Food Control 86 (2018) 117e125

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Broad-spectrum antimicrobial activity, chemical composition and

mechanism of action of garlic (Allium sativum) extracts
Cun Chen a, Chun-Hong Liu b, Jing Cai c, Wei Zhang d, Wei-Liang Qi a, Zheng Wang e,
Zhi-Bin Liu b, *, Yi Yang b, **
a
College of Chemistry and Life Science, Chengdu Normal University, Chengdu 611130, Sichuan, PR China
b
Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, Sichuan, PR
China
c
West China School of Pharmacy, Sichuan University, Chengdu 610041, Sichuan, PR China
d
College of Bioengineering, Sichuan University of Science & Engineering, Zigong 643000, Sichuan, PR China
e
Chengdu jiaweita Agriculture CO., LTD, PR China

a r t i c l e i n f o a b s t r a c t

Article history: In many cultures, garlic (Allium sativum) has a reputation as a therapeutic panacea. In this work, a
Received 20 July 2017 range of plant pathogenic bacteria and fungi were inhibited by garlic bulb extracts that were ob-
Received in revised form tained under various conditions. The conditions included different solvents (distilled water, meth-
3 November 2017
anol and ethanol), and water at different pH values (pH 3.0, 5.0, 7.0, 9.0 and 10.7). Water extraction
Accepted 5 November 2017
Available online 8 November 2017
produced the best antimicrobial activity, compared to methanol and ethanol, and the greatest ac-
tivity was obtained by extraction under strongly acidic condition (pH 3.0). Subsequent analysis using
HPLC and GCeMS revealed that the major active ingredients were 3-vinyl-1,2-dithiacyclohex-5-ene
Keywords:
Garlic (Allium sativum) extracts
and 3-vinyl-1,2-dithiacyclohex-4-ene. In addition, changes observed in membrane permeability,
Antimicrobial activity protein leakage and by scanning electron microscopy suggested that the antimicrobial activity of
HPLC garlic extracts may be due to destruction of the structural integrity of cell membranes, leading to cell
GCeMS death.
Antibacterial mechanism © 2017 Elsevier Ltd. All rights reserved.

1. Introduction esculentus) (Okwu, Awurum, & Okoronkwo, 2007); ethanol extracts

Many plant diseases caused by bacteria or fungi cause huge
economic loss by affecting the quality and quantity of plant mate-
rial during both growth and storage. A variety of chemical bacte-
ricides and fungicides are used to reduce plant diseases in
agriculture. These chemicals, however, cause environmental
pollution (Zhang, Jiang, & Ou, 2011), which has prompted plant
pathologists to search for natural antimicrobial substances that can
replace them. There is a wide diversity of sources among botani-
cals: extracts of citrus species inhibit the growth of Fusarium oxy-
sporum, which causes damping-off diseases of okra (Hibiscus
* Corresponding author.
** Corresponding author.
E-mail addresses: liuzhibin@scu.edu.cn (Z.-B. Liu), yangyi528@scu.edu.cn
(Y. Yang).
of Lantana camara, lemon and avocado leaves have significant
fungicidal effects on radial growth of Colletotrichum gloeosporioides
(Prasad & Anamika, 2015); essential oils and plant extracts of
peppermint (Mentha
piperita L.), coriander (Coriandrum sativum
L.), and anise (Pimpinella anisum L.) have in vitro anti-biofilm ac-
tivities against Staphylococcus aureus and Escherichia coli (Bazargani
& Rohloff, 2016).
Garlic (Allium sativum) has attracted considerable attention
because of its storage characteristics and durability during ship-
ping. It has been known as a flavoring agent with important me-
dicinal properties for centuries (Martins, Petropoulos, & Ferreira,
2016). Studies have revealed that garlic has activity against bac-
teria and fungi. Research has shown that aqueous garlic and
eucalyptus extracts are effective against seed-borne infections that
cause bacterial spot disease of tomato and pepper (Mirik & Aysan,
2005). Garlic extracts inhibit Pseudomonas syringae pv. tomato,
Xanthomonas vesicatoria and Clavibacter michiganensis subsp.
michiganensis, causal agents of bacterial speck, bacterial spot and

https://doi.org/10.1016/j.foodcont.2017.11.015
0956-7135/© 2017 Elsevier Ltd. All rights reserved.
118 C. Chen et al. / Food Control 86 (2018) 117e125
bacterial canker, respectively (Balestra, Heydari, Ceccarelli, Ovidi,
& Quattrucci, 2009). Four different phytopathogenic fungi (Fusa-
rium oxysporum, Botrytis cinerea, Verticillium dahliae and Phy-
tophthora capsici) are inhibited by 28 garlic cultivars (Sikandar
et al., 2016). The distinctive odor of garlic is known to be associ-
ated with organic sulfur compounds, such as allicin (Cavallito,
Buck, & Suter, 1944; Oommen, Anto, Srinivas, & Karunagaran,
2004).
The use of garlic has been proposed by many authors because
of its antibacterial and antifungal properties, and most of the
active ingredients are present in the essential oil obtained by
hydrodistillation (El-Sayed, Chizzola, Ramadan, & Edris, 2017; Li
et al., 2016; Sung et al., 2014; Tsao & Yin, 2001). Distillation
has some disadvantages, however, since some highly reactive
sulfur molecules in the garlic volatile fraction are thermally un-
stable and may degrade during thermal distillation. Some re-
searchers have obtained extracts from plants using supercritical
fluid extraction (SFE) as a mild extraction processes. Dean
extracted deoxyschisandrin and paeonol from two Chinese
herbal medicines, Schisandra chinensis and Paeonia suffruticosa
using SFE (Dean & Liu, 2015). Michalak used SFE to extract ma-
rine macroalgae from the Baltic Sea in order to screen for plant
growth biostimulant properties (Michalak et al., 2016). The
choice of SFE conditions has largely been determined empirically,
which is time consuming, and the equipment is expensive. We
therefore attempted to extract active ingredients from garlic
under different conditions and determine the chemical compo-
sition of the extracts.
The purpose of this investigation was first to compare the
inhibitory effects of garlic extracts, obtained under different
conditions, on plant pathogenic bacteria and fungi. Subsequently,
the active ingredient of the extracts showing the highest inhib-
itory activity was separated and concentrated. The principal
active ingredient was determined by gas chromatographyemass
spectrometry (GCeMS). Finally, the mechanism of action of the
antibacterial activity was explored by evaluation of cell perme-
ability, cell membrane integrity and scanning electron
microscopy.
2. Materials and methods
2.1. Garlic bulb extracts preparation
Garlic bulbs were purchased from the local market in Chengdu
and were stored at 4  C until used. The garlic bulbs were peeled, cut
into small pieces, and then crushed in a mortar. Portions of the
garlic (10 g) were added to 20 mL deionized water at different pH
values (3.0, 5.0, 7.0, 9.0 and 10.7 adjusted with 50 mmol L1 tris-HCl
buffer), methanol or ethanol. The mixtures were kept for 2 h at
room temperature and then centrifuged at 10,000 rpm for
20 min at 4  C. The supernatants were filtered through membrane
filters (0.22 mm) and the extracts were stored at 4  C in the dark
until required.
2.2. Microbial strains and culture
Erwinia carotovora (BNCC138474), Pseudomonas syringae
(BNCC134219), Xanthomonas campestris pv. malvacearum
(BNCC138498), Magnaporthe grisea (BNCC144711), Fusarium pro-
liferatum (BNCC143058) and Alternaria brassicicola (CICC264) were
obtained from Beina Chuanglian Biological Research Institute.
2.3. Assay of antimicrobial activity against phytopathogenic
bacteria
Plant pathogenic bacteria (E. carotovora, X. campestris pv. mal-
vacearum, P. syringae) were grown to OD600 z 0.6 in nutrient broth
(NB) at 200 rpm and 28  C on an orbital shaker.
Antibacterial tests were carried out using Oxford cup assays
with some modifications (Shang et al., 2014). After autoclaving, the
nutrient agar medium was cooled to 50  C. Agar (60 mL) was mixed
with bacterial solution (0.6 mL) and poured into 15 cm diameter
plates. Oxford cups (6 mm diameter) were placed at equal distances
on the agar surface and garlic extracts (20 mL) were added with a
micropipette. The size of the inhibition halo was measured after
24 h at 28  C. The same procedure was repeated in triplicate.
2.4. Assay of antimicrobial activity against phytopathogenic fungi
Plant pathogenic fungi (F. proliferatum, A. brassicicola, M. grisea)
were cultivated on potato dextrose agar (PDA) plates at 28  C. The
fungi were maintained on PDA plates at 4  C and periodically
subcultured. The spores were washed with 10 mL of sterile water
and centrifuged at 5000 rpm for 5 min to remove the supernatant.
Spores were mixed with sterile water to a count of approximately
2
107 mL1 by hemocytometer. Spore suspensions were stored at
4  C until used (Yin & Cheng, 1998).
Antifungal tests were carried out using Oxford cup assays,
similarly to the antibacterial tests in section 2.3. After autoclaving,
the potato dextrose agar medium was cooled to 50  C. Agar (60 mL)
was mixed with spore suspension (0.2 mL) and poured into 15 cm
diameter plates. Oxford cups (6 mm diameter) were placed at equal
distances on the agar surface and garlic extracts (20 mL) were added
with a micropipette (the garlic extracts added to M. grisea were
diluted to 25% with distilled water). The different fungi were
cultured for 2e4 days, and the size of the inhibition halo was
measured. The same procedure was repeated in triplicate.
2.5. UVevis spectroscopy and high performance liquid
chromatography (HPLC)
Garlic extracts were diluted with distilled water to produce 2%
solutions. Spectra were recorded over a wavelength range of
190e500 nm using a TU-1901 UVeVIS spectrophotometer (Beijing
Purkinje General Instrument Co., Ltd.)
In the HPLC method, a C18 column (250
4.5 mm) at 25  C was
injected with 20 mL of sample and monitored at 210 nm. Acetoni-
trile:water (30:70 v/v) was the mobile phase, with a flow rate of
0.8 mL min1. The analyses were performed on a Shimadzu LC-20A
liquid chromatograph with a diode array detector (CBM20A, Shi-
madzu, Tokyo, Japan).
2.6. Gas chromatographyemass spectrometry (GCeMS)
The peak appearing at about 16.0 min in the HPLC was collected,
concentrated, and then analyzed using a Shimadzu GCMS-QP2010
system equipped with a TG-5MS capillary column
(30 m
0.25 mm, 0.25 mm). Helium was the carrier gas in constant
flow mode at 1 mL min1. The ionization voltage was 70eV. The
column temperature was initially kept at 60  C for 5 min, and then
increased by 10  C min1 up to 280  C. The injection port temper-
ature was maintained at 220  C. The spectrum was analyzed and
compounds identified using the MS data libraries NIST05.LIB and
NIST05s.LIB.
 C. Chen et al. / Food Control 86 (2018) 117e125
2.7. Changes in membrane permeability and integrity of bacterial
cells
Bacterial cell membrane permeability was determined by
relative conductivity (Diao, Hu, Zhang, & Xu, 2014). After incu-
bation at 28  C overnight, E. carotovora was treated with garlic
extracts. Each 10 mL culture was treated with 200, 400 or
800 mL of extracts, and the conductance was recorded using a
conductivity meter (DDS-307) after incubation for 1, 2, 4, 6, 8
and 12 h.
Breakdown of bacterial cell membrane integrity is manifested
by leakage of protein. The bacterial culture media treated with 200,
400 and 800 mL garlic extracts were centrifuged at 6000 rpm for
10 min and the supernatants removed. Samples of each superna-
tant (200 mL) were treated with 800 mL Coomassie Brilliant Blue G-
250. After 10 min the mixtures were subjected to colorimetric
analysis on a T6-spectrophotometer (Beijing Purkinje General In-
strument Co., Ltd.) at 595 nm. Parallel determinations in triplicate
were used to calculate the mass concentration of protein in the
suspension.
2.8. Scanning electron microscopy (SEM)
The E. carotovora cells were incubated in NB at 28  C overnight.
Culture samples (10 mL) were treated with 400 or 800 mL of garlic
extracts and the mixtures were incubated at 28  C for 6 and 12 h,
respectively. The mixtures were centrifuged at 6000 rpm for 10 min
and the precipitated cells were washed with 0.1 mol L1 phosphate
buffered saline (PBS) at pH 7.4, and then fixed with 2.5% glutaral-
dehyde overnight at 4  C. Following a brief rinse in PBS, cells were
dehydrated in an ethanol series (30%, 50%, 70%, 80% and 90%) for
15 min at each graduation, and then twice for 15 min in 100%
ethanol. The cells were then critical point dried in liquid CO2 and
coated with gold by cathodic spraying. The final samples were
examined in a scanning electron microscope (JSM-750 0F, JEOL Ltd.,
Tokyo, Japan).
3. Results
3.1. Activity of different garlic extracts against plant pathogenic
bacteria
Growth of E. carotovora, X. campestris pv. malvacearum, and
P. syringae was inhibited when garlic extracted with water (pH 7.0),
methanol and ethanol was placed on seeded agar plates. The size of
the inhibition halos clearly showed that the water extracts had the
highest activity against the plant pathogenic bacteria and the
methanolic extracts showed higher antibacterial activity than the
ethanolic extracts (Fig. 1a).
Based on these results, the effects of garlic extracted with
deionized water at different pH values (3.0, 5.0, 7.0, 9.0 and 10.7)
were explored. Zones of inhibition showed that extracts with water
at pH 3.0, 5.0, and 7.0 had good activity against the plant pathogenic
bacteria (pH 3.0 > pH 5.0 > pH 7.0), but the extracts at pH 9.0 and
10.7 had almost no inhibitory effect (Fig. 1b).
3.2. Activity of different garlic extracts against plant pathogenic
fungi
As observed in the bacterial experiments, the water extracts had
the highest activity against plant pathogenic fungi (F. proliferatum,
A. brassicicola, M. grisea), and the methanolic extracts showed
higher antimicrobial activity than the ethanol extracts (Fig. 2a).
119
Consistent with the antibacterial data, extracts with water at pH
3.0, 5.0, and 7.0 had good activity against the fungi (pH 3.0 > pH
5.0 > pH 7.0), but the extracts at pH 9.0 and 10.7 had almost no
inhibitory effect. In contrast to Fig. 1, the effect of garlic extracts on
fungi was significantly better than that against bacteria (Fig. 2b). It
is worth noting that it was necessary to dilute the extracts used
against M. grisea, otherwise the inhibition zone was too large.
3.3. HPLCeUV analysis of garlic extracts
HPLCeUV chromatograms were recorded to determine the
active ingredient of the extracts. In order to confirm the wavelength
for HPLCeUV, the UVeVis spectrum of 2% garlic extracts solution
was recorded over 190e500 nm. The absorption peak appeared at a
wavelength near 210 nm (Appendix A.). The HPLCeUV210nm chro-
matograms for garlic extracted with deionized water (pH 3.0, 7.0,
9.0), methanol, and ethanol were shown in Fig. 3. The absorption
peak appearing at about 16.0 min was much stronger in the water
extracts (pH 3.0, 7.0) than the methanol extracts, and the level in
the methanol extracts were slightly higher than that in the ethanol
extracts. Interestingly, the chromatogram of the water extracts (pH
9.0) did not contain a peak in this position (Fig. 3). The results
indicated that the size of the inhibition zone was positively corre-
lated with the height of the peak appearing at about 16.0 min in the
chromatogram. Consequently, we speculated that this peak was the
active substance. The peak was collected, concentrated, and sub-
sequent experiments confirmed its antimicrobial activity
(Appendix B.).
3.4. GCeMS analysis
GCeMS is considered to be of great value in the study of com-
pounds having moderate thermal stability. In this work, the active
substance isolated and purified from garlic extracts was analyzed
by GCeMS. The results identified 3-vinyl-1,2-dithiacyclohex-5-ene
(CAS:62488-53-3) and 3-vinyl-1,2-dithiacyclohex-4-ene
(CAS:62488-52-2) as the two major compounds and the levels of
these two organosulfur compounds were 29.96% and 52.10%,
respectively (Fig. 4). The mass spectra of the identified compounds
are shown in Fig. 5.
3.5. Changes in membrane permeability and integrity of bacterial
cells
A membrane permeability test was performed to study the ef-
fect of garlic extract on the bacterial cell surface. Fig. 6 showed the
effect of garlic extracts on the membrane permeability of
E. carotovora. There was little change in the relative conductivity of
all samples during the first 2 h, but then an increase was observed.
The relative conductivity increased rapidly as the treatment time
and concentration of garlic extracts increased, indicating that the
bacterial membrane permeability was correspondingly increased.
Similarly, garlic extracts elevated the leakage of proteins through
the membrane of E. carotovora (Fig. 7).
3.6. Scanning electron microscopy study
E. carotovora bacteria were treated with 400 and 800 mL of garlic
extracts for 6 and 12 h, respectively. The morphological and phys-
ical changes were then observed by SEM. The SEM images showed
that the cells in the control group were short, rod-shaped, regular
and complete (Fig. 8, A-a, B-a), while cells treated with garlic ex-
tracts became broken and adhered to each other (Fig. 8, A-b, A-c, B-
120 C. Chen et al. / Food Control 86 (2018) 117e125

Fig. 1. Zones of inhibition caused by garlic extracts on seeded bacteria agar plates. E. carotovora and X. campestris pv. malvacearum were inhibited by 20 mL garlic extracts.
a. Garlic extracted with water, methanol and ethanol (I: water extracts; II: methanol extracts; III: ethanol extracts; IV: ethanol; V: methanol; VI: water).
b. Garlic extracted with deionized water at different pH (I: pH 3.0; II: pH 5.0; III: pH 7.0; IV: pH 9.0; V: pH 10.7).

Fig. 2. Zones of inhibition caused by garlic extracts on seeded fungi agar plates. F. proliferatum and A. brassicicola were inhibited by 20 mL garlic extracts; M. grisea was inhibited by
5 mL garlic extracts (the garlic extracts were diluted with distilled water to produce a 25% solution, and then 20 mL of the diluted solution was used).
a. Garlic extracted with water, methanol and ethanol (I: water extracts; II: methanol extracts; III: ethanol extracts; IV: ethanol; V: methanol; VI: water).
b. Garlic extracted with deionized water at different pH (I: pH 3.0; II: pH 5.0; III: pH 7.0; IV: pH 9.0; V: pH 10.7).
 C. Chen et al. / Food Control 86 (2018) 117e125
Fig. 3. HPLCeUV210nm chromatograms of garlic extracts.
121
b, B-c). It could be inferred that garlic extracts caused the
destruction of the bacterial cell membrane and led to leakage of cell
material.
4. Discussion
In this work, we made a change on the basis of previous
studies(Curtis, Noll, Sto € rmann, & Slusarenko, 2004; Slusarenko,
Patel, & Portz, 2008), and chose a range of plant pathogenic bac-
teria and fungi for study. E. carotovora is the bacterium most often
associated with soft rot of many economically important crops,
such as potato (Claderaolivera, Caron, Motta, Souto, & Brandelli,
2006). P. syringae has been reported to induce a variety of plant
diseases, including leaf spots, blights, and galls (Young, 1991).
X. campestris is the causal agent of black rot disease in cruciferous
plants, which is a continuing threat to world crucifers (Williams,
1980). M. grisea causes rice blast, which is the most serious dis-
ease in a wide variety of grasses including rice, barley and wheat
(Talbot, 2003; Wilson & Talbot, 2009). F. proliferatum is one of the
main causal agents of maize ear rot (Logrieco, Moretti, Ritieni,
Bottalico, & Corda, 1995). A. brassicicola causes black leaf spot,
which is a common disease of crucifers worldwide and can cause
significant losses in the yield and quality of crucifer crops (Chen,
Price & Park-Ng, 2003).
A water extract exhibited higher antibacterial activity than an
ethanol extract of leaves and bark from Cassia alata, when tested
against Candida albicans and S. aureus (Somchit, Reezal, Nur, &
Mutalib, 2003). A study with whole body tissue of Lambis lambis
found that a water extract had activity against P. aeruginosa, Pro-
teus vulgaris and Klebsiella aerogenes, while a methanol extract
was effective against K. aerogenes (Rohini, Priya, Lavanya, Kalpana,
& Karthika, 2012). Yeoh used different extraction periods and
solvents at various pH values to extract pectin from orange peel,
finding that most of the pectin was extracted under strongly acidic
(pH 1.5) conditions (Yeoh, Zhang, Shi, & Langrish, 2008). Research
showed that alginate and other polysaccharides were better
extracted from the brown alga, Macrocystis pyrifera, at higher pH
(8e12) and temperature (40  C, 60  C, and 80  C). The extracts
promoted growth of tomato plants and adventitious root forma-
tion in the mung bean cutting bioassay (Bricen ~ o-Domínguez,
Herna ndez-Carmona, Moyo, Stirk, & Staden, 2014). All of these
studies show that the quantities and types of substance extracted
depend on the extraction conditions, solvent and pH. In this study,
we explored the activity of garlic extracted with methanol, ethanol
and water at different pH values (3.0, 5.0, 7.0, 9.0 and 10.7) against
a number of plant pathogens. The methanol and ethanol extracts
had little inhibitory effect, while the water extracts under acidic
conditions had a significant effect. The water extracts under
alkaline conditions, however, had almost no effect (Fig. 1; Fig. 2).
Curtis(Curtis et al., 2004) and Portz(Portz, Koch, & Slusarenko,
2008) used a juicer to extract the garlic juice and quantified the
amount of allicin contained in garlic extracts, and made a series of
experiments to inhibit microbes. But our HPLCeUV determination
showed that the peak appearing at about 16.0 min was consistent
with the inhibition zone experiment (Fig. 3). After collection and
concentration of this peak, it was found to have the inhibitory ac-
tivity (Appendix B), and it was not allicin (Appendix C). We spec-
ulate that there are a variety of antibacterial compounds in garlic,
and the quantities and types of compounds extracted depend on
different extraction methods. Subsequent experiments showed
that 3-vinyl-1,2-dithiacyclohex-5-ene and 3-vinyl-1,2-
dithiacyclohex-4-ene were the two major components (Fig. 4;
122
Fig. 5. Mass spectra of major organosulfur compounds.
a: 3-Vinyl-1,2-dithiacyclohex-4-ene (CAS:62488-52-2)
b: 3-Vinyl-1,2-dithiacyclohex-5-ene (CAS:62488-53-3).
Fig. 6. Effect of garlic extracts on membrane permeability of E. carotovora. Values
represent means of three independent replicates; error bars indicate SD.
C. Chen et al. / Food Control 86 (2018) 117e125
Fig. 4. GCeMS chromatograms.
Fig. 7. Leakage of protein from E. carotovora. Values represent means of three inde-
pendent replicates; error bars indicate SD.
Fig. 5). The sequence and interval time of the peak containing the
organosulfur compounds and allicin in the HPLC is consistent with
the reports from Iberl (Iberl, Winkler, Müller, & Knobloch, 1990)
and Ryvak (Rybak, Calvey, & Harnly, 2004).
Zoghbi reported 3-vinyl-1,2-dithiacyclohex-5-ene and 3-vinyl-
1,2-dithiacyclohex-4-ene in the analysis of volatile oil from Ade-
nocalymma alliaceum leaves, which is a species of garlic bush that
exudes a strong odor similar to that of garlic (Zoghbi, Ramos, Maia,
Silva, & Luz, 1984). Chen, in the analysis of herbicides in garlic,
found that the two compounds were present at high levels when
the garlic was untreated, but the levels appeared to be greatly
reduced when the garlic was pretreated in a microwave oven (Chen
et al., 2007). A more recent article reports that levels of the two
compounds decreased during storage (Fei, Tong, Wei, & Yang,
2015). On the other hand, it has been reported that 3-vinyl-1,2-
dithiacyclohex-5-ene and 2-vinyl-4H-1,3-dithiin (CAS:80028-57-
5) are formed from allicin during thermal gas-chromatographic
analysis (Eric Block, 1985; E. Block, Naganathan, Putman, & Zhao,
1993; Tolstikov, 1997). In contrast, another article reports that the
two compounds were the major organosulfur components present
when the garlic bulbs were cut (Abu-Lafi, Dembicki, Goldshlag
PHanus, & Dembitsky, 2004). Keleş found that different garlic
 C. Chen et al. / Food Control 86 (2018) 117e125 123

Fig. 8. SEM images of E. carotovora (a. untreated; b. addition of 400 mL garlic extracts per 10 mL culture; c. addition of 800 mL garlic extracts per 10 mL culture). Bacteria were
cultured for 6 and 12 h, respectively.
A. Bacteria cultured for 6 h.
B. Bacteria cultured for 12 h.

types contained different proportions of 3-vinyl-1,2- Programs of Science and Technology of Sichuan Province (No.
dithiacyclohex-5-ene, 3-vinyl-1,2-dithiacyclohex-4-ene and 2- 2017JY0178), National Natural Science Foundation of China (NSFC
vinyl-4H-1,3-dithiin (Keleş, Taşkin, Baktemur, Kafkas, & Grant No. 31600993) and Scientific Research Fund of Sichuan Pro-
Büyükalaca, 2014). Similarly, Molinacalle reported that 3-vinyl- vincial Education Department (No.17ZB0071).
1,2-dithiacyclohex-5-ene and 3-vinyl-1,2-dithiacyclohex-4-ene
were present at different ratios in three garlic varieties Appendices
(Molinacalle, Priegocapote, & de Castro, 2016). From our point of
view, Chen's results are confirmed by our results: when the garlic Appendix A. UVeVis spectra of garlic water (pH 3.0) extracts, 2%
has not been treated by microwave oven, 3-vinyl-1,2- solution
dithiacyclohex-5-ene and 3-vinyl-1,2-dithiacyclohex-4-ene are
present at high levels.
The bacterial cell membrane provides a barrier to the passage of
small ions such as Kþ and Naþ (Harold & Altendorf, 1974; Lanyi,
1979). The observed changes in membrane conductivity indicated
that the permeability of the membrane was altered by the garlic
extracts (Fig. 6). The SEM images showed that samples treated with
garlic extracts at different concentrations and for different times,
were ruptured to different degrees (Fig. 8). The leakage of proteins
also suggested that the garlic extracts could destroy the structural
integrity of cell membranes (Fig. 7). Studies have found that losses
of the cell contents can cause cell death, which supports our results
(Cui, Zhao, & Lin, 2015; Ming et al., 2008). The mechanism by which
the garlic extracts kill the microorganism might be due to disrup-
tion of the cytoplasmic membrane.

5. Conclusions

In this study, garlic extracts had excellent performance as an

antimicrobial agent against both plant pathogenic bacteria and
fungi. The results suggest the potential to develop treatment using
these two organosulfur compounds for application in agriculture.

Acknowledgments

This work was financially supported by Applied Basic Research

124 C. Chen et al. / Food Control 86 (2018) 117e125
Appendix B. The pH 3.0 HPLC-UV peak appearing at about 16.0 min was collected, concentrated and found to have the inhibitory activity. I, II,
III indicate three different quantities of concentrate. All the microorganisms were inhibited by 30, 20, and 10 mL of concentrate except M. grisea,
which was inhibited by 7.5, 5, and 2.5 mL garlic extracts (the concentrate was diluted with distilled water to produce a 25% solution, and then
30, 20, and 10 mL of the diluted solution was used)
Appendix C. HPLCeUV210nm chromatograms of allicin and the
collected peak
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