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Food Microbiology 41 (2014) 19e26

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Food Microbiology

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Purification and characterization of antifungal compounds fromLactobacillus plantarum HD1 isolated from kimchi

Eun Hye Ryu a, Eun Ju Yang a, Eun Rhan Woo b, Hae Choon Chang a,*

aDepartment of Food and Nutrition, Kimchi Research Center, Chosun University, Seoseok-dong, Dong-gu, Gwangju 501-759, Republic of KoreabDepartment of Pharmacy, Chosun University, Gwangju 501-759, Republic of Korea

a r t i c l e i n f o

Article history:Received 6 February 2013Received in revised form15 January 2014Accepted 19 January 2014Available online 25 January 2014

Keywords:Lactobacillus plantarumAntifungal activity3-Hydroxy fatty acids5-Oxododecanoic acidKimchi

* Corresponding author. Tel.: þ82 62 230 7345; faxE-mail address: hcchang@chosun.ac.kr (H.C. Chang

0740-0020/$ e see front matter � 2014 Elsevier Ltd.http://dx.doi.org/10.1016/j.fm.2014.01.011

a b s t r a c t

Strain HD1 with antifungal activity was isolated from kimchi and identified as Lactobacillus plantarum.Antifungal compounds from Lb. plantarum HD1 were active against food- and feed-borne filamentousfungi and yeasts in a spot-on-the-lawn assay. Antifungal activity of Lb. plantarum HD1 was strongeragainst filamentous fungi than yeast. Antifungal compounds were purified using solid phase extraction(SPE) and recycling preparative-HPLC. Structures of the antifungal compounds were elucidated byelectrospray ionization-mass spectrometry and nuclear magnetic resonance. Active compounds from Lb.plantarum HD1 were identified as 5-oxododecanoic acid (MW 214), 3-hydroxy decanoic acid (MW 188),and 3-hydroxy-5-dodecenoic acid (MW 214). To investigate the potential application of these antifungalcompounds for reduction of fungal spoilage in foods, Korean draft rice wine was used as a food model.White film-forming yeasts were observed in control draft rice wine after 11 days of incubation. However,film-forming yeasts were not observed in draft rice wine treated with SPE-prepared culture supernatantof Lb. plantarum HD1 (equivalent to 2.5% addition of culture supernatant) until 27 days of incubation. Theaddition of antifungal compounds to Korean draft rice wine extended shelf-life up to 27 days at 10 �Cwithout any sterilization process. Therefore, the antifungal activity of Lb. plantarum HD1 may lead to thedevelopment of powerful biopreservative systems capable of preventing food- and feed-borne fungalspoilage.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Molds and yeasts are able to grow on most foods, includingnatural foods, processed foods, and fermented foods. Molds andyeasts play a central role in the spoilage of food products andfeed systems (Loureiro and Malfeito-Ferreira, 2003; Filtenborget al., 1996). Such spoilage can cause considerable economic loss,and further contamination by mycotoxins can cause publichealth problems (Schnürer and Magnusson, 2005). The currentneed for biopreservation has spurred the search for food-compatible antimicrobials produced by microorganisms. Lacticacid bacteria (LAB) are promising alternatives to chemical pre-servatives, as antimicrobial compounds from LAB show potentialin suppressing food-borne yeasts and molds (De Muynck et al.,2004). Additionally, LAB have a long history of use as bio-preservatives for food and feed storage (Stiles, 1996). Antifungal

: þ82 62 222 8086.).

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compounds from LAB include metabolites containing proteina-ceous (Atanassova et al., 2003; Magnusson and Schnürer, 2001;Okkers et al., 1999) as well as low molecular mass compounds(less than 100 Da) such as reuterin, carboxylic acids and theirderivatives, fatty acids and their derivatives, cyclic dipeptides,and nucleosides (Talarico et al., 1988; Corsetti et al., 1998; Niku-Paavola et al., 1999; Lavermicocca et al., 2000; Ström et al., 2002;Sjögren et al., 2003; Dal Bello et al., 2007; Prema et al., 2010;Yang and Chang, 2010; Yang et al., 2011; Ryan et al., 2011; Wanget al., 2012; Li et al., 2012). The number of reports characterizingof antifungal compounds from LAB is still low, whereas therehave been numerous investigations on antibacterial compoundsfrom LAB (Reis et al., 2012). An antifungal compounds from LABhave been shown to be applicable to the control of food-borneyeasts and molds. However, to date, the antifungal activity ofLAB remains poorly understood. Thus, new food-grade antifungalcompounds should be continuously identified along with thedevelopment of commercial formulations.

Korean draft rice wine (makgeolli) is a traditional Korean fer-mented rice wine using cooked rice as a main material and a

E.H. Ryu et al. / Food Microbiology 41 (2014) 19e2620

mixture of molds and yeasts (nuruk) as a fermentation starter. Afterthe fermentation process, its alcohol content reaches 6e8% (Kanget al., 2012; Lee et al., 2012). Rice wine is bottled without anysterilization process, even though this has changed in recent yearsdue to heat-sterilization.

This paper reports the isolation and identification of antifungalactivity of Lactobacillus plantarum HD1 from kimchi, a traditionalKorean fermented vegetable product. Furthermore, the presentstudy describes the purification and characterization of the lowmolecular mass antifungal compounds obtained from this isolatedstrain. In addition, it evaluates the antifungal capacity of Lb. plan-tarum HD1 as a biopresevative in a food model of Korean draft ricewine.

2. Materials and methods

2.1. Cultures and media

The microbial strains used in this study and their culture mediawith culture conditions are listed in Table 1. LAB were grown in deMan Rogosa Sharpe (MRS) broth (Difco, Detroit, MI, USA) at 30 �Cfor 24 h. Yeast strains were grown on yeast extractepeptoneedextrose agar (YPD; Difco) or yeast mold agar (YM; Difco). Moldswere grown on malt extract agar (MEA; Difco) or potato dextroseagar (PDA; Difco).

2.2. Isolation of antifungal activity of LAB

Kimchi samples were collected from a home, restaurant, andtemple located in South Korea. Screening for antifungal activity ofLAB was performed as previously described (Yang and Chang,2008). Kimchi samples were macerated using a hand blender

Table 1Microbial strains used in this study.

Strain Mediuma Culturecondition

Reference

LABLactobacillusplantarum HD1

MRS,MRS þ 2% CaCO3

30 �C, 24 h This study

YeastsPichia kudriavzeviiGY1

YPD 30 �C, 24 h Chang and Yang, 2012

Saccharomycesservazzii GY2

YPD 30 �C, 24 h Chang and Yang, 2012

Saccharomycesbulderi HY

YPD 30 �C, 24 h Chang and Yang, 2012

Kazachstaniaexigua WY3

YPD 30 �C, 24 h Chang and Yang, 2012

Candida albicansATCC 11006

YM 25 �C, 48 h Rudek, 1978

MoldsAspergillus flavusATCC 22546

MEA 30 �C, 48 h Richard et al., 1969

Aspergillus fumigatusATCC 96918

MEA 30 �C, 48 h Pettit et al., 1998

Aspergilluspetrakii PF-1

PDA 30 �C, 48 h Yang and Chang, 2008

Aspergillusochraceus PF-2

PDA 30 �C, 48 h Yang and Chang, 2008

Aspergillusnidulans PF-3

MEA 30 �C, 48 h Yang and Chang, 2008

Cladosporiumgossypiicola KF-2

PDA 25 �C, 72 h Yang and Chang, 2008

Penicillium roquefortiATCC 10110

PDA 25 �C, 72 h Pillai and Weete, 1975

a MRS: de Man Rogosa Sharpe (Difco, Detroit, MI, USA); YPD: yeast extract-peptone-dextrose agar (Difco); YM: yeast mold agar (Difco); MEA: malt extractagar (Difco); PDA: potato dextrose agar (Difco).

(Hanil, Seoul, Korea) for 2 min. The obtained kimchi juice wasfiltered through a sterile thin cloth, after which the filtrate wasserially diluted with sterile-distilled water and then spread ontoMRSþ2% CaCO3 agar. The plates were incubated at 30 �C for 2 days,and the tentatively considered LAB strains were selected. Amongthe selected strains, rod-type LAB were selected. Their antifungalactivities against a food-borne spoilage fungus, Aspergillus fumiga-tus ATCC 96918, and a film-forming yeast, Pichia kudriavzevii GY1,were determined according to a previously described method(Yang and Chang, 2008). Thereafter, cell-free culture supernatantsof LAB were concentrated 5-fold and their antifungal activitiesassayed using paper disc assay.

2.3. Identification of the isolate

The isolate was identified based on its morphological charac-teristics under a microscope, biochemical properties using an API50 CHL (BioMérieux, Marcy-I’Etoile, France), and determination of16S rRNA gene sequences using an ABI prism 3730 DNA analyzer(Applied Biosystems, Foster city, CA, USA) according to the methoddescribed by Yang and Chang (2008). The determined 16S rRNAgene sequences were compared with sequences available in theGenBank database (http://blast. ncbi.nlm.nih.gov/Blast.cgi) usingthe BLASTN program.

2.4. Antifungal activity assays

The paper disc assay (Yang and Chang, 2008) and spot-on-the-lawn assay (Hoover and Harlander, 1993) were used to detectantifungal activities. Plates were prepared by adding the mold (106

spores per 20mL ofMEA) to 1.5% (w/v) bactoagar (Duchefa, Harlem,The Netherlands) or by spreading the yeast (106 CFU/mL) onto YPDagar, as listed in Table 2. A spore solution was prepared as previ-ously reported (Yang and Chang, 2010). For the paper disc assay,paper discs (diameter 8 mm; Advantec, Tokyo, Japan) on MEAplates were spotted with 100 mL of sample. The plates were incu-bated at 30 �C for 48 h and examined for inhibition zones. For thespot-on-the-lawn assay, 10e25 mL of sample was spotted onto thesensitive mold and yeast plates. Antifungal activity, expressed asarbitrary units (AU) per milliliter, was defined as the reciprocal ofthe highest dilution at which fungal growth was inhibited. Theantifungal titer was calculated as (1000/d) D, where D is the dilu-tion factor and d is the dose (amount of antifungal samples pipettedonto each spot). The above experiment was done in triplicate.

Table 2Antifungal activity of Lb. plantarum HD1 against yeasts and molds.

Microorganism Indicator species Activitya (AU/mL)

Yeasts Pichia kudriavzevii GY1 32Saccharomyces servazzii GY2 16Saccharomyces bulderi HY 8Kazachstania exigua WY3 128Candida albicans ATCC 11006 8

Molds Aspergillus flavus ATCC 22546 640Aspergillus fumigatus ATCC 96918 640Aspergillus petrakii PF-1 320Aspergillus ochraceus PF-2 320Aspergillus nidulans PF-3 320Cladosporium gossypiicola KF-2 160Penicillium roqueforti ATCC 10110 160

a Activity was determined using spot-on-the-lawn assay, as described in the text,and calculated in arbitrary units (AU) per milliliter. Measurement was repeated atleast two times using three independent Lb. plantarum HD1 culture preparations.

E.H. Ryu et al. / Food Microbiology 41 (2014) 19e26 21

2.5. Purification of antifungal compounds from Lb. plantarum HD1

Solid phase extraction (SPE) of the culture supernatant wascarried out according to a previously described method (Yang andChang, 2010). Briefly, a 24 h culture of Lb. plantarum HD1 grownin MRS broth at 30 �C was centrifuged (9500 � g, 15 min) and thenfilter-sterilized through a 0.45 mm pore filter (Advantec). Super-natant (2.5 L) of Lb. plantarumHD1was loaded onto the SPE column(Isolute, C18 EC, 10 g; International Sorbent Technology, Hengoed,UK), after which the column was washed with 5% (v/v) aqueousacetonitrile and eluted with 30 mL of 95% (v/v) aqueous acetoni-trile. The eluted sample was then concentrated by vacuum evapo-ration in a Speed Vac apparatus (VS-802, Vison, Daejeon, Korea).The SPE-prepared sample containing the active substance wasfurther separated using a HPLC. We used a recycling preparative-HPLC system (LC9104; Japan Analytical Industry, Tokyo, Japan)fitted with a 3702 UV detector (Japan Analytical Industry). Thecolumn used was a JAIGEL-W252 gel permeation chromatographycolumn (20 � 500 mm, Japan Analytical Industry). The first andsecond HPLC elution solvent was 50% (v/v) aqueous acetonitrilewhile the third and fourth HPLC elution solvent was 40% (v/v)aqueous acetonitrile applied at a flow rate of 3 mL/min. For thefourth HPLC, active fractions obtained from the third HPLC werereseparated by recycling preparative-HPLC until a peak with anti-fungal activity was obtained. Elution was monitored using UV de-tector at 210 nm. All peaks were measured for antifungal activityusing the spot-on-the-lawn method (Hoover and Harlander, 1993).

2.6. Identification of antifungal compounds

Structures of the antifungal compounds were determined usingnuclear magnetic resonance (NMR) spectroscopy, gaschromatography-mass spectrometry (GCeMS), and electrosprayionization-mass spectrometry (ESI-MS).

NMR spectra were recorded on samples in CD3CN and D2O on aBruker Advance-500 NMR spectrometer (Bruker Biospin GmBH,Rheinstetten, Germany). All spectra were recorded at 24 �C. One-dimensional 1H and 13C NMR experiments in combination withDEPT (Distortionless Enhancement by Polarization Transfer), two-dimensional 1He13C heteronuclear multiple quantum coherence(HMQC), 1He13C heteronuclear multiple bond correlation (HMBC),and 1He1H correlation spectroscopy (COSY) experiments wereperformed.

GCeMS was performed using a Clarus 680 GC/MS 600T model(PerkineElmer, Boston, MA, USA) equipped with a DB-1701 column(30 m � 0.25 mm; film thickness, 0.25 mm; Agilent, Folsom, CA,USA). The carrier gas was helium at a constant flow rate of 1 mL/min. The oven temperature was held at 40 �C for 5 min, raised to280 �C at a rate of 15 �C/min, and then held for 15 min. The injectorand GC transfer line temperatures were 250 �C and 260 �C,respectively. The mass detector was operated in electron impactmode at an ionization energy of 70 eV. Compound identificationswere made by comparison of the GC-retention index (SadtlerResearch Laboratories, 1986) with the NIST mass spectral searchprogram (NIST/EPA/NIH mass spectral library ver. 2.0 f).

To acquire the mass spectra in positive-ion and negative-ionmodes, ESI-MS was performed using a quadrupole orthogonaltime-of-flight (QTOF) mass spectrometer (synapt G2, Waters,Manchester, UK).

2.7. Application of antifungal compounds as biopreservatives infood

Application of antifungal compounds from Lb. plantarumHD1 asbiopreservatives in food was carried out. First, the antifungal

activity of Lb. plantarum HD1 was compared to those of threecommonly used antifungal preservatives. Potassium sorbate (0.1%,w/v; Sigma, St. Louis, MO, USA), sodium benzoate (0.1%, w/v;Aldrich, Milwaukee, WI, USA), and natamycin (pimaricin 20 ppm;Sigma) were used at their maximum approved concentrations, asset by the Food and Drug Administration (FDA). The three pre-servatives were dissolved in 20 mM sodium acetate (pH 4.0). Pre-pared cell-free supernatant of Lb. plantarum HD1 was concentrated(5-fold) in 20mM sodium acetate (pH 4.0). A. fumigatus ATCC 96918and P. kudriavzevii GY1 were used as sensitive strains. Antifungalactivities were examined by paper disc assay.

The antifungal compounds from Lb. plantarum HD1 as bio-preservatives were added to Korean draft rice wine. An SPE samplecontaining active substances prepared as in Materials and methodsSection 2.5 was dissolved in 20 mM sodium acetate (pH 4.0), afterwhich 10 mL of the SPE sample (equivalent to 5 mL of culture broth)was added to 200 mL of Korean draft rice wine, which was notsterilized after fermentation. Korean draft rice wine was incubatedat 10 �C for 30 days, and growth of film-forming yeasts, whichinduce decay of Korean draft rice wine, was observed.

2.8. Replication of experiments

Antifungal activity assays as well as application of antifungalcompounds from Lb. plantarum HD1 to food were carried out induplicate, with three independent sample preparations each.

3. Results and discussion

3.1. Isolation and identification of antifungal activity of LAB

Ninety-one rod-type LAB were isolated from approximately 80collected kimchi samples. Among them, strain HD1 showed thestrongest antifungal (anti-mold and anti-yeast) activity. Strain HD1was gram-positive and catalase-negative. Assessment of biochem-ical characteristics using the API CHL system found that strain HD1belonged to Lb. plantarum (data not shown). When the 16S rRNAgene sequences (1368 bp) of isolate HD1 were determined (Gen-Bank accession No. JQ343914) and compared with those of typestrains of LAB in GenBank, the sequences of strain HD1 showed99.9% homology with those of Lb. plantarum NBRC 15891T. Thus,isolate HD1 was finally designated as Lb. plantarum HD1.

3.2. Spectrum of antifungal activity

Antifungal activities of Lb. plantarum HD1 against various yeastsand molds were determined (Table 2). Lb. plantarum HD1 showedstronger activities against molds (160e640 AU/mL) than yeasts (8e128 AU/mL). The strongest activities (640 AU/mL) of Lb. plantarumHD1 were against Aspergillus flavus and A. fumigatus, whereas theweakest activities (8 AU/mL) were against Saccharomyces bulderiand Candida albicans.

3.3. Purification of antifungal compounds

Twenty-one fractions were acquired in the first injection intopreparative-HPLC (Fig. 1A), and their antifungal activities (anti-mold activity against A. fumigatus; anti-yeast activity againstP. kudriavzevii) were assayed using the spot-on-the-lawn method(Fig. 1a). Among the 21 fractions in Fig. 1, active fractions 11 to 15were collected, combined together, and injected into the secondpreparative-HPLC. The injected sample was then separated into 11fractions (Fig. 1B), with fractions 9 to 11 showing the highest ac-tivities (Fig. 1b). These three fractions were then combined andinjected into the third preparative-HPLC. This procedure yielded 22

Fig. 1. Purification of antifungal compounds from culture supernatant of Lb. plantarum HD1 by preparative-HPLC. Chromatogram of preparative-HPLC after solid-phase extraction(SPE) by the first injection (A), chromatogram of recycling preparative-HPLC of fractions 11e15 by the second injection (B), chromatogram of recycling preparative-HPLC of fractions9e11 by the third injection (C). Fractions collected by HPLC purification were concentrated by vacuum evaporation and dissolved in 20 mM sodium acetate (pH 4.0), and theirantifungal activities were assayed on A. fumigatus and P. kudriavzevii lawn plates.

Fig. 2. Purification of antifungal compounds from partially purified fractions through preparative-HPLC by recycling preparative-HPLC. Chromatograms of recycling preparative-HPLC of fraction 7 (A), fraction 11 (B), and fraction 12 (C). Left-and-right arrows (4) indicate each recycling section. Fractions collected by HPLC purification were concentratedby vacuum evaporation and dissolved in 20 mM sodium acetate (pH 4.0), and their antifungal activities were assayed on A. fumigatus and P. kudriavzevii lawn plates.

E.H. Ryu et al. / Food Microbiology 41 (2014) 19e2624

fractions, of which three were selected displaying both high anti-mold and anti-yeast activities with one peak shape: fractions 7,11, and 12 (Fig. 1C and c).

The selected fractions 7, 11, and 12 were separately injected intothe fourth HPLC and then reseparated by recycling preparative-HPLC. Fraction 7 was reseparated into 13 fractions after six runsof recycling preparative-HPLC (Fig. 2A), with fraction 7e7 showingthe highest activities against A. fumigatus and P. kudriavzevii(Fig. 2a). Fraction 11 was reseparated two times by recyclingpreparative-HPLC, whereas fraction 11 showed one neat peak andcould not be further separated (Fig. 2B). Fraction 12 was resepa-rated into seven fractions by four runs of recycling preparative-HPLC (Fig. 2C), with fraction 12e5 showing the highest activitiesagainst both mold and yeast (Fig. 2c). Recovery of the antifungalcompounds and determination of their activities according to thepurification steps are shown in Table 3. Purified fractions 11, 7e7,and 12e5 showed total activities of 6.4 � 103 AU, 3.8 � 103 AU, and8.0 � 102 AU, and their total activities were 0.02%, 0.012%, and0.003% of the culture supernatant, respectively.

3.4. Identification of antifungal compounds

When the three purified active compounds in fractions 11, 7e7,and 12e5 were analyzed by GCeMS, one peak per fraction wasdetected with retention time of 18.06, 16.70, and 17.90 min,respectively. However, compound identification using the NISTmass spectral search program failed; we could only obtain infor-mation that all three purified compounds contained C-chain and e

COOH ligand structures through GCeMS analysis (data not shown).Therefore, the structures of the three purified active compounds

in fractions 11, 7e7, and 12e5 were elucidated by ESI-MS and NMR.Based on the [ESþ] and [ES�] spectra, the molecular masses offractions 11, 7e7, and 12e5 were determined to be ESI-MS(m/z);214, 188, and 214, respectively. According to the NMR spectra, the1H NMR spectrum of fraction 11 showed one primary methyl groupat d 0.89 (3H, t, J¼ 7.2 Hz) as well as ninemethylene groups at d 1.30(8H, m), 1.53 (2H, qui, J ¼ 7.2 Hz), 1.81 (2H, qui, J¼ 7.2 Hz), 2.28 (2H,br s), 2.43 (2H, t, J ¼ 7.2 Hz), and 2.51 (2H, t, J ¼ 7.2 Hz). In addition,the 13C NMR and HSQC spectral data revealed 12 carbons, includingtwo carbonyl groups at d 213.5 and 178.8, one methyl group atd 14.6, and nine methylene groups at d 43.7, 42.6, 34.4, 33.0, 30.4,30.3, 25.1, 23.8, and 20.4. The 1H NMR spectrum of fraction 7e7showed one primary methyl group at d 0.89 (3H, t, J ¼ 7.2 Hz), oneoxymethine proton at d 3.97 (1H, br s), and sevenmethylene groupsat d 1.32 (10H, br s), 1.47 (2H, s), and 2.39 (2H, m). In addition, the13C NMR and HSQC spectral data revealed 10 carbons, including onecarboxylic group at d 175.9, one oxygenated carbon at d 69.0, one

Table 3Purification of antifungal compounds produced by Lb. plantarum HD1.

Purification stage Vol. (mL) Activitya

(AU/mL)Total activityb

(AU)Recovery (%)

Culture supernatant 50000 640 3.2 � 107 100.000Solid phase extraction 40 51200 2.1 � 106 6.4001st HPLC fractions 11e15 40 6400 2.6 � 105 0.8002nd HPLC fractions 9e11 25 3200 8.0 � 104 0.2503rd HPLCFraction 7 3.5 6400 2.2 � 104 0.070Fraction 11 2.0 3200 6.4 � 103 0.020Fraction 12 1.5 1600 2.4 � 103 0.0084th HPLC (recycling process)Fraction 7e7 1.2 3200 3.8 � 103 0.012Fraction 12e5 1.0 800 8.0 � 102 0.003

a Activity was determined against A. fumigatus ATCC 96918 and was calculated inAU/mL, as described in the text.

b Total activity was calculated as the total AU within the volume of the sample(mL).

methyl group at d 14.6, and seven methylene groups at d 43.0, 38.3,33.2, 30.8, 30.6, 26.8, and 23.9. The 1H NMR spectrum of fraction12e5 showed one primary methyl group at d 0.89 (3H, t, J¼ 7.2 Hz),one oxymethine proton at d 4.00 (1H, qui, J ¼ 6.5 Hz), two olefinicprotons at d 5.49 (1H, m), 5.43 (1H, m), and sevenmethylene groupsat d 1.32 (8H, m), 2.05 (2H, qua, J ¼ 7.0 Hz), 2.27 (2H, t-like,J ¼ 7.5 Hz), 2.51 (1H, dd, J ¼ 8.4, 15.6 Hz), and 2.34 (1H, dd, J ¼ 8.3,15.4 Hz). In addition, the 13C NMR and HSQC spectral data revealed12 carbons, including one carboxylic group at d 176.3, oneoxygenated carbon at d 69.7, two olefinic carbons at d126.2, 133.6,one methyl group at d 14.6, and seven methylene groups at d 42.8,36.0, 33.1, 30.9, 30.3, 28.5, and 23.9. Thus, according to the ESI-MSand NMR analyses, the active compounds in fractions 11, 7e7, and12e5 were elucidated as 5-oxododecanoic acid, 3-hydroxy dec-anoic acid, and 3-hydroxy-5-dodecenoic acid, respectively.

3-Hydroxy fatty acid compounds, such as 3-hydroxy decanoicacid and 3-hydroxy-5-dodecenoic acid in this study, have alreadybeen reported as antifungal substances by Sjögren et al. (2003).They reported four antifungal substances, 3-hydroxy decanoic acid,3-hydroxy-5-dodecenoic acid, 3-hydroxydodecanoic acid, and 3-hydroxytetradecanoic acid, from Lb. plantarum MiLAB 14. Theyfurther reviewed that the mechanisms behind the antifungal ef-fects of 3-hydroxy fatty acids are due to detergent-like properties ofthe compounds that alter cellular membrane structure in the targetorganisms. However, to date, 5-oxododecanoic acid has never beenreported as an antifungal compound of LAB. 5-Oxododecanoic acidis a peach- and cream-like aromatic compound accepted as agenerally recognized as safe (GRAS) flavoring substance by the FDA(Smith et al., 2009). It can be chemically synthesized and used inimitation dairy and milk products (http://www.fao.org/ag/agn/jecfa-flav/details.html?printable¼true&flavId¼6964). We believethat our study is the first report regarding the natural production of5-oxododecanoic acid by LAB Lb. plantarum HD1 with anti-yeast aswell as anti-mold activities.

3.5. Application of antifungal compounds from Lb. plantarum HD1to food

Fig. 3 shows the antifungal activities of Lb. plantarum HD1 cul-ture supernatant along with those of other widely used antifungalpreservatives, sodium benzoate, potassium sorbate, and pimaricin.Sodium benzoate is approved by the FDA as a food preservative andwas first adapted by the food industry for use at 0.05e0.1% (w/v)(Jay, 1992). Potassium sorbate is used at 0.05e0.1% (w/v), whereaspimaricin is used at less than 20 ppm (Davidson, 2001). In this

Fig. 3. Comparison of antifungal activity of Lb. plantarum HD1 with those of com-mercial preservatives against A. fumigatus and P. kudriavzevii. 1, 5-fold-concentratedMRS broth; 2, 5-fold-concentrated cell-free supernatant of Lb. plantarum HD1; 3, So-dium benzoate (0.1%); 4, Potassium sorbate (0.1%); 5, Pimaricin (20 ppm). Paper discassay was used for antifungal activity; 100 mL of each sample was spotted onto discwith sensitive lawn.

Fig. 4. Growth of film-forming yeasts on Korean draft rice wine treated with SPE-prepared culture supernatant of Lb. plantarum HD1 (equivalent to 5 mL of culture) (A) and controlKorean draft rice wine (B) during incubation at 10 �C for 30 days.

E.H. Ryu et al. / Food Microbiology 41 (2014) 19e26 25

study, the filamentous fungus A. fumigatus was much more sensi-tive to all antifungal agents compared to the film-forming yeastP. kudriavzevii. Antifungal activity of the 5-fold-concentrated cul-ture supernatant of Lb. plantarum HD1 (equivalent to 0.5 mL ofculture broth) was significantly higher compared to those of otherfood preservatives, even at their maximum approved concentra-tions. Anti-yeast activity of the 5-fold-concentrated culture super-natant of Lb. plantarum HD1was clearly observed, whereas those ofother food preservatives could hardly be detected. Five-fold-concentrated MRS broth itself as a control did not show any anti-mold or anti-yeast activity (Fig. 3).

The ability to prevent or to retard the growth of food spoilagefungi on foods considered to be the most important regardingbiopreservation for human health and economy. Therefore,partially purified antifungal compounds from Lb. plantarum HD1,as a novel biopreservative, were tested in a food model of Koreandraft rice wine. To prepare the partially purified antifungal com-pounds from Lb. plantarum HD1, the acetonitrile-eluted culturesupernatant of Lb. plantarum HD1 from the SPE column (C18-column) was vacuum evaporated, after which the dried samplewas dissolved in 20 mM sodium acetate buffer (pH 4.0). The SPE-prepared sample contained the three identified antifungal com-pounds as well as other undefined antifungal compounds pro-duced by Lb. plantarum HD1, as we observed antifungal activitiesin other fractions (Figs. 1 and 2). The shelf-life of draft rice winewas 10 days below 10 �C due to the growth of film-forming yeastssuch as P. kudriavzevii or P. membranifaciens. Further, growth offilm-forming yeasts in Korean rice wine is associated with off-flavors, which compromises beverage quality. When we treatedthe partially purified antifungal compounds mixture (equivalent to2.5% addition of culture supernatant) to Korean draft rice wine, asshown in Fig. 4, film-forming yeasts were not observed in ricewine treated with the SPE-prepared culture supernatant of Lb.plantarum HD1 until 27 days, after which thin film spots wereobserved from 28 to 29 days. On the other hand, film-formingyeasts were observed in control rice wine after 11 days of incu-bation at 10 �C, and they completely covered the surface of controlrice wine by 27 days. These results show that Lb. plantarum HD1

extended the shelf-life of Korean draft rice wine from 11 days upto 27 days at 10 �C. Therefore, the use of Lb. plantarum HD1 mayinhibit growth of film-forming yeasts. Based on our observationthat the antifungal activity of Lb. plantarum HD1 was strongeragainst filamentous fungi than yeast, use of Lb. plantarum HD1may lead to the development of powerful biopreservative systemscapable of preventing fungal spoilage and mycotoxin formation inthe food and feed industries.

Acknowledgment

This research was supported by the Technology DevelopmentProgram for Food, Ministry of Agriculture, Food and Rural Affairs,Republic of Korea.

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