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Food Control 37 (2014) 277e283

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Food Control

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Antifungal activity of citral, octanal and a-terpineol againstGeotrichum citri-aurantii

Haien Zhou, Nengguo Tao*, Lei JiaCollege of Chemical Engineering, Xiangtan University, Xiangtan 411105, PR China

a r t i c l e i n f o

Article history:Received 27 April 2013Received in revised form17 September 2013Accepted 24 September 2013

Keywords:Antifungal activityCell membrane integrityGeotrichum citri-aurantiiVolatile compounds

* Corresponding author. Tel.: þ86 731 58292246; faE-mail address: nengguotao@126.com (N. Tao).

0956-7135/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodcont.2013.09.057

a b s t r a c t

This study investigated the antifungal activity and potential antifungal mechanisms of three volatilecompounds (i.e., citral, octanal, and a-terpineol) against Geotrichum citri-aurantii, one of the mainpostharvest pathogens in citrus. Results showed that the volatile compounds exhibited strong antifungalactivity against the targeted pathogens, with minimum inhibitory concentration (MIC) and minimumfungicidal concentration (MFC) of 0.50 mL/mL and 1.00 mL/mL for citral, 0.50 mL/mL and 2.00 mL/mL foroctanal, and 2.00 mL/mL and 4.00 mL/mL for a-terpineol. The volatile compounds alter the morphology ofG. citri-aurantii hyphae by causing loss of cytoplasm content and distortion of the mycelia. The mem-brane permeability of the G. citri-aurantii increased with increasing concentrations of the three volatilecompounds, as evidenced by cell constituent release, extracellular conductivity, and pH. Moreover, thevolatile compounds induced a decrease in the total lipid content of G. citri-aurantii cells, indicating thedestruction of cell membrane structures. These results suggest that the antifungal activity of citral,octanal, and a-terpineol against G. citri-aurantii could be attributed to the disruption of cell membraneintegrity and leakage of cell components.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

China is one of the world’s largest citrus producers, with aproduction quantity of 4,886,900 T and a harvest area of 174,750 hain 2010 (Zhu et al., 2013). In China, citrus fruits are commonlyproduced in remote mountainous areas that are distant from con-sumer markets. Thus, the fruits must often be stored. Months-longdelay between harvest and consumption may result in postharvestlosses due to pathological diseases such as green molds, bluemolds, and sour rot (Scora & Scora, 1998).

Sour rot, despite being less common than green and blue molds,is a potentially devastating postharvest citrus disease, especiallyduring years with high rainfall (Hao et al., 2010; Smilanick,Mansour, Gabler, & Sorenson, 2008). This disease is caused byGeotrichum citri-aurantii. The incidence of sour rot in some areas inChina has recently been increasing (Liu, Liu, Wang, & Zheng, 2010).Sour rot cannot be efficiently controlled by registered fungicidessuch as imazalil, thiabendazole, pyrimethanil, or fludioxonil, whichcan adequately control the incidence of green or blue molds (Liuet al., 2009). Sodium o-phenylphenate (SOPP) is the only com-mercial fungicide that can partially control sour rot, but the use of

x: þ86 731 58293284.

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SOPP is very limited owing to the risk of fruit damage (Feng, Wu, Li,Jiang, & Duan, 2011; Liu et al., 2009). Consequently, the need for theselection of new compounds and/or additives that can help managesour rot in citrus is imperative. Considerable scientific interest hasrecently been directed toward substances that capable of control-ling pathological diseases because of the increasing resistance ofpostharvest fungal pathogens to fungicides as well as the growingconcern for human safety and environmental protection (Talibiet al., 2012).

A group of such substances may be plant essential oils. Essentialoils are a mixture of volatile constituents such as terpenes and theiroxygenated derivatives, as well as some aliphatic compounds (i.e.,octanal, propanol, and ethyl acetate). The antifungal activities ofsuch oils have also been widely documented and are suggested tobe the effect of their major compounds or a synergistic or antago-nistic effect of various compounds (Alma, Mavi, Yildirim, Digrak, &Hirata, 2003; Bajpai, Sharma, & Baek, 2013; Deba, Xuan, Yasuda, &Tawata, 2007; Fisher & Phillips, 2008). Liu et al. (2009) reportedthat the arthroconidia germination and germ tube elongation ofG. citri-auranti were significantly inhibited by thyme oil at a con-centration of 600 mL/L, and that thyme oil applied to Satsumamandarins inoculated with G. citri-aurantii reduced decay from 76%in the untreated control fruits to 35% after 30 d at 20 �C. Thetreatment did not harm the Satsuma mandarin.

H. Zhou et al. / Food Control 37 (2014) 277e283278

Among the volatile compounds that constitute plant essentialoils, citral, octanal, and a-terpineol show a broad spectrum ofantifungal activity against some citrus postharvest pathogens suchas Penicillium italicum and Penicillium digitatum (Caccioni,Guizzardi, Biondi, Renda, & Ruberto, 1998; Daferera, Ziogas, &Polissiou, 2000; Droby et al., 2008; Linde, Combrinck, Regnier, &Virijevic, 2010; Scora & Scora, 1998; Wolken, Tramper, & Werf,2002; Wuryatmo, Klieber, & Scott, 2003). However, knowledgeabout the antifungal activity of these volatile compounds againstG. citri-aurantii is rather limited. Therefore, this study aims todetermine the antifungal efficacy of citral, octanal, and a-terpineolagainst the mycelial growth of G. citri-aurantii. The antifungalmechanism will be investigated by determining (i) themorphology of the cell membranes using scanning electron mi-croscopy (SEM), (ii) the release of 260 nm absorbing cellularcomponents, (iii) the extracellular conductivity and pH, and (iv)the total lipid content.

2. Materials and methods

2.1. Fungal species

The mold of G. citri-aurantii was provided by the Department ofBiotechnology and Food Engineering, Xiangtan University, Xiang-tan, China. The fungus was purified and preserved at 28 � 2 �C onpotato dextrose agar (PDA). The spores’ concentrationwas adjustedto 5 � 105 cfu/mL using a hamacytometer.

2.2. Chemicals

Citral (95%), octanal (99%), and a-terpineol (90%) were obtainedfrom SigmaeAldrich (St. Louis, MO, USA). Cholesterol (95%)and phosphovanillin (98%) were purchased from TCI Shanghai(Shanghai, China). All the chemicals were analytic grade.

2.3. Antifungal activity of the three volatile compounds

Effects of citral, octanal and a-terpineol on the mycelialgrowth of G. citri-aurantii were tested in vitro by agar dilutionmethod (Yahyazadeh, Omidbaigi, Zare, & Taheri, 2008). PDA(20 mL) was poured into sterilized Petri dishes (90 mm diameter)and measured amounts of citral, octanal and a-terpineol wereadded to PDA mediums (plus with 0.05% Tween-80) to givedesired concentrations of 0.00, 0.25, 0.50, 1.00, 2.00, 4.00, 8.00 mL/mL. A 6 mm diameter disc of inocula was cut from the peripheryof an actively growing culture on PDA plates with a punchingbear, and then was placed at the center of each new Petri plate.The plates were incubated at 28 � 2 �C for 48 h. Each treatmentwas performed in triplicates. The percentage of inhibition ofmycelial growth (MGI) was calculated according to the followingformula:

MGI ð%Þ ¼ ½ðdc� dtÞ=dc� � 100

where dc (cm) is the mean colony diameter for the control sets anddt (cm) is the mean colony diameter for the treatment sets. Thelowest concentration that completely inhibited the growth of thefungus was considered the minimum inhibitory concentration(MIC). The minimum fungicidal concentration (MFC) was regardedas the lowest concentration that prevented growth of the pathogenafter a following 72 h incubation at 28 � 2 �C in a fresh PDA plate,indicating more than 99.5% killing of the original inoculums (Talibiet al., 2012).

2.4. Scanning electron microscopy (SEM)

The 4-day-old fungal cultures on potato dextrose agar (PDA)treatedwith three volatile compounds at various concentrations (0,MIC and MFC) were used for all SEM observations (Helal, Sarhan,Abu, & Abou, 2007; Yahyazadeh et al., 2008). About 5 � 10 mmsegments were cut from cultures growing on PDA plates andpromptly placed in vials containing 3% (v/v) glutaraldehyde in0.05 mol/L phosphate buffer (pH 6.8) at 4 �C. Samples were kept inthis solution for 48 h for fixation and then washed with distilledwater three times for 20 min each. Following which they weredehydrated in an ethanol series (30%, 50%, 70%, and 95%, v/v), for20min in each alcohol dilution and finally with absolute ethanol for45 min. Samples were then critical point dried in liquid carbondioxide. Fungal segments were placed in desiccators until furtheruse. Following drying, samples prepared were mounted on stan-dard 1/2 in SEM stubs using double-stick adhesive tabs and coatedwith gold-palladium electroplating (60 s, 1.8 mA, 2.4 kV) in aPolaron SEM Coating System sputter coater. All samples wereviewed in a JEOL JSM-6360LV SEM (JEOL, Tokyo, Japan) operating at25 kV at 5000� level of magnification.

2.5. Release of cellular constituents

The release of cellular constituents into the supernatants wasmeasured according to the method described previously (Paul,Dubey, Maheswari, & Kang, 2011) with minor modifications.Briefly, the 2-day-old mycelia from 100 mL potato dextrose broth(PDB) were collected by centrifugation at 4000g for 20min, washedthree times, and resuspended in 100 mL phosphate buffered saline(pH 7.0). The resulting suspensions were treated with the threevolatile compounds at various concentrations (0, MIC and MFC) for0, 30, 60 and 120 min. Then, 2 mL of samples was collected andcentrifuged at 12,000 g for 2min. To determine the concentration ofthe released constituents, 1 mL of supernatant was used to measurethe absorbance at 260 nm with the UV-2450 UV/Vis Spectropho-tometer (SHIMADZU international trade (Shanghai) Co. Ltd).

2.6. Measurement of extracellular conductivity and extracellular pH

The extracellular conductivity was measured according to themethod described previously (Shao, Cheng, Wang, Yu, & Mungai,2013) using a DDS-W conductivity meter (Shanghai Precision Sci-entific Instrument Co., Ltd. Shanghai, China). The extracellular pH ofG. citri-aurantii cells was determined by a Delta 320 pH meter(Mettler-Toledo Instruments (Shanghai) Co., Ltd., Shanghai, China)following the instructions. Initially, 100 mL fungal suspensions(105 cfu/mL) were added to 20 mL PDB and incubated in a moistchamber at 28 � 2 �C for 2 d. The mixtures were centrifuged at4000 g for 20min and the resulting pellet was collected, washed for2e3 times with sterilized double distilled water, and resuspendedin 20 mL sterilized double distilled water. After the addition of thethree volatile compounds at MIC or MFC, the extracellular con-ductivity and extracellular pH of G. citri-aurantii cells were deter-mined at 0, 30, 60 and 120 min of treatment. Control flasks withoutvolatiles were also tested. Results were expressed as amount ofextracellular conductivity (ms/cm) and pH in the growth media ineach interval of incubation.

2.7. Determination of lipid content

Total lipid content of G. citri-aurantii cells with the three volatilecompounds at various concentrations (0, MIC and MFC) weredetermined using phosphovanillin method (Helal et al., 2007;Wang et al., 2009). The 2-day-old mycelia from 50 mL PDB was

Table 1Antifungal activity of citral, octanal, and a-terpineol.

Concentration (mL/mL) Mycelial growth inhibition (%)*

Citral Octanal a-Terpineol

0.00 0.00 � 0.00a 0.00 � 0.00a 0.00 � 0.00b

0.25 73.53 � 2.31b 28.66 � 1.62b �8.87 � 0.93a**0.50 100.00 � 0.00c 100.00 � 0.00c �3.88 � 1.35b**1.00 100.00 � 0.00c 100.00 � 0.00c 14.35 � 2.74c

2.00 100.00 � 0.00c 100.00 � 0.00c 100.00 � 0.00d

4.00 100.00 � 0.00c 100.00 � 0.00c 100.00 � 0.00d

8.00 100.00 � 0.00c 100.00 � 0.00c 100.00 � 0.00d

*Values are presented as mean � SD.aedSignificant differences at P < 0.05 level.**Promotion of mycelial growth.

H. Zhou et al. / Food Control 37 (2014) 277e283 279

collected and centrifuged at 4000 g for 10 min. Then the sampleswere dried with a vacuum freeze drier for 4 h. About 0.1 g of drymyceliawere homogenized with liquid nitrogen and extractedwith4.0 mL of methanolechloroformewater mixture (2:1:0.8, v/v/v) ina clean dry test tube with vigorous shaking for 30 min. The tubeswere centrifuged at 4000 g for 10 min. The lower phase containinglipids was thoroughly mixed with 0.2 mL saline solution andcentrifuged at 4000 g for 10 min. Then, an aliquot of 0.2 mL chlo-roform and lipidmixturewas transferred to a novel tube and 0.5mLH2SO4 was added, heated for 10 min in a boiling water bath. Afterthat, 3 mL phosphovanillin was added and shake vigorously, andthen incubated at room temperature for 10 min. The absorbance at520 nm was utilized to calculate total lipid content from the stan-dard calibration curve using cholesterol as a standard.

2.8. Statistical analysis

All data were expressed as the mean � SD by measuring threeindependent replicates. Analysis of variance using one-way ANOVAfollowed by Duncan’s test was performed to test the significance ofdifferences between means obtained among the treatments at the5% level of significance using SPSS statistical software packagerelease 16.0 (SPSS Inc., Chicago, IL, USA).

3. Results

3.1. Antifungal activity

The effects of citral, octanal, and a-terpineol on the mycelialgrowth of G. citri-aurantii in vitro are shown in Table 1. Theminimum inhibitory concentration (MIC) and minimum fungi-cidal concentration (MFC) values of citral, octanal, and a-terpineolare presented in Table 2. When citral concentration reached0.25 mL/mL, nearly three quarters of mycelial growth wasinhibited, with a radial growth inhibition of 73.53%. At 0.50 mL/mL(MIC), citral completely inhibited the growth of G. citri-aurantii,whereas at 1.00 mL/mL (MFC), it induced fungicidal activity. Whenoctanal concentration reached 0.25 mL/mL, only 28.66% of myce-lial growth was inhibited. The MIC and MFC values of octanalwere 0.50 and 2.00 mL/mL, respectively. With respect to

Table 2MICs and MFCs of citral, octanal, and a-terpineol.

Treatment MIC (mL/mL)a MFC (mL/mL)b

Citral 0.50 1.00Octanal 0.50 2.00a-Terpineol 2.00 4.00

a Fungistatic concentration.b Fungicidal concentration.

a-terpineol, the mycelial growth of G. citri-auranti was slightlystimulated at concentrations between 0.25 and 0.50 mL/mL butwas partially inhibited at a concentration of 1.00 mL/mL. The MICand MFC values of a-terpineol were 2.00 and 4.00 mL/mL,respectively. Collectively, the MIC values of citral and octanalwere equal (0.50 mL/mL) but were significantly lower than that ofa-terpineol (2.00 mL/mL). Citral was supposed to be the mostpotent antifungal substance because it has the lowest MFC valueamong the three volatile compounds.

3.2. Scanning electron microscopy

The effect of the three volatile compounds on themorphology ofG. citri-aurantii as examined through SEM is shown in Fig. 1. Thecontrol fungus grown on PDA had normal tubular hyphae withregular, homogenous hyphae (Fig. 1a and b). After 2 d of treatmentusing the MIC and MFC of the three volatile compounds, all fungalmycelia showed considerable alterations with respect to hyphalmorphology. Treatment with citral at a concentration of 0.50 mL/mL(MIC) distorted and partly squashed the mycelia (Fig. 1c). Thisphenomenon was not observed in octanal (Fig. 1e) and a-terpineol(Fig. 1g). Moreover, shrunken and distorted mycelia were observed(Fig. 1d) after treatment with citral at a concentration of 1.00 mL/mL(MFC). A similar phenomenon was also observed on the hyphae ofG. citri-aurantii cells treated with octanal (Fig. 1f) and a-terpineol(Fig. 1h).

3.3. Release of cell constituents

The results of the release of cell constituents when G. citri-aurantii was treated with citral, octanal, and a-terpineol at threedifferent concentrations (0, MIC, and MFC) for 0, 30, 60, and120 min, respectively, is shown in Fig. 2. An immediately signif-icant (P < 0.05) increase in the release of cell constituents wasobserved when the fungi were treated with the volatile com-pounds at different concentrations. As the process timeincreased, a significant (P < 0.05) increase in the release of260 nm absorbing material was observed. The OD260 value in theG. citri-aurantii suspensions treated with citral at MIC (0.50 mL/mL) for 120 min was 0.295, the value was much higher (P < 0.05)than that of the control (0.103), but significantly lower (P < 0.05)than that of citral (0.636) at MFC (1.00 mL/mL). A similar phe-nomenon in octanal- and a-terpineol-treated samples was alsoobserved. The OD260 value of G. citri-aurantii suspensions treatedwith octanal at MIC and MFC maintained a moderate ascendingtrend, whereas the OD260 of G. citri-aurantii suspensions treatedwith a-terpineol at MIC and MFC maintained a smooth ascendingtrend.

3.4. Extracellular conductivity

The results of extracellular conductivity from G. citri-aurantiicells treatedwith citral, octanal, and a-terpineol at MIC andMFC fora period of 0 mine120 min are shown in Fig. 3. The values ofextracellular conductivity after 30min of exposure to citral MIC andMFC were 118.0 and 203.3 ms/cm, respectively, significantly higherthan that of the control (81.2 ms/cm) (P < 0.05). The change becamemore evident with the increase in exposure time. At 120 min ofexposure, the conductivity of G. citri-aurantii suspensions treatedwith citral, octanal, and a-terpineol at MIC and MFC markedlyincreased, with conductivity reaching 165.60,136.51, and 154.70 ms/cm for MIC and 307.03, 492.00, and 279.00 ms/cm for MFC,respectively.

Fig. 1. Scanning electron microphotography of G. citri-aurantii: a and b: untreated; c and d: treated with 0.50 and 1.00 mL/mL citral, respectively; e and f: treated with 0.50 and2.00 mL/mL octanal, respectively; g and h: treated with 2.00 and 4.00 mL/mL a-terpineol, respectively.

H. Zhou et al. / Food Control 37 (2014) 277e283280

3.5. Extracellular pH

The extracellular pH of G. citri-aurantii cells exposed to citral,octanal, and a-terpineol is presented in Fig. 4. A gradual increasein extracellular pH was observed in the control. Before reaching30 min of exposure to the three volatile compounds at MIC andMFC, a sharp reduction in the extracellular pH of the G. citri-aurantii suspensions occurred, followed by a gradual increase,reaching stable levels at 120 min of exposure. The extracellularpH values in the G. citri-aurantii suspensions after incubation

with citral at MIC and MFC for 30 min were 4.80 and 4.55,respectively, which were significantly lower than that of thecontrol (5.85) (P < 0.05). The extracellular pH in G. citri-aurantiisuspensions after incubation with octanal at MIC and MFC for30 min were 4.60 and 4.30, respectively, which were significantlylower than that of the control (5.94) (P < 0.05). The extracellularpH values in G. citri-aurantii suspensions after incubation with a-terpineol at MIC and MFC for 30 min were 4.84 and 4.53,respectively, which were significantly lower than that of thecontrol (5.89) (P < 0.05).

Fig. 2. Effects of the three volatile compounds on the 260 nm absorbing materialrelease of G. citri-aurantii ((-): control; (:): MIC; (C): MFC). Fig. 3. Effects of the three volatile compounds on the extracellular conductivity of

G. citri-aurantii ((-): control; (:): MIC; (C): MFC).

H. Zhou et al. / Food Control 37 (2014) 277e283 281

3.6. Total lipid content

The effect of citral, octanal, and a-terpineol on the total lipidcontent ofG. citri-aurantii cells is shown inTable 3. By increasing theconcentration of three volatile compounds, a significant (P < 0.05)decrease in the total lipid content of G. citri-aurantii cells wasobserved. The total lipid contents of G. citri-aurantii after incubationwith citral at MIC and MFC for 120 min were 111.1 � 3.9 and78.1 � 6.6 mg/g dry weight, respectively, which were significantlylower than that of control (151.6 � 3.8 mg/g dry weight). The totallipid content of G. citri-aurantii cells significantly decreased from151.6� 3.8mg/g dryweight in nonfumigated cells to 123.5� 9.3 and99.8 � 4.9 mg/g dry weight in cells fumigated with octanal at MICandMFC, respectively. The total lipid content of G. citri-aurantii cellsalso significantly decreased from 151.6 � 3.8 mg/g dry weight innonfumigated cells to 128.8� 1.7 and 111.2� 4.9mg/g dryweight incells fumigated with a-terpineol at MIC or MFC, respectively.

4. Discussions

In this study, citral, octanal, and a-terpineol were found to beeffective in inhibiting the growth of G. citri-aurantii in vitro. The

inhibitory effect was positively correlated with the concentration ofcitral, octanal, and a-terpineol. These results are consistent withthose of previous studies describing the antifungal activity of thesevolatile compounds (Caccioni et al., 1998; Daferera et al., 2000;Droby et al., 2008; Linde et al., 2010; Scora & Scora, 1998; Wolkenet al., 2002; Wuryatmo et al., 2003). Among the three volatile com-pounds, citral showed themost potent antifungal effect, followed byoctanal and a-terpineol. The MIC values of citral, octanal, and a-terpineol obtained in this study were 0.50, 0.50, and 2.00 mL/mL,respectively, which were as low as those obtained with the fungusreported in previous studies (Tyagi & Malik, 2011; Wolken et al.,2002). This finding suggests that these volatile compounds are po-tential biological fungicides. a-Terpineol, at concentrations of 0.25and 0.50 mL/mL, promoted the mycelial growth of G. citri-aurantii.However, it completely inhibited the mycelial growth of G. citri-auranti at a concentration of 2.00 mL/mL. This phenomenonwas notobserved in citral and octanal, indicating that the aldehyde com-pounds are more effective than alcohols in controlling citrus post-harvest pathogens, as reported by another study (Droby et al., 2008).

The potential mechanisms of the antimicrobial activity of vola-tile compounds remain unclear, but a number of mechanisms have

Fig. 4. Effects of the three volatile compounds on the extracellular pH of G. citri-aur-antii ((-): control; (:): MIC; (C): MFC).

H. Zhou et al. / Food Control 37 (2014) 277e283282

been proposed (Burt, 2004). Some studies suggest that volatilecompounds can penetrate the cell, where such compounds inter-fere with the cellular metabolism (Marino, Bersani, & Comi, 2001).Other studies suggest that volatile compounds disturb the cellularmembrane, react with active sites of enzymes, or act as Hþ carriers,thus depleting the adenosine triphosphate pool (Bajpai et al., 2013;Chang, Cheng, & Chang, 2001; Farag, Daw, Hewedi, & El-Baroty,

Table 3Effect of the three volatile compounds on the total lipid contents of G. citri-aurantii.

Concentration (mL/mL) Total lipid contents (mg/g dry weight)*

Citral Octanal a-Terpineol

Control 151.6 � 3.8a 151.6 � 3.8a 151.6 � 3.8aMIC 111.1 � 4.2b 123.5 � 9.3b 128.8 � 1.7bMFC 78.1 � 6.6c 99.8 � 4.9c 111.2 � 4.9c

*Values are presented as mean � SD.aecSignificant differences at P < 0.05 level.

1989; Ultee, Bennik, & Moezelaar, 2002). In the current experi-ment, in vitro antifungal activity results enabled us to hypothesizethat the potential antifungal activity of citral, octanal, and a-terpineol against G. citri-aurantii could be closely correlated withthe physiology of the hyphae. To verify this hypothesis, G. citri-aurantii cells treated with citral, octanal, and a-terpineol weresubjected to SEM observations. As expected, SEM analysis showedthat the volatile compounds could alter the morphology of G. citri-aurantii hyphae, disrupting the membrane integrity. Our results arein agreement with those reported by Yahyazadeh et al. (2008) andTyagi andMalik (2011). These changes may generally occur becauseof an increase in the permeability of cells, and such changescommonly result in the leakage of small molecular substances, ions,and lesions as well as discrepancies in cell metabolism (Bajpai et al.,2013). Therefore, the antifungal activity of the three volatile com-pounds against G .citri-aurantii could be the result of attacks on thecell membrane and disruption of the cytoplasm in the hyphae, ul-timately leading to the death of the mycelia.

To confirm this hypothesis, the absorbance at 260 nm of thesupernatant, extracellular conductivity, and extracellular pH weredetermined to explain the release of the cellular material andthe changes of membrane permeabilization. The release ofultraviolet-absorbing cell materials is an index of cell lysis. Themarked leakage of cytoplasmic material is commonly used as anindication of gross and irreversible damage to the cytoplasmicand plasma membranes (Bajpai et al., 2013; Paul et al., 2011; Shaoet al., 2013; Turgis, Han, Caillet, & Lacroix, 2009). Results showedthat, after the addition of the three volatile compounds, therelease of cell constituents in the fungi suspensions visiblyincreased with increasing volatile compound concentration. Themaximum release of cell constituents was observed in the G. citri-aurantii cell suspensions treated with citral at MFC, showing anabsorbance of 0.636. The extracellular conductivity in the cellsuspensions with the three volatile compounds was higher thanthat of the control at all times, and the values increased with theincreasing concentrations of the compounds. Meanwhile, after30 min of exposure, the three volatile compounds apparentlyinduced the leakage of intracellular protons, as evidenced by thedecrease in the values of extracellular pH. These findings suggestthat irreversible damage to the cytoplasmic membranes ofG. citri-aurantii occurred, and the ions inside the cells leaked andan imbalance in osmotic pressure of the intra- and extra-cellularmembrane occurred.

Lipids are the major component of the cell membrane and havemany important functions, which include adjusting the liquidity ofthe membrane, increasing the stability of the membrane, andreducing the permeability of water-soluble materials (Heaton &Randall, 2011). The decrease in lipid content suggests that mem-brane stability decreased while the permeability of water-solublematerials increased (Helal et al., 2007). Volatile compounds suchas terpenes have been reported to disrupt or penetrate the lipidstructures of the cells (Prashar, Hili, Veness, & Evans, 2003). Thetotal lipid content of Aspergillus niger cells significantly decreased(P < 0.01) from 130 � 5.6 mg/g dry weight in nonfumigated cells to110 � 4.4 mg/g dry weight in cells fumigated with 1 mL/mL ofCymbopogon citratus essential oil (Helal, Sarhan, Abu Shahla, &Abou Elkair, 2006). Helal et al. (2007) also reported that the totallipid content of Aspergillus flavus cells significantly decreased from117 � 6.2 mg/g dry weight in nonfumigated cells to 90 � 5.6 mg/gdry weight in cells fumigated with 1 mL/mL of C. citratus essentialoil. In the present study, the addition of citral, octanal, and a-terpineol significantly decreased the lipid content of G. citri-aur-antii. This result suggests that the three volatile compounds arecapable of acting on the cell membrane structure and disrupt thecell membrane integrity.

H. Zhou et al. / Food Control 37 (2014) 277e283 283

In conclusion, this study showed that citral, octanal, and a-terpineol can significantly inhibit the mycelial growth of G. citri-auranti. The antifungal activity can be attributed to the disruptionof the cell membrane integrity and the leakage of cell components.

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (Grant No. 31271964) and Research Founda-tion of Education Bureau of Hunan Province (No. 12B126).

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