LWT - Food Science and Technology 165 (2022) 113718

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LWT
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Essential oil obtained from Thlaspi arvense L. leaves and seeds using
microwave-assisted hydrodistillation and extraction in situ by vegetable oil
and its antifungal activity against Penicillium expansum
Ru Zhao a, b, Ailing Ben b, Mengxia Wei a, c, Ming Ruan b, Huiyan Gu d, **, Lei Yang a, e, *
a
Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin, 150040, China
b
Key Laboratory of Quality and Safety of Agricultural Products of Nanjing, Nanjing Xiaozhuang University, Nanjing, 211171, China
c
State Key Laboratory of Esophageal Cancer Prevention & Treatment and Henan Key Laboratory for Esophageal Cancer Research of the First Affiliated Hospital,
Zhengzhou University, Zhengzhou, 450052, China
d
School of Forestry, Northeast Forestry University, Harbin, 150040, China
e
Heilongjiang Provincial Key Laboratory of Ecological Utilization of Forestry-based Active Substances, Harbin, 150040, China

A R T I C L E I N F O A B S T R A C T

Keywords: An improved approach was developed to obtain essential oil from Thlaspi arvense L. leaves (TAL) and seeds (TAS)
Essential oil using microwave-assisted hydrodistillation and extraction in situ by vegetable oil. Some potential parameters that
Microwave-assisted distillation and extraction affected the yield of essential oils were systematically investigated. Under the obtained optimization conditions,
in situ
the maximized yields of TAL and TAS essential oils were 0.77 ± 0.02 mg/g and 2.69 ± 0.08 mg/g, respectively.
Vegetable oil
The results analyzed by GC–MS revealed that the main component of TAS essential oil was allyl isothiocyanate
Antifungal activity
Penicillium expansum. (AITC), which accounted for 44.69% of the total essential oil content. The proportion of AITC in TAL essential oil,
however, was only 9.66%. Results showed that they all had significant inhibition on P. expansum, with AITC
exhibiting the greatest inhibition on P. expansum. The study of antifungal activity indicates that AITC has po­
tential application value and broad application prospects in food preservation-related fields in the future.

1. Introduction
Penicillium expansum has a wide host range and is one of the most
common and riskiest fungal pathogens, leading to several critical dis­
eases of post-harvest fruits and vegetables, such as apples, pears,
strawberries, avocados, mangos and tomatoes (Frisch, Mann, Marek, &
Niessen, 2021). It produces resistant, asexual spores during reproduc­
tion, which increases the challenges to its elimination (Ma et al., 2020).
During the past decades, due to the toxic effects of metabolites produced
by P. expansum to human health and its impact on economic losses, some
preventive strategies to prevent the contamination of these mycotoxins
have attracted widespread attention. Currently, synthetic fungicides and
preservatives are considered the main methods to control fungal rot in
post-harvest fruits and vegetables (Ge et al., 2019; He, Zhang, Li, Xu, &
Tian, 2019). However, it has been reported that they also significantly
increase the resistance of mildew, which is a potential threat to human
health and the environment (Fisher, Hawkins, Sanglard, & Gurr, 2018).
Therefore, in order to deal with the above food safety problems, it is
necessary and urgent to find an environmentally friendly and non-toxic
natural product.
Essential oils are natural and potential substitute value for the pre­
vention of pests and diseases of fruits and vegetables, mainly because
essential oils have the advantages of low cost, environmental friendli­
ness, biodegradability, easy decomposition and low toxicity (Soliman &
IBadea, 2002; Xiao et al., 2021). In recent years, essential oils are
commonly encapsulated into nanoparticles, microcapsules, nano­
emulsions and biodegradable films for the preservation of foods (Liu
et al., 2017; Sani, Geshlaghi, Pirsa, & Asdagh, 2021; Wen et al., 2016).
Thlaspi arvense L., native to North America and Europe, is an annual herb
of Cruciferae (Moser, Shah, Winkler-Moser, Vaughn, & Evangelista,
2009). T. arvense L. contains several active components, such as essential
oils, flavonoids, glucosinolates and proteins. Among these, essential oils
are worthy of paying more attention to because the hydrolysate products
of glucosinolates have biological activities (Zhao et al., 2021). The

* Corresponding author. Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin, 150040, China.
** Corresponding author.
E-mail addresses: ghuiyan@nefu.edu.cn (H. Gu), yanglei@nefu.edu.cn (L. Yang).

https://doi.org/10.1016/j.lwt.2022.113718
Received 10 April 2022; Received in revised form 21 June 2022; Accepted 26 June 2022
Available online 28 June 2022
0023-6438/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
R. Zhao et al. LWT 165 (2022) 113718

extraction of essential oils from T. arvense L. leaves (TAL) and seeds

(TAS) and the study of their antifungal activity on Penicillium dilatatum
could lay a theoretical foundation for the prevention of spoilage of
harvested fruits and vegetables.
Traditionally, steam distillation, hydrodistillation methods are
commonly used for the isolation essential oils extracted from plants
(Golmakani & Rezaei, 2008). However, several studies have reported
that these traditional methods have some disadvantages (Desai, Parikh,
& De, 2014; Spadi et al., 2021). Recently, several emerging technolo­
gies, such as supercritical fluid extraction (Zhao & Zhang, 2014),
microwave-assisted hydrodistillation (Gonzalez-Rivera et al., 2021),
microwave-assisted steam distillation (Xiao et al., 2021) and
ohmic-assisted hydrodistillation (Manouchehri, Saharkhiz, Karami, &
Niakousari, 2018), have been applied to obtain essential oils due to the
obvious shortcomings of some traditional methods. Of these processes,
microwave-mediated heating is a promising technique that has recently
been applied to obtain various essential oils from plants (Yingngam
et al., 2021). The wide use of microwave-mediated heating for obtaining
volatile compounds is attributed to its desirable advantages, such as
shorter distillation time, convenient operating condition, faster heating
speed and higher product quality (Marković et al., 2018). However, the
inner cavity size of microwave oven is limited, which can only accom­
modate 1–2 L of round bottom flasks. Thus, for many plant species with
lower content of essential oil, only tens of microliters of essential oil can
be obtained in a single experiment of hydrodistillation. It is difficult to
quantify such a trace amount of essential oil for researchers who need
Fig. 1. Schematic diagram of microwave-assisted vegetable oil in-situ extrac­
process parameters optimized. Therefore, how to quickly and relatively
tion apparatus.
accurately quantify the trace of essential oils is an objective technical
bottleneck. For this bottleneck, Some researchers add certain volatile
organic solvents such as n-hexane, dichloromethane or diethyl ether to 2. Materials and methods
the tube of Clevenger-Type Essential Oil Extractor (Chen et al., 2018;
Ferhat, Tigrine-Kordjani, Chemat, Meklati, & Chemat, 2007). Essential 2.1. Materials and chemicals
oil in hydrogel are extracted into an organic solvent by Liquid-liquid
extraction. However, these petroleum-based solvents (n-hexane, Leaves and seeds of T. arvense were bought from Hebei materials
dichloromethane or diethyl ether) have certain toxicity, biological in­ wholesale market. They were crushed with a plant crusher, graded with
compatibility, the health of operators is susceptible to the environment, 60 mesh sieve and then stored in a sealed desiccator. The same batch of
and the environmental hazard may also be caused. As reported in the raw materials was used to extract the essential oils. P. expansum was
literature (Li, Fabiano-Tixier, Ginies, & Chemat, 2014; Yara-Varón et al., purchased from the China Center of Industrial Culture Collection (strain
2017), Chemat’s team attempted to use vegetable oil as an alternative number: CICC 40658, Beijing, China) and was isolated from rotted fruits.
solvent of the petroleum-based solvents and even for direct extraction of Six kinds of vegetable oils, including olive oil (Beijing Century
volatile aromatic compounds. However, these attempts still failed to Kangxin Trading Co., Ltd., Beijing, China), coconut oil (Shanghai Pan­
solve the quantitative problem of trace essential oil. chen Trading Co., Ltd., Shanghai, China), palm oil, palm kernel oil
In the Clevenger device, the volatile components in plant cells (Tianjin Julong Cereals and Oils Co., Ltd., Tianjin, China; Country of
become vapor with water vapor, cooled by the condenser, and extracted origin of raw materials), corn oil (Qinhuangdao Jinhai Food Industry
by vegetable oil. These volatile components consist of monotenoid, Co., Ltd., Qinhuangdao, China), rapeseed oil (Jiangxi Qinglong Hi-Tech
sewerpene and aromatic compounds, which is no non-volatile compo­ Grease Co., Ltd., Yichun, China) were purchased from Jingdong Mall
nent due to the condensate of volatile components. Our pre-experiments (Harbin, China). Allyl isothiocyanate (AITC) was purchased from Sig­
have shown that the essential oil of a plant has a similar ultraviolet ma–Aldrich Trading Co., Ltd. (Shanghai, China).
absorption spectrum, there is a specific absorption peak or shoulder
peaks. Therefore, it is feasible to use vegetable oil as a reference for the 2.2. Apparatus
quantitative determination of the content of essential oil dissolved in
vegetable oil. The extraction apparatus was designed based on the Clevenger
In the present work, the new approach of microwave-assisted apparatus and its schematic diagram is presented in Fig. 1. The appa­
hydrodistillation and vegetable oil in situ extraction (MHD-VOE) was ratus for the MHD-VOE process consisted of a reflux condenser, a Cle­
developed to obtain essential oils from TAL and TAS. In this method, venger extractor and a P70D20N1P-G5(W0) microwave oven
vegetable oil is used as a green and environmentally friendly extractant. (Guangdong Galanz Microwave Life Electric Manufacturing Co., Ltd.,
The potential parameters affecting the MHD-VOE process (the type of Shunde, China). The power of the microwave oven had a continuously
vegetable oil, oil volume, liquid-solid ratio, microwave irradiation adjustable function. The microwave irradiation frequency and the
power and microwave irradiation time) by single factor experiments and maximum output power were 2450 MHz and 700 W, respectively. The
Box–Behnken design (BBD). The composition of the obtained essential interior cavity size of the microwave oven was 195 mm × 315 mm ×
oils was analyzed by GC-MS. Finally, the antifungal activity against 350 mm. Vegetable oil was used for the organic phase and was also
P. expansum of essential oils from TAL and TAS and their main compo­ added to fill the apparatus solvent return loop.
nent of AITC was evaluated.

2
R. Zhao et al.
2.3. Microwave-assisted hydrodistillation and vegetable oil in situ
extraction (MHD-VOE) procedure
A portion of 20.0 g of TAL and TAS powders was weighed accurately
and placed into a round-bottom flask, after which a certain volume of
distilled water was added to the flask. The flask was put into the mi­
crowave oven with an essential oil extractor on it. Before distillation, a
certain volume of vegetable oil was added to the essential oil extractor.
The timer was started when the first drop of water dripped from the
lower end of the condenser and distillation was allowed to proceed for a
period of time under different microwave irradiation powers. After the
distillation process, the mixture of essential oils and the vegetable oil
were collected and then stored in the refrigerator at 4 ◦ C. Besides, the
process of microwave-assisted extraction (MAE) was the same as MHD-
VOE except adding vegetable oil.
The UV full-wavelength scans of various vegetable oils and essential
oils from the TAL and TAS were performed by a UV-5500PC UV–visible
spectrophotometer (Shanghai, China). Various vegetable oils and the
essential oils were then subjected to UV full-wavelength scanning
(210–450 nm) to select suitable vegetable oils for extracting the essen­
tial oils. In each group of single factor and BBD experiments, 200 μL of a
mixture of essential oil and vegetable oil was removed and diluted to the
right concentration with n-hexane, and then assessed at a certain
wavelength. Each experiment and the number of measurements were
repeated 3 times and the yield of essential oil was calculated according
to the following formula:
Y =
C × V ×n
(1)
m0
where Y is the yield of the essential oil, mg/g; C is the concentration of
the essential oil, mg/mL; V is the volume of the vegetable oil, mL; n is the
dilution multiple of the essential oil; and m0 is the weight of TAL and
TAS powders, g.
2.4. Conventional hydrodistillation method
The conventional hydrodistillation procedure was carried out with
reference to a previous study with slight modification (Ferhat et al.,
2007). TAL and TAS powders (20.0 g) were weighed accurately and
placed into a round-bottom flask, after which a certain volume of
distilled water was added to the flask, followed by attachment to a
Clevenger apparatus. In addition, 1.5 mL n-hexane was added to the
essential oil extractor to extract the essential oils. When the first drop of
condensed water dripped, the count started and the flask was heated
with a 600 W heating jacket for 4 h. After the distillation process, the
essential oils were collected, dehydrated and stored in the refrigerator at
4 ◦ C until analysis.
2.5. Box–Behnken design
Based on the single factor experiments, three operational parameters
including liquid-solid ratio (X1: 7.5, 10.0, 12.5 mL/g), microwave irra­
diation power (X2: 385, 540, 700 W) and microwave irradiation time
(X3: 20, 30, 40 min) were assigned to the further optimization by Box-
Benhnken design using Design-Expert 2018 (Stat-Ease Inc. MN, USA)
without any block. As showed in Table S1, the design consists of three
impact factors and a total of 17 actual experiments. Other than this, the
actual and predicted values corresponding to each set of experiments are
presented in Table S1.
2.6. Gas chromatography–mass spectrometry analysis (GC-MS)
The compositions of the separated essential oils were analyzed by a
Shimadzu GC-MS TQ8040 instrument (Shimadzu Corporation, Kyoto,
Japan). The GC section equipped with a HP-5 MS capillary column (30
3
LWT 165 (2022) 113718
mm × 250 μm × 0.25 μm film thickness). The essential oils of TAL and
TAS were diluted 100 times with chromatographic grade n-hexane,
dried with anhydrous sodium sulfate, and filtered with a 0.45 μm
microporous filter membrane before GC-MS analysis. The GC-MS oper­
ating conditions referred to the previous paper (Chen et al., 2018).
2.7. Antifungal activity against Penicillium expansum
2.7.1. Activation and cultivation of Penicillium expansum
Given that the strain has been in a dormant state over a long period of
time after lyophilization, the first strain generation requires appropriate
extension of its culture time and subsequent transfer to 2–3 generations
to regain vitality. First, 0.5 mL of liquid medium was placed into the
lyophilized tube with a sterile pipette to dissolve the lyophilized pow­
der. Then, the dissolved bacterial suspension was put into a tube with
4–5 mL of liquid medium. The 1–2 drops of residual bacterial suspension
were transferred to solid medium. The liquid test tube and the slant test
tube were incubated in a BJPX biochemical incubator (Shandong
Brocade Biological Industry Co., Ltd., Zibo, China) at 25 ◦ C. After seven
days of strain culture, an appropriate amount of mycelium was removed
using a glass rod and transferred onto petri dishes containing solid
culture medium and then placed into a biochemical incubator to
continue culturing.
2.7.2. Antifungal inhibition of TAL essential oil and TAS essential oil
Antifungal inhibition of P. expansum by TAL essential oil, TAS
essential oil and their main component, AITC, was assessed. First, TAL
essential oil, TAS essential oil or AITC was dissolved in 0.5% Tween-80
(Tween-80: 500 mg/100 mL). Then, TAL essential oil and TAS essential
oil were separately diluted in potato dextrose agar (PDA) medium to the
required concentrations (0.25, 0.50, 0.75, 1.00, 1.50, and 2.00 mg/mL),
and AITC was diluted to 25, 50, 100, 125, 250, and 500 μg/mL. A 0.8 cm
diameter P. expansum mycelium disc was removed using a sterile pipette
tip and inoculated onto the solid medium containing different concen­
trations of samples. Nystatin was added to the PDA medium, which was
considered as positive control to observe the growth of P. expansum. The
blank control had the same volume added of sterile water containing
0.5% Tween-80 to the PDA medium. Each medium was cultured for
seven days in a biochemical incubator at 25 ◦ C. The colony diameter of
P. expansum was measured three times by the cross method. The inhi­
bition rate of the sample on Penicillium expansum was calculated using
the following formula:
Anifungal inhibition (%) =
d0 − dt
(3)
d0
where d0 is the mean diameter (mm) of colony growth in the negative
control group and dt is the mean diameter (mm) of colony growth in the
experimental group.
2.7.3. Effect on the spore germination of P. expansum
The minimal inhibitory concentration (MIC) and the minimal
bactericidal concentration (MBC) were determined by the double dilu­
tion method to explore the effect of TAL essential oil, TAS essential oil
and AITC on spore germination of P. expansum. P. expansum in the
logarithmic growth phase was selected to prepare a spore suspension:
10 mL of sterile water containing 0.5% Tween-80 was added to a petri
dish; the P. expansum mycelium and spores were scraped off with a
triangular coating rod; and then the sterile water solution was placed
into an XK96-A vortex mixer (Jiangsu Xinkang Medical Equipment Co.,
Ltd., Jiangyan, China) for 30 s. Subsequently, the mycelium was filtered
by sterile filter paper, and the spore suspension was counted with a
hemocytometer of 25 × 16 type (Shanghai Qiujing Biochemical Reagent
Instrument Co., Ltd., Shanghai, China) under an optical microscope to
calculate the concentration. Then, the spore suspension was diluted with
liquid medium to between 105-106 CFU/mL. TAL essential oil, TAS
R. Zhao et al.
Fig. 2. Optimization of the yield of TAL essential oil and TAS essential oil using Box-Behnken design (BBD). Interaction of liquid-solid ratio and microwave irra­
diation power on the yield of TAL essential oil and TAS essential oil (a, d), interaction of liquid-solid ratio and microwave irradiation time on the yield of TAL
essential oil and TAS essential oil (b, e), interaction of microwave irradiation power and microwave irradiation time on the yield of TAL essential oil and TAS
essential oil (c, f).
essential oil and AITC were diluted with medium to the required con­
centrations: namely TAL essential oil and TAS essential oil concentra­
tions were 0.25, 0.50, 0.75, 1.00, 1.50, 2.00 mg/mL and AITC
concentrations were 25, 50, 100, 125, 250, 500 μg/mL 100 μL of
essential oil sample solutions of different concentrations were placed
into a 96-well plate, after which 100 μL of P. expansum suspension was
added and incubated at 25 ◦ C for 24 h to observe whether there were
white precipitates, or flocs, at the bottom of the 96-well plate. The same
volume of 0.5% Tween-80 solution and spore suspension were added to
the blank control group.
2.7.4. Time-killing curve study
The dynamic time-killing process of TAL essential oil, TAS essential
oil and AITC on P. expansum was monitored and plotted. The prepared
spore suspension of P. dilatatum was placed into test tubes. Next,
essential oil or AITC samples of different concentrations (0, 1/2 × MIC,
1 × MIC, 2 × MIC, MBC) were added and the tubes were cultured in a
biochemical incubator at 25 ◦ C. At different culturing times of 2, 4, 6, 8,
12, 16, 24 and 36 h, 50 μL of P. expansum culturing solution was
removed and diluted 10 folds. Next, 50 μL of the diluted solution was
placed onto the surface of the agar medium and subsequently incubated
in a biochemical incubator at 25 ◦ C for 24 h. Spore count was performed
with a hemocytometer. The dynamic killing curve of the sample against
P. expansum was plotted by time on the abscissa and the logarithm of
spores on the ordinate axis.
2.7.5. Scanning electron microscopy analysis of the mycelium morphology
of P. expansum
The mycelium morphology and spore growth of P. expansum were
observed by a JSM-7500F scanning electron microscope (Tokyo, Japan)
after culturing for seven days under MIC conditions. The sample was
separated from the culture medium, and the effect of each sample on the
hyphal structure and spore growth of P. expansum. The 8 mm diameter of
each P. expansum sample was separated from the culture medium
(experimental group, blank control group or positive control group) and
4
LWT 165 (2022) 113718
placed into a beaker. The process of sample preparation was performed
according to a previously reported method with slight modification
(Endo, Cortez, Ueda-Nakamura, Nakamura, & Dias, 2010). First, sam­
ples were washed with PBS buffer at pH 7.4 and then washed with sterile
water. They were fixed at room temperature for 6 h with 2.5% glutar­
aldehyde solution (for electron microscopy) and then washed with
sterile water. Finally, samples were successively dehydrated with
different concentrations of ethanol solution (30%, 50%, 70%, 90% and
100%). The dehydrated samples were air dried on a sterile operating
table. In addition, the sample from the blank control group was also
treated by placing mycelium in a desiccator for air drying, which was
considered as a comparison for observation. All samples were fixed on
the specimen holder for gold treatment.
2.8. Statistical analysis
A series of BBD experiments were designed by the Design Expert and
actually the related values of each run for BBD was expressed as an
average. All experiments were conducted for three times (n = 3), the
results were given as the mean values ± SD (standard deviations).
3. Results and discussion
3.1. Type selection of vegetable oil
The selection of vegetable oil has a greater impact on the calculation
of the yield of TAL and TAS essential oils. Therefore, six kinds of vege­
table oils (olive, coconut, palm, palm kernel, corn and rapeseed oil), TAL
and TAS essential oils were assessed by ultraviolet full-wavelength
scanning. The results and specific analysis process are shown in sup­
plementary materials. The coconut oil was selected as the solvent for
extracting TAL and TAS essential oils.
R. Zhao et al. LWT 165 (2022) 113718

Table 1
GC-MS results for the chemical compositions of essential oil extracted from Thlaspi arvense L.
No. Compounds of TAL essential oil RI ID RA d (%) Compounds of TAS essential oil RI ID RA d (%)
a b c
f g
MHD- MAE HD MHD- MAEf HDg
VOEe VOEe

1 t-Butylcyclohexane MS 4.12 4.02 3.98 t-Butylcyclohexane MS 7.12 7.22 7.25

2 3-Furaldehyde MS 0.31 - 0.28 Allyl isothiocyanate 890 RI, 44.69 44.52 44.86
MS
3 1,1-bis(Methylthio)ethane MS 0.92 1.01 0.99 4-Methylhexanal MS 0.41 - -
4 Cyclohexylmethane MS 0.33 0.32 0.35 2-Heptenal, (E)- 953 RI, 0.18 - 0.15
MS
5 Allyl isothiocyanate 890 RI, 9.66 9.41 9.45 Benzaldehyde 961 RI, 0.48 0.46 0.5
MS MS
6 Benzaldehyde 961 RI, 0.53 - 0.52 5-Ethylthiazole MS 1.22 1.13 1.02
MS
7 Furan, 2-pentyl 996 RI, 1.80 1.44 1.81 2,4-Dimethylthiazole MS 1.22 1.34 1.25
MS
8 2-ethylhex-2-enol MS 0.53 0.45 0.55 Furan, 2-pentyl 996 RI, 3.57 3.26 3.42
MS
9 o-Cymene 1026 RI, 0.19 0.23 0.16 2-Piperidinethione MS 0.39 0.4 0.41
MS
10 Tetrahydrocarvone MS 0.20 0.15 - 2-ethylhex-2-enol MS 0.44 0.41 -
11 Benzeneacetaldehyde 1043 RI, 1.23 1.22 1.21 2,4-Heptadienal, (E,E)- 1008 RI, 0.42 0.45 0.4
MS MS
12 trans-2,4-Decadienol MS 0.60 0.54 0.57 o-Cymene 1026 RI, 0.15 0.16 0.22
MS
13 Nonanal 1102 RI, 1.86 1.76 1.78 D-Limonene 1030 RI, 0.18 0.2 0.19
MS MS
14 Bornylene MS 0.22 0.28 0.35 Tetrahydrocarvone MS 0.24 - 0.25
15 Benzyl nitrile 1141 RI, 7.97 7.44 8.06 Benzeneacetaldehyde 1043 RI, 3.69 3.58 3.26
MS MS
16 Terpinen-4-ol 1177 RI, 0.28 0.20 0.29 2-Ethylhexyl acrylate MS 0.90 0.91 0.92
MS
17 Cyclodecanol MS 0.45 0.45 0.41 3,5-Octadien-2-one 1093 RI, 1.65 1.48 1.62
MS
18 (E,E)-2,4-Decadienol MS 1.07 0.88 1.00 Nonanal 1102 RI, 1.87 1.79 1.81
MS
19 Tetrahydrocarvone MS 0.50 0.49 0.47 Benzyl nitrile 1141 RI, 1.17 1.2 1.24
MS
20 Cyclohexene, 3-pentyl- MS 0.38 0.33 0.44 (+)-2-Bornanone 1144 RI, 0.37 0.22 0.38
MS
21 Furan, 2-hexyl MS 0.46 0.41 0.38 trans-5-Tetradecene MS 0.53 0.58 0.65
22 Benzene, 1359 RI, 2.00 2.05 2.15 3-Anisaldehyde 1196 RI, 0.22 - 0.20
(isothiocyanatomethyl)- MS MS
23 n-Decanoic acid 1402 RI, 2.98 2.66 3.22 Carvone 1231 RI, 0.45 0.39 0.43
MS MS
24 Undecanoic acid 1465 RI, 0.29 0.27 - trans-2,4-Decadienol MS 0.92 0.88 1.21
MS
25 Methanone, dicyclohexyl- MS 2.30 2.06 2.22 Adamantane MS 1.01 1.22 1.35
26 Megastigmatrienone MS 0.74 0.77 0.72 2-Hexylfuran MS 3.03 2.94 3.15
27 1-Decanol, 2-hexyl- MS 0.78 0.69 0.71 Phenol, 4-(1-methylpropyl)- 1279 RI, 0.50 0.23 -
MS
28 Cyclopentadecanone MS 0.29 - 0.26 2-Ethylhexyl acrylate MS 1.05 0.97 1.11
29 4-Hexadecanol MS 0.55 0.47 0.35 Benzene, 1359 RI, 0.89 0.95 0.92
(isothiocyanatomethyl)- MS
30 2-Pentadecanone 1697 RI, 17.66 15.82 17.89 Dodecanoic acid 1568 RI, 0.76 - 0.68
MS MS
31 trans-Geranylgeraniol MS 0.46 0.41 0.42 Tetradecanoic acid 1765 RI, 0.74 0.55 0.72
MS
32 n-Hexadecanoic acid 1977 RI, 2.77 2.84 2.80 2-Heptadecanone 1900 RI, 0.77 0.64 0.75
MS MS
33 Methyl stearidonate 2088 RI, 0.54 0.53 0.50 n-Hexadecanoic acid 1971 RI, 9.13 8.89 8.98
MS MS
34 Pentacosane MS 0.48 0.50 0.55 9,12-Octadecadienoic acid (Z, 2104 RI, 3.57 3.25 3.61
Z) MS
35 Tetracosane MS 0.18 0.19 - Oleic acid 2140 RI, 2.34 2.03 2.24
MS
36 Triacontane MS 0.96 1.02 0.89 Octadecanoic acid 2188 RI, 0.30 - 0.28
MS
37 Oxirane, hexadecyl MS 0.36 0.33 0.34 /
38 Tetratriacontane MS 2.44 2.42 2.13 /
39 Hentriacontane MS 22.64 22.70 23.45 /
Total identified (%) 93.19 86.76 91.65 Total identified (%) 96.57 92.25 95.43
Oxygenated compounds (%) 41.42 34.84 38.70 Oxygenated compounds (%) 38.53 32.92 36.07
Sulfur compounds (%) 11.66 11.46 11.60 Sulfur compounds (%) 48.41 48.34 48.46
Others (%) 51.77 40.46 52.95 Others (%) 9.63 10.99 10.90
a
Compounds listed in order of elution from HP-5MS capillary column.

5
R. Zhao et al.
b
TAL essential oil: essential oil extracted from Thlaspi arvense L. leaves.
c
TAS essential oil: essential oil extracted from Thlaspi arvense L. seeds.
d
Relative area percentage (peak area relative to the total peak area, %).
e
MHD-VOE: microwave-assisted hydrodistillation and vegetable oil in-situ extraction.
f
MAE: microwave-assisted extraction.
g
HD: conventional hydrodistillation.
3.2. Single-factor experiments of the MHD-VOE process
The effect of coconut oil volume, liquid-solid ratio, microwave
irradiation power, microwave irradiation time on the yield of TAL and
TAS essential oils were systematically investigated, and the results and
specific analysis process are shown in supplementary materials.
3.3. Parameter optimization by RSM
RSM was empleyed to further investigate the interactions on the
yield of TAL and TAS essential oils between the factors, some parameters
based on the univariate experimental results above. Three factors
including X1: liquid-solid ratio (7.5, 10.0 and 12.5 mL/g); X2: microwave
irradiation power (385, 540 and 700 W); and X3: microwave irradiation
time (20, 30 and 40 min) were selected for optimization by BBD. The
actual and predicted values for the yields of TAL and TAS essential oils
are presented in Table S1. The maximum predicted values of TAL and
TAS essential oil yields were 0.77 and 2.59 mg/g, respectively.
The BBD fitting results of the response surface quadratic model are
presented in Table S2. The final equations of the yield of TAL essential
oil (Y1) and TAS essential oil (Y2) in terms of actual factors were
expressed as Equations (4) and (5):
Y1 = 0.740 + 0.033 X1 + 0.073 X2 + 0.058 X3 – 0.013 X1X2 – 0.022 X1X3 +
0.013 X2 X3 – 0.092 × 21 – 0.052 × 22 – 0.062 × 23
Y2 = 2.54 + 0.10 X1 + 0.17 X2 + 13.01 X3 – 0.030 X1X2 – 0.060 X1X3 – 0.015
X2 X3 – 0.33 × 21 – 0.18 × 22 – 0.14 × 23
As Table S2 presents, the determination coefficients (R2) for TAL and
TAS essential oils were 0.9847 and 0.9906, respectively, which indi­
cated that the model could be applied to interpretation for all of the
variations and that there was good concordance of the actual and pre­
dicted responses. Table S2 shows that the models for TAL and TAS
essential oils with F-values of 50.21 and 81.62, respectively, both had a
higher significance level with a P value < 0.0001. This implied that the
model was precise and had an extremely low probability (less than
0.01%) for the occurrence of a Model F-value that was large due to
Fig. 3. The schematic diagram of inhibition activities of different samples on Penicillium expansum. TAL essential oil, the concentration was successively 0, 0.25, 0.50,
0.75, 1.00, 1.5, 2.00 mg/mL (a); TAS essential oil, the concentration was successively 0, 0.25, 0.50, 0.75, 1.00, 1.5, 2.00 mg/mL (b); AITC, the concentration was
successively 25, 50, 100, 125, 250, 500 μg/mL (c).
6
LWT 165 (2022) 113718
statistical noise. The adjusted determination coefficient (Adjust. R2)
values for the predicted model were 0.9651 and 0.9960, respectively,
which showed a high degree of correlation between the experimental
data and predicted values. The low coefficients of variation (2.85 and
2.01) of TAL and TAS essential oils, respectively, indicated that all ex­
periments had high reproducibility and reliability.
The interactive influence of three parameters on the yields of TAL
and TAS essential oils was analyzed by 3D surface graphs, which are
shown in Fig. 2. As presented in Table S2, the linear terms, X1, X2 and X3,
the interaction terms, X1 and X3, and the quadratic terms, X21, X22 and X23,
significantly affected the yields of TAL and TAS essential oils. Fig. 2a
shows the interactive influences of the liquid-solid ratio and microwave
irradiation power while keeping the microwave irradiation time at the
central level (30 min). As shown in Fig. 2a, the yield of TAL essential oil
gradually improved with increasing microwave irradiation power, a
similar trend was observed in Fig. 2b. Fig. 2b and e further portray the
interactive effects of the liquid-solid ratio and microwave irradiation
time that significantly improved the yields of TAL and TAS essential oils.
In Fig. 2c and f, the interactive effects of the liquid-solid ratio and mi­
crowave irradiation time and the yields of TAL and TAS essential oils
gradually improved and then remained constant with X2 and X3
increasing.
To evaluate the accuracy and consistency between the actual results
and the predictive model, a verification experiment was performed
under optimized conditions (n = 3). The optimization conditions for
maximized yields of TAL and TAS essential oils were obtained by soft­
ware: a liquid-solid ratio of 10.10 mL/g; a microwave irradiation power
of 624.45 W; a microwave irradiation time of 34.25 min. The resulting
maximum yields of TAL and TAS essential oils were 0.78 mg/g and 2.65
mg/g, respectively. Considering practical convenience, the actual
extraction conditions consisted of a liquid-solid ratio of 10 mL/g, mi­
crowave irradiation power of 624 W and microwave irradiation time of
34 min. The resulting maximum yields of TAL and TAS essential oils
were 0.77 ± 0.02 mg/g and 2.69 ± 0.08 mg/g, respectively, which were
close to the predicted values. Compared with the conventional hydro­
distillation (0.73 ± 0.02 mg/g and 2.23 ± 0.04 mg/g), the developed
microwave-assisted hydrodistillation and vegetable oil in situ extraction
R. Zhao et al. LWT 165 (2022) 113718

(MHD-VOE) method obtained higher the yields of TAL and TAS essential
oils, which was higher than MAE (0.76 ± 0.02 mg/g and 2.44 ± 0.05
mg/g).

3.4. Composition analysis of TAL and TAS essential oils

The composition of TAL and TAS essential oils was determined by

GC-MS. The results are shown in Table 1. As we can see from Table 1, the
TAL essential oil obtained by the two different methods had a similar
composition. The TAL essential oil detected 39, 36 and 36 components
by the MHD-VOE, MAE and HD methods, which accounted for 93.19%,
86.76% and 91.65% of the total components of the essential oil,
respectively. Among these, oxygen-containing chemicals accounted for
a large proportion at 41.42%, 34.84% and 38.70%, respectively. From
Table 1, 36 kinds of components were identified in the TAS essential oil
obtained by the MHD-VOE method, which accounted for 96.99% of the
total essential oil components. Allyl isothiocyanate (AITC) were the
main components in the TAS essential oil at a high proportion of
44.69%, which was attributed to TAS containing a large amount of
sinigrin (Zhao et al., 2021), which itself could be hydrolyzed into AITC
and then steamed out using hydrodistillation (Wu, Xue, Hou, Feng, &
Zhang, 2015). From Table 1, it can be seen that the proportion of AITC in
the TAL essential oil obtained by the MHD-VOE method was only 9.66%,
which may have been caused by the differing contents of sinigrin in TAL
and TAS oils. AITC is a potential antibacterial agent that can prevent
food spoiling. GC-MS analysis will play an important role in the further
development and utilization of TAL and TAS essential oils.

3.5. Antifungal activity analysis

3.5.1. Analysis of the inhibitory activity of TAL and TAS essential oils and
AITC on P. expansum
A schematic diagram of the inhibitory activity of different samples
against P. expansum is shown in Fig. 3. Fig. 3 shows the growth diameter
of P. expansum in a petri dish after the action of different concentrations
of samples. The diameter of the P. expansum zone gradually decreased as
the sample concentration increased, which indicated that TAL and TAS
essential oils and AITC also had varying degrees of inhibition on
P. expansum. In addition, the analysis of the data of the inhibitory ac­
tivity of different samples against P. expansum is presented in Table S3.
Table S3 indicates that, when the concentration of TAS essential oil was
0.75 mg/mL, the inhibition rate on P. expansum reached 100%, while the
inhibition rate of TAL essential oil on P. expansum was 98.60% ± 2.32%.
When the concentration of TAL essential oil was 1.00 mg/mL, it also
completely inhibited the growth of P. expansum, which indicates that
P. expansum was more sensitive to TAS essential oil. AITC exhibited a
strong inhibitory effect, with the inhibitory activity on P. expansum as
high as 96.48% ± 2.45% with AITC at a concentration of 100 μg/mL. At
a concentration of 125 μg/mL, the growth of P. expansum was
completely inhibited.

3.5.2. Analysis of minimal inhibitory concentration and minimal

bactericidal concentration
The double dilution method is often used to detect the antibacterial
activity of different substances. The MIC and MBC values of TAL and
TAS essential oils and AITC for P. expansum are shown in Table S4. We
found that P. expansum exhibited a higher sensitivity to AITC given that
the MIC and MBC values were only 100 μg/mL and 125 μg/mL,
respectively. However, under the action of TAL essential oil, the MIC and
MBC values for P. expansum were 0.75 mg/mL and 1.00 mg/mL,
respectively. Compared with TAL essential oil, TAS essential oil had a
stronger inhibitory effect on P. expansum, and the TAS essential oil had
the same MIC and MBC values, which were both 0.75 mg/mL.
Fig. 4. The time killing curve of TAL essential oil (a), TAS essential oil (b),
3.5.3. Analysis of the time killing curve AITC (c) on Penicillium expansum.
To fully study the inhibitory effect of TAL essential oil, TAS essential

7
R. Zhao et al.
Fig. 5. Mycelial morphology of Penicillium expansum treated by different samples after 7 days. Treated by TAL essential oil (a); b, treated by TAS essential oil (b);
treated by AITC (c); treated by nystatin (d); the blank control 1 fixed by 2.5% glutaraldehyde solution (e); the blank control 2 of dried naturally in the desiccator (f).
oil and their main component AITC on P. expansum and predict their
potential for the prevention and control of fruit spoilage, experiments
were carried out at 0, 1/2MIC, MIC and 2MIC and at MBC concentrations
on P. expansum. The logarithmic changes on P. expansum spores over
time are shown in Fig. 4.
Fig. 4a shows the time-killing curve of TAL essential oil on
P. expansum. Under the action of 1/2MIC concentration, the growth of
P. expansum was clearly inhibited when P. expansum was cultured for 6
h, which resulted in a significant decrease in the number of spores.
However, the number of spores gradually increased and reached the
logarithmic growth phase after 6 h. Under the action of MIC concen­
trations, the number of spores of P. expansum decreased rapidly within
the first 4 h, and then the rate of spore reduction within 4–8 h slowed
down. The number of spores of P. expansum basically remained at a
relatively low level until 16 h later.
Under the action of 0, 1/2MIC, MIC, 2MIC and MBC concentrations,
the time-killing curves of TAS essential oil on P. expansum are presented
in Fig. 4b, which showed different inhibitory effects on P. expansum. The
number of spores of P. expansum rapidly decreased in the first 8 h into
the decay period at a concentration of 2MIC, and P. expansum was
completely killed within 8 h. In addition, the inhibitory effect of TAS
essential oil on P. expansum continued to improve with increasing
concentration.
The time-killing curve of different AITC concentrations (0, 1/2MIC,
MIC, 2MIC and MBC) to P. expansum was plotted and presented in
Fig. 4c. There was a meaningful phenomenon, in which P. expansum
rapidly entered the decay period under the action of MIC, 2MIC and
MBC concentrations and was completely killed within four, six and 12 h,
8
LWT 165 (2022) 113718
respectively. The results of the time-killing curve indicated that the
inhibitory activity of TAL and TAS essential oils and that of AITC on
P. expansum was concentration dependent. In particular, AITC had a
more significant inhibitory effect on P. expansum, thus, we inferred that
component analysis of the TAL and TAS essential oils and the antifungal
activity study of AITC on P. expansum were conducive to the expansion
and development of AITC in food preservation. Moreover, it has also
been reported that AITC has antimicrobial activity against spoilage
lactic acid bacteria (Takahashi et al., 2021), and encapsulated AITC has
been applied to postharvest fruits and vegetables, such as strawberries
and tomatoes (Colussi et al., 2021; Wu et al., 2015).
3.5.4. Observation of P. expansum mycelial morphology
The mycelial morphology of P. expansum untreated or treated with
different samples after seven days was observed by scanning electron
microscopy (SEM), and the mycelial pictures are presented in Fig. 5. It
can be seen from the figure that the surface of the hyphae of the un­
treated blank control group was smooth and had no obvious creases and
there was a large number of spores. The spores were oval, full and
regular. The mycelial morphology after TAL essential oil treatment is
shown in Fig. 5a. The mycelial morphology became thin and irregular,
the surface of the hyphae was more wrinkled and there was no spore
growth. Fig. 5b describes the mycelial morphology of P. expansum
treated with TAS essential oil. The hyphae exhibited a shriveled, curled
state and varied in thickness. The hyphae after AITC treatment showed
serious distortion, collapse, breakage and entanglement phenomena,
and there also was not any spore growth (Fig. 5c). The mycelial
morphology treated with TAL essential oil, TAS essential oil or AITC all
R. Zhao et al.
presented similar destruction compared with that of the positive control
of nystatin (Fig. 5d), and the hyphae were thin, shriveled and shrunken
in shape with many wrinkles on the surface. In the blank control groups,
P. expansum was not treated with any samples. The P. expansum hyphae
were fixed with 2.5% glutaraldehyde solution (Fig. 5e) or were air dried
in a desiccator (Fig. 5f). The SEM observation results indicated that the
hyphae of the blank control group were thick, smooth and densely
distributed. Moreover, there were clusters of spherical spores on the
surface of the hyphae. Compared with that of the untreated P. expansum
hyphae, the P. expansum mycelial structures after sample treatments
were completely destroyed. TAL essential oil, TAS seed essential oil and
AITC all had significant inhibitory effects on P. expansum. SEM results
showed that the antifungal effects of the samples may be the result of
inhibiting the germination of P. expansum spores and destroying myce­
lial structure.
4. Conclusions
A novel extraction method of MHD-VOE was developed to obtain
desirable yields of TAL and TAS essential oils. This method replaced
organic solvents with vegetable oils for extracting the essential oils,
which is conducive to environmentally friendly development. Under
optimal conditions, the maximum yields of TAL and TAS essential oils
were 0.77 ± 0.02 mg/g and 2.69 ± 0.08 mg/g, respectively. Subse­
quently, the compositions of the essential oils from TAL and TAS were
assessed by GC-MS analysis. AITC had a high proportion of TAL essential
oil at 44.69% and exhibited superior antifungal activity against
P. expansum. Therefore, AITC may be a desirable alternative compared
with traditional synthetic fungicides and preservatives for the preser­
vation and safty control of foods. However, the impact of AITC on the
preservation and physicochemical properties of foods, such as vegeta­
bles, fruits and meat, needs further investigation and research.
CRediT authorship contribution statement
Ru Zhao: Formal analysis, Investigation, Writing – original draft,
Writing – review & editing. Ailing Ben: Formal analysis, Validation,
Methodology, Funding acquisition. Mengxia Wei: Formal analysis.
Ming Ruan: Investigation, Validation. Huiyan Gu: Writing – review &
editing, Conceptualization, Writing - Review, Funding acquisition. Lei
Yang: Conceptualization, Writing – Review, Writing – review & editing.
Declaration of competing interest
The authors have no declaration of interest statement.
Acknowledgments
The authors thank the Natural Science and Major Research Project of
Jiangsu Province (18KJA180007) and the Fundamental Research Funds
for the Central Universities (2572019AA13) for financial support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.lwt.2022.113718.
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