreover, hot air drying was found to reduce various monoterpene concentra- monoterpene
concentrations
tions by 38–95% bywhile
38–95% while simultaneously
simultaneously increasingincreasing various sesquiterpene
various sesquiterpene concen-
concentrations by
trations by 210–290% as compared to fresh inflorescence [26]. These results align
210–290% as compared to fresh inflorescence [26]. These results align with our observations with our
observations
indicating anindicating
increase inan increase
both in both
terpene terpene and concentrations
and cannabinoid cannabinoid concentrations dur-
during the drying
ing the drying
process. This process.
suggestsThis
thatsuggests thatactivities
biological biological activitiestocontinue
continue to occur
occur within thewithin the
cannabis
cannabis inflorescences
inflorescences throughout throughout
the drying the drying period.
period.

2.4.2. Hybrid
2.4.2. Hybrid Chemovar—Gen12
Chemovar—Gen12
The highest
The highest concentrations
concentrationsofofmonoterpenes
monoterpenes in the Gen12
in the inflorescence
Gen12 were were
inflorescence observed
ob-
under C5O5
served underdrying conditions
C5O5 drying (Figure(Figure
conditions 7). Specifically, α-pinene
7). Specifically, concentration
α-pinene increased
concentration in-
creased by 7–37%, while (−)-β-pinene concentration increased by 8–46% (Figure 7a,b and
Table 2). Regarding these two specific terpenes, C5O5 drying conditions revealed statisti-
cally significant differences when compared to the other drying conditions and t0 (Figure
7a,b and Table 2). β-myrcene lost 16–30% of its initial concentration under atm, N2, and
open-air drying conditions, whereas drying under C5O5 drying conditions yielded a 15%
Plants 2024, 13, 1049 14 of 22

by 7–37%, while (−)-β-pinene concentration increased by 8–46% (Figure 7a,b and Table 2).
Regarding these two specific terpenes, C5O5 drying conditions revealed statistically signif-
icant differences when compared to the other drying conditions and t0 (Figure 7a,b and
Table 2). β-myrcene lost 16–30% of its initial concentration under atm, N2 , and open-air
drying conditions, whereas drying under C5O5 drying conditions yielded a 15% relative
concentration increase (Figure 7c and Table 2). Consequently, C5O5 drying conditions
were the best for β-myrcene and (−)-β-pinene preservation in both studied chemovars
(Figures 4 and 7 and Table 2). d-Limonene exhibited a relative concentration reduction
ranging from 15–58%, with the smallest reduction observed under C5O5 drying conditions
(not statistically significant from t0 , Figure 7d and Table 2). In contrast, linalool concentra-
Plants 2024, 13, x FOR PEER REVIEW 15 of 23
tion remained stable throughout the drying process, except under C5O5 drying conditions,
which led to a notable 70% relative increase (Figure 7e and Table 2).

Figure 7. Mean monoterpene

monoterpeneconcentrations
concentrations(in DW%,y-axis,
(inDW%, y-axis,for
foreach
each drying
drying procedure
procedure and
and t0 ; tn0; =n 5)
=
5)
of of
thethe Gen12
Gen12 chemovar
chemovar determinedunder
determined underfourfourdifferent
differentdrying
dryingconditions
conditionsby byGC/MS:
GC/MS: controlled
atmospheric
atmospheric conditions
conditions (atm),
(atm), controlled N22≥≥99%
controlled N 99%conditions
conditions (N(N2), controlled CO2 5%/O2 5%/N2 90%
2 ), controlled CO2 5%/O2 5%/N2
conditions (C5O5), and open-air drying process used as a reference system
90% conditions (C5O5), and open-air drying process used as a reference system (air). (air). (a) α-pinene,
(a) α-pinene, (b)
(−)-β-pinene, (c) β-myrcene, (d) d-limonene and (e) linalool. Statistical significance between the dif-
(b) (−)-β-pinene, (c) β-myrcene, (d) d-limonene and (e) linalool. Statistical significance between
ferent drying conditions and t0 for each compound was calculated using one-way ANOVA followed
the different drying conditions and t0 for each compound was calculated using one-way ANOVA
by Tukey’s post hoc test (p value < 0.0332 (*), p < 0.0021 (**), p < 0.0002 (***), p < 0.0001 (****)).
followed by Tukey’s post hoc test (p value < 0.0332 (*), p < 0.0021 (**), p < 0.0002 (***), p < 0.0001 (****)).
In the Gen12 chemovar, the concentration of seven of the fourteen sesquiterpenes,
In the Gen12 chemovar, the concentration of seven of the fourteen sesquiterpenes,
namely β-caryophyllene, α-humulene, (−)-guaiol, (−)-α-bisabolol, eudesma-3,7(11)-diene,
namely β-caryophyllene, α-humulene, (−)-guaiol, (−)-α-bisabolol, eudesma-3,7(11)-diene,
γ-eudesmol,
γ-eudesmol, and andβ-eudesmol,
β-eudesmol,increased
increased during
during thethe
drying process
drying (Figure
process 8a–g).
(Figure Notably,
8a–g). No-
the open-air drying conditions produced a greater increase in their concentrations
tably, the open-air drying conditions produced a greater increase in their concentrations com-
pared
comparedto alltocontrolled atmosphere
all controlled drying
atmosphere conditions
drying (Figure
conditions 8a–g).8a–g).
(Figure Among these seven
Among these
sesquiterpenes, β-caryophyllene and α-humulene demonstrated
seven sesquiterpenes, β-caryophyllene and α-humulene demonstrated the smallest the smallest relativerela-
in-
crease (21%) (21%)
tive increase underunder
open-air dryingdrying
open-air conditions (Table(Table
conditions 2). These two sesquiterpenes
2). These also
two sesquiterpenes
exhibited similar drying patterns under the other conditions examined,
also exhibited similar drying patterns under the other conditions examined, reinforcing reinforcing the
observed strong correlation between them (Figure 8a,b and Table 2).
the observed strong correlation between them (Figure 8a,b and Table 2). The other five The other five ses-
quiterpenes
sesquiterpenes previously mentioned
previously mentionedexhibited a relative
exhibited increase
a relative ranging
increase fromfrom
ranging 42–59% un-
42–59%
der open-air drying conditions (Figure 8c–g and Table 2). In contrast, the
under open-air drying conditions (Figure 8c–g and Table 2). In contrast, the highest rel- highest relative
increase observed
ative increase for these
observed for sesquiterpenes under under
these sesquiterpenes all controlled atmosphere
all controlled drying drying
atmosphere condi-
tions was 33% (Figure 8c–g and Table 2). Under open-air drying conditions, the concen-
tration of bulnesol remained similar to t0, whereas all controlled atmosphere drying con-
ditions resulted in a relative concentration increase of 30–39% (Figure 8h and Table 2). In
the case of γ-gurjunene, β-eudesmene, α-selinene, β-bisabolene, and cis-α-bisabolene, the
highest concentrations were achieved under N2 drying conditions (19–30% relative in-
crease, Figure 8i,k–n). γ-elemene was the only sesquiterpene that displayed a concentra-
Plants 2024, 13, 1049 15 of 22

conditions was 33% (Figure 8c–g and Table 2). Under open-air drying conditions, the
concentration of bulnesol remained similar to t0 , whereas all controlled atmosphere drying
conditions resulted in a relative concentration increase of 30–39% (Figure 8h and Table 2).
In the case of γ-gurjunene, β-eudesmene, α-selinene, β-bisabolene, and cis-α-bisabolene,
the highest concentrations were achieved under N2 drying conditions (19–30% relative
increase, Figure 8i,k–n). γ-elemene was the only sesquiterpene that displayed a concentra-
tion reduction, ranging between 8–18%, across all drying conditions (Figure 8j and Table 2).
Notably, the γ-elemene concentration reduction observed under open-air drying conditions
was the only one to show a statistically significant difference when compared to t0 (Figure 8j
and Table 2).
An analysis of the overall monoterpene content in the Gen12 chemovar revealed
that all drying conditions yielded a relatively stable monoterpene content, with a relative
statistically non-significant decrease of only 3–14% (Figure 9a). Notwithstanding, under
C5O5 drying conditions, a notable relative increase of 26% in the total monoterpene content
was observed as compared to t0 (Figure 9a). This increase was also statistically significant
in comparison to all other drying conditions (Figure 9a). Differences of up to 0.38 DW%
in monoterpene content were observed across various drying conditions, which could
potentially influence the overall aroma of the inflorescence (Figure 9a).
Taken altogether, the sesquiterpene content increased by 15–23% across all tested
drying conditions (Figure 9b and Table 2).
In both the Gen12 and 240 chemovars, it was observed that monoterpenes were more
sensitive to the drying process than sesquiterpenes, which could be explained by the higher
monoterpene volatility as compared to the sesquiterpenes [35].
Regarding the total terpene content in the Gen12 chemovar, there was an increase
during the drying process (ranging from 0.34–0.75 DW%), with C5O5 drying conditions
being the only ones statistically significant compared to t0 (Figure 9c and Table 2). The
higher total terpene content under C5O5 drying conditions was largely attributed to the
increase in monoterpene content and the exceptional preservation of β-myrcene in this
chemovar (Figures 7 and 9).
The present findings underscore the significant potential of employing drying pro-
cesses tailored to specific chemovars, in order to optimally preserve the aroma and cannabi-
noid components as well as reduce mold infestation. In the 240 chemovar, the best drying
conditions for both cannabinoid and sesquiterpene preservation were under N2 drying con-
ditions, while in the Gen12 chemovar, all drying conditions were suitable for cannabinoid
preservation and C5O5 drying conditions were optimal for the overall terpene preservation.
Drying under controlled atmosphere conditions provides a faster alternative to tra-
ditional methods while preserving the total terpene content. However, it can modify the
concentrations of specific terpenes, potentially impacting the overall aroma profile. The
alterations observed in terpene concentrations during the drying process are attributed
to degradation, evaporation, and oxidation phenomena [29]. Uziel et al. observed differ-
ences in terpene preservation when comparing traditional drying methods to microwave
drying at 40 ◦ C [19]. These observations align with our findings and with findings from
other drying techniques such as freeze-drying, convection drying, and vacuum-microwave
drying [19,28,29]. While hot air drying is effective in retaining CBD content, it can lead to a
significant reduction in terpene levels, by 80–90%, particularly at higher temperatures [24].
Moreover, infrared or microwave drying can led to a 75% reduction in total volatile content,
specifically due to massive monoterpene degradation during drying [26]. Our study high-
lights the importance of minimizing drying duration while simultaneously maintaining
both cannabinoid and volatile terpene concentrations.
In summary, significant chemical alterations occur in cannabis inflorescences post-
harvest, with changes during drying potentially exceeding those in the month before
harvest, as suggested by prior studies [36,37]. Given the shorter duration of drying com-
pared to flowering, it is crucial for growers to account for post-harvest changes.
Plants 2024,
Plants 13, x1049
2024, 13, FOR PEER REVIEW 1616ofof23
22

Figure
Figure8.8.MeanMeansesquiterpene
sesquiterpene concentrations
concentrations (in(in
DW%,
DW%, y-axis, forfor
y-axis, each drying
each procedure
drying procedure andand
t0; n t=0 ;
5) of the Gen12 chemovar measured under the four different drying conditions
n = 5) of the Gen12 chemovar measured under the four different drying conditions by GC/MS: by GC/MS: con-
trolled atmospheric conditions (atm), controlled N2 ≥ 99% conditions (N2), controlled CO2 5%/O2
controlled atmospheric conditions (atm), controlled N2 ≥ 99% conditions (N2 ), controlled CO2
5%/N2 90% conditions (C5O5), and open-air drying process used as a reference system (air). (a) β-
5%/O2 5%/N2 90% conditions (C5O5), and open-air drying process used as a reference system
caryophyllene, (b) α-humulene, (c) (−)-guaiol, (d) (−)-α-bisabolol, (e) eudesma-3,7(11)-diene, (f) γ-
(air). (a) β-caryophyllene,
eudesmol, (g) β-eudesmol,(b) bulnesol, (i) (c)
(h)α-humulene, (−)-guaiol,(j)(d)γ-elemene,
γ-gurjunene, (−)-α-bisabolol, (e) eudesma-3,7(11)-
(k) β-eudesmene, (l) α-se-
diene, (f) γ-eudesmol, (g) β-eudesmol, (h) bulnesol, (i) γ-gurjunene, (j) γ-elemene,
linene, (m) β-bisabolene and (n) cis-α-bisabolene. Statistical significance between the (k) different
β-eudesmene,dry-
(l) α-selinene,
ing conditions(m) t0 for each and
andβ-bisabolene (n) cis-α-bisabolene.
compound was calculated Statistical
using significance
one-way ANOVAbetweenfollowed
the different
by
drying conditions
Tukey’s and
post hoc test (p tvalue
0 for each compound
< 0.0332 was calculated
(*), p < 0.0021 using(***),
(**), p < 0.0002 one-way ANOVA
p < 0.0001 followed by
(****)).
Tukey’s post hoc test (p value < 0.0332 (*), p < 0.0021 (**), p < 0.0002 (***), p < 0.0001 (****)).
 tistically non-significant decrease of only 3–14% (Figure 9a). Notwithstanding, under
C5O5 drying conditions, a notable relative increase of 26% in the total monoterpene con-
tent was observed as compared to t0 (Figure 9a). This increase was also statistically signif-
icant in comparison to all other drying conditions (Figure 9a). Differences of up to 0.38
Plants 2024, 13, 1049 DW% in monoterpene content were observed across various drying conditions, which 17 of 22
could potentially influence the overall aroma of the inflorescence (Figure 9a).

Figure 9.
Figure Meantotal
9. Mean totalterpene
terpenecontent
content(in(inDW%, y-axis,
DW%,y-axis, forfor each
each drying
drying procedure
procedure andand
t0; nt0=; n5)=of5)the
of
the Gen12
Gen12 chemovar
chemovar measured
measured under under the different
the four four different
dryingdrying conditions
conditions by GC/MS:
by GC/MS: controlledcontrolled
atmos-
pheric conditions
atmospheric (atm), (atm),
conditions controlled N2 ≥ 99%
controlled N2 conditions (N2), controlled
≥ 99% conditions CO2 5%/O
(N2 ), controlled CO225%/N
5%/O 2 90%
2 5%/N con-2
ditions (C5O5), and
90% conditions open-air
(C5O5), anddrying process
open-air used
drying as a reference
process used assystem (air). (a)
a reference total monoterpenes,
system (air). (a) total
(b) total sesquiterpenes
monoterpenes, (b) total and (c) total terpenes.
sesquiterpenes and (c)Statistical significance
total terpenes. between
Statistical the different
significance between dryingthe
conditions and t0 for each compound was calculated using one-way ANOVA followed by Tukey’s
different drying conditions and t0 for each compound was calculated using one-way ANOVA
post hoc test (p value < 0.0332 (*), p < 0.0021 (**), p < 0.0002 (***), p < 0.0001 (****)).
followed by Tukey’s post hoc test (p value < 0.0332 (*), p < 0.0021 (**), p < 0.0002 (***), p < 0.0001 (****)).

Taken altogether,
3. Materials and Methods the sesquiterpene content increased by 15–23% across all tested
drying conditions (Figure 9b and Table 2). In both the Gen12 and 240 chemovars, it was
3.1. Chemicals
observed that monoterpenes were more sensitive to the drying process than sesquiter-
Acetonitrile, anhydrous ammonium formate, ethanol, and formic acid were obtained
penes, which could be explained by the higher monoterpene volatility as compared to the
from Sigma-Aldrich (HPLC grade, Saint Louis, MO, USA). Ultra-pure water was provided
sesquiterpenes
by the Milli-Q [35].
Plus system (Millipore Corp., Billerica, MA, USA). Cannabinoid analyti-
Regardingwere
cal standards the purchased
total terpene fromcontent
RESTEKin the Gen12 chemovar,
(RESTEK, Bellefonte,there was an
PA, USA): increase
cannabidi-
during the drying process (ranging from 0.34–0.75 DW%), with C5O5
varinic acid (CBDVA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabigerol drying conditions
being
(CBG),the only ones
cannabidiol statistically
(CBD), significant compared to t0 (Figure
(−)-∆9-trans-tetrahydrocannabivarinic acid 9c and Table
(THCVA), 2). The
cannabinol
higher total terpene content under C5O5 drying conditions was
(CBN), (−)-∆9-trans-tetrahydrocannabinol (∆-9-THC), (−)-∆8-trans-tetrahydrocannabinol largely attributed to the
increase in monoterpene content and the exceptional preservation
(∆-8-THC), (−)-∆9-trans-tetrahydrocannabinolic acid (THCA), and cannabichromenic acid of β-myrcene in this
chemovar
(CBCA). Each (Figures 7 andstandards
of those 9). was obtained at a stock concentration of 1000 µg/mL
The
except present
CBLA, which findings underscore
was obtained the significant
at a stock concentrationpotential
of 500of employing
µg/mL. Terpenedrying pro-
standard
cesses
mix, at a stock concentration of 2500 µg/mL from each terpene, and which contains thecan-
tailored to specific chemovars, in order to optimally preserve the aroma and fol-
nabinoid components as well
lowing terpenes—α-pinene, as reduce(−
camphene, mold infestation.
)-β-pinene, In the 240
β-myrcene, chemovar,
δ-3-carene, the best
α-terpinene,
drying
p-cymene, conditions
d-limonene, for both cannabinoid
ocimene, andterpinolene,
γ-terpinene, sesquiterpene preservation
linalool, were under
(−)-isopulegol, geraniol,N2
drying conditions,α-humulene,
β-caryophyllene, while in the nerolidol,
Gen12 chemovar,
(−)-guaiol, all drying conditions were suitable
and (−)-α-bisabolol—was obtainedfor
cannabinoid
from RESTEKpreservation and C5O5PA,
(RESTEK, Bellefonte, drying
USA).conditions were optimal for the overall ter-
pene preservation.
3.2. Plant
Drying Material
under controlled atmosphere conditions provides a faster alternative to tra-
ditional methods
Fresh medicinal while preserving
Cannabis sativathe
L. total
female terpene content. However,
inflorescences it can modify
from two different commer- the
concentrations
cially available of specific terpenes, potentially
chemovars—namely, “Gen12” (hybridimpacting the overall
chemovar) and aroma profile. The
“240” (high-THCA
alterations
chemovar)—wereobserved in terpene
provided byconcentrations
the Barlev farm during the and
in May drying process
October are attributed
2023, respectively to
degradation, evaporation,
(Bar-Lev Agricultural Crops, and oxidation
Kfar 32◦ 15′ 21.2
phenomena
Hess, Israel, ′′ ]N
[29 34◦ 57et
. Uziel ′ 01.0
al.′′ observed
E). differ-
encesBoth
in terpene
chemovarspreservation
represent when comparing
prevalent traditional
chemovars drying
in Israel methods toand
(high-THCA microwave
hybrid).
Furthermore,
drying at 40 °Cboth chemovars
[19]. were accessible
These observations align towithus throughout
our findingsthe and duration of the study.
with findings from
Both chemovars were analyzed for their cannabinoid and terpene content at the Agricultural
Research Organization, the Department of Food Science, Israel. The cannabis inflorescence,
which harbors significant concentrations of cannabinoids and terpenes, is the only part of
the plant utilized for both medical and recreational purposes [1,6]. Consequently, it is the
focal point of analysis in all pertinent studies within this domain. The sampling method
employed in our study aligns with the most widely accepted procedures among researchers
and industry practitioners [1,6].
Plants 2024, 13, 1049 18 of 22

3.3. Drying Process

Controlled atmosphere drying chambers were used for the initial drying and curing
processes of the fresh cannabis inflorescences. Three different drying environments were
examined using the controlled atmosphere chambers—controlled atmospheric conditions
(atm), controlled CO2 5%/O2 5%/N2 90% conditions (C5O5), and controlled N2 ≥ 99%
conditions (N2 ). To attain a relative humidity of less than 10%, we used gases with humidity
levels below 0.1%. Additionally, within the controlled atmosphere drying chambers,
we inserted 500 g dried silica gel pearls in each chamber (Drying pearls orange, Merck,
Darmstadt, Germany) to absorb the moisture during the drying process. The temperature
in the drying chambers and open air was set to 15 ◦ C, within the commonly used drying
temperature range of 15–21 ◦ C [1]. The controlled atmosphere drying chambers restored
the desired drying and curing conditions within the chambers every 30 min. After six days
the cannabis inflorescences from both chemovars were completely dry, since no additional
inflorescence weight loss was observed (the weight change was within the error range of the
water content of 10% ± 1% for the dried inflorescence) and therefore no additional curing
step was needed [26]. As a reference system, an open-air drying process was employed for
14 days, followed by one day of curing within a confined breathable tray in the presence
of 500 g dried silica gel pearls. This reference system imitated the commonly applied
slow industrial drying procedure during the first 14 days of drying. During the drying
procedure, all of the cannabis inflorescences were placed in breathable trays within the
controlled atmosphere chambers, while the open-air reference samples were placed in
an air-ventilated room containing 50–55% relative humidity. In order to determine the
water content of the cannabis inflorescence samples, three plates containing weighted
cannabis inflorescences from each chemovar were placed in the drying chambers and the
weight was recorded before, during, and after drying. Each plate contained three cannabis
inflorescences weighing 2–4 g. For calculating the cannabinoid and terpene content, the
fresh cannabis inflorescences weight at the initial time point (t0 ) was normalized to dry
weight using the average weight loss for each chemovar.

3.4. Sample Preparation

Fresh or dried cannabis inflorescences from the 240 and Gen12 chemovars were ground
homogenously with a mortar and pestle in the presence of liquid nitrogen, providing
5 replicates from each treatment group per chemovar. The homogenously ground cannabis
samples (500 ± 0.5 mg for fresh inflorescences and 100 ± 0.1 mg for dried inflorescences)
were extracted with 4 mL of ethanol in 15 mL Falcon tubes and shaken (Digital Orbital
Shaker, MRC, Holon, Israel) in the dark for 15 min at 500 rpm. One mL of the extract
was transferred to an Eppendorf tube and centrifuged for 4 min at 12,000 rpm. For the
determination of cannabinoid levels, a dilution of 1:11 of the supernatant with ethanol
was carried out, and 1 mL aliquot was transferred to an HPLC vial and subjected to
high-pressure liquid chromatography–photodiode arrays (HPLC–PDA) analysis. For the
determination of terpene levels, 0.25 mL of the supernatant was inserted into a GC vial and
analyzed via gas chromatography–mass spectroscopy (GC/MS).

3.5. Quantification of Cannabinoids by HPLC–PDA and Terpenes by GC/MS

The ethanolic cannabis extracts were analyzed as described in Birenboim et al., utiliz-
ing HPLC–PDA (Acquity Arc FTN-R; Model PDA-2998, Waters Corp., Milford, MA, USA)
equipped with Kinetex® 1.7 µm XB-C18 100 A LC column (150 × 2.1 mm i.d. and 1.7 µm
particle size; Phenomenex, Torrance, CA, USA) for the cannabinoids analysis [18]. The
cannabinoids were quantified by comparing the integrated peak area with the correspond-
ing cannabinoid calibration curve ranging from 1 to 1000 µg/mL (Table S1) [18].
The terpene analysis was carried out by GC/MS (Agilent, Santa Clara, CA, USA) as
recently reported by Birenboim et al. utilizing a DB-5 capillary column (5% phenyl, 95%
dimethylpolysiloxane, 30 m × 0.250 mm, 0.25 m; Agilent, Santa Clara, CA, USA) for analyte
separation [18]. The terpenes were quantified by comparing the integrated peak area with
Plants 2024, 13, 1049 19 of 22

the corresponding terpene calibration curve ranging from 0.5 to 250 µg/mL (Table S2) [18].
The methods’ analytical validation parameters (i.e., R2 , limit of detection, limit of quantifi-
cation, repeatability, and accuracy) were recently published in Birenboim et al. [18].

3.6. Microbiological Assay

Wet inflorescence at t0 , dry inflorescence from CA drying chambers at day 6, and
wet open-air inflorescence at day 12 of the 240 chemovar were tested for microbiological
contamination using the colony forming unit (CFU) of total yeasts and molds and real-time
PCR. For total yeasts and molds analysis, fresh or dried cannabis inflorescences from the
240 chemovar were ground homogenously with a mortar and pestle in the presence of liquid
nitrogen, providing three replicates from each sample. For the CFU test, 50 ± 0.1 mg of
the homogenously ground cannabis samples were inserted into 1 mL of sterilized distilled
water solution in a 1.5 mL Eppendorf tube and vortexed for 10–15 s. Thereafter, 100, 1000,
and 10,000 times dilutions of the solutions were prepared. Subsequently, 100 µL of the
diluted solutions were spread on Potato Dextrose Agar (PDA) plates supplemented with
0.025% chloramphenicol and incubated at room temperature for 4 days to develop yeasts
and molds. The microbial count of each plate was then reported as the CFU per dry gram
of each sample (CFU/g dry).
The relative fungal biomass was determined as described previously in Li et al. [38].
Briefly, each cannabis inflorescence was ground homogenously with a mortar and pestle in
the presence of liquid nitrogen. From each inflorescence, three samples of approximately
50 mg were taken for DNA extraction using Wizard® genomic DNA purification kits
(Promega, Madison, WI, USA) according to the manufacturer’s instructions. DNA quantity
and quality were determined by the NanoDrop One (Thermo Fisher Scientific, Waltham,
MA, USA) spectrophotometer. The extracted DNA was diluted for 10 ng/µL and 1 ng/µL
for further analysis. The relative biomass of Botrytis cinerea and Alternaria alternata was
evaluated by an RT-qPCR analysis conducted with a Step One Plus Real-Time PCR (Applied
Biosystems, Waltham, MA, USA). PCR amplification was performed with 2.5 µL of a
diluted DNA template (10 ng/µL) in a 10 µL reaction mixture containing 5 µL Syber
Green (Applied Biosystems, Waltham, MA, USA) and 250 nM primers. The qRT-PCR
analysis was conducted with the corresponding primer sets of the selected fungi: forward,
5′ -TGCTCCAGAAGCTTTGTTCCAA-3′ , and reverse, 5′ -TCGGAGATACCTGGGTACATAG-
3′ , for the B. cinerea actin gene, and forward, 5′ -TTGGACTGCTCTAGCCTGGT-3′ , and
reverse, 5′ -GTCAAACACGTGCGATAACC-3′ , for the A. alternata actin gene. The PCR
cycling program included: 10 min at 94 ◦ C, followed by 40 cycles at 94 ◦ C for 10 s, 60 ◦ C for
15 s, and 72 ◦ C for 20 s. The relative biomass of the selected genes was normalized using
Ct values of the Cannabis 18S rRNA gene (forward, 5′ -TTCGTCCTCCCCCAAAAGT-3′ ,
and reverse, 5′ -CCGAGCGTTTTGTTCTTTCG-3′ ) as the reference gene, and expression
values were calculated relatively to the uninfected sample using Step One software v2.2.2
(Applied Biosystems, Waltham, MA, USA). Each treatment consisted of three biological
repeats and three technical replicates.

3.7. Statistical Analysis and Cannabinoid/Terpene Content Calculations

For each compound analyzed, one-way ANOVA followed by Tukey’s post hoc test
was used to determine the differences in cannabinoid and terpene concentrations between
the different drying conditions and t0 , for both chemovars, at α = 0.05 using GraphPad
PRISM 10 (San Diego, CA, USA).
Relative concentration was defined as the dry concentration at any specified time (in
DW%) divided by the concentration at t0 (normalized to DW%) and was calculated for
each compound as described in Equation (1).

DW% after drying

Relative concentration = (1)
normalized DW% at t0
Plants 2024, 13, 1049 20 of 22

Total THC and total CBD content were calculated according to Equations (2) and (3),
respectively [24].

total THC [DW%] = (THCA DW%) ∗ 0.877 + THC DW% (2)

total CBD [DW%] = (CBDA DW%) ∗ 0.877 + CBD DW% (3)

4. Conclusions
This study explored the impact of varying fast drying conditions on the chemical
composition of cannabis inflorescences. The drying environment notably affected the
chemical composition of cannabis inflorescences. Compared to traditional methods, con-
trolled atmosphere chambers reduced the drying and curing time by at least 60%, without
reducing the total volatile terpene content and without encouraging mold growth. On the
other hand, inflorescences from the 240 chemovar subjected to traditional drying condition
were highly infested by Alternaria alternata and also revealed low infestation of Botrytis
cinerea; consequently, they are less ideal for routine commercial use. The different drying
conditions employed in the present study affected the cannabinoid composition in both
chemovars differently. In the 240 chemovar, controlled N2 and atm drying conditions were
able to preserve THCA levels comparable to t0 , while in the Gen12 chemovar all of the
employed drying conditions preserved THCA and CBDA content. On the other hand, in
both chemovars, open-air drying conditions resulted in a larger extent of decarboxylation
of the major cannabinoids as compared to the controlled atmosphere drying conditions,
resulting in 3–8-fold higher THC and CBD concentrations compared to t0 . A decrease in
CBGA concentration was observed in both chemovars, and the lowest CBGA concentration
after the drying process was observed under open-air drying conditions.
Regarding the aroma components, C5O5 drying conditions were optimal for pre-
serving monoterpenes. On the other hand, N2 drying conditions were the only drying
conditions yielding a statistically significant increase in sesquiterpene content in both
chemovars.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/plants13071049/s1, Table S1: Analytical parameters of cannabinoids
analyzed by HPLC-PDA; Table S2: Analytical parameters of terpenes analyzed by GC/MS.
Author Contributions: Conceptualization, M.B., D.K. and J.A.S.; methodology, M.B., D.K. and J.A.S.;
validation, D.K. and J.A.S.; formal analysis, M.B.; investigation, M.B., N.B., D.D.-A., D.M., D.K. and
J.A.S.; resources, D.C., D.K. and J.A.S.; data curation, M.B.; writing—original draft preparation, M.B.;
writing—review and editing, D.K. and J.A.S.; visualization, M.B.; supervision, D.K. and J.A.S.; project
administration, D.K. and J.A.S.; funding acquisition, D.K. and J.A.S. All authors have read and agreed
to the published version of the manuscript.
Funding: This research was funded by the Israeli Ministry of Agriculture grant number 421046020.
The funders had no role in the study design, data collection, and analysis, decision to publish, or
preparation of the manuscript.
Data Availability Statement: Data will be made available on request.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
Controlled atmosphere (CA); (−)-∆9-trans-tetrahydrocannabinolic acid (THCA); cannabidiolic
acid (CBDA); (−)-∆9-trans-tetrahydrocannabinol (THC); cannabidiol (CBD); dry weight% (DW%);
cannabidivarinic acid (CBDVA); cannabigerolic acid (CBGA); cannabigerol (CBG); (−)-∆9-trans-
tetrahydrocannabivarinic acid (THCVA); cannabinol (CBN); (−)-∆8-trans-tetrahydrocannabinol (∆-8-
THC); cannabichromenic acid (CBCA); (−)-∆9-trans-tetrahydrocannabiorcolic-C4 acid (THCA-C4);
controlled atmospheric conditions (atm); controlled CO2 5%/O2 5%/N2 90% conditions (C5O5);
controlled N2 ≥ 99% conditions (N2 ); initial time point (t0 ); high-pressure liquid chromatography–
Plants 2024, 13, 1049 21 of 22

photodiode arrays (HPLC–PDA); gas chromatography–mass spectroscopy (GC/MS); weight% (wt%);

colony forming unit (CFU).

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