Food Microbiology 41 (2014) 19e26

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Purification and characterization of antifungal compounds from Lactobacillus plantarum HD1 isolated from kimchi Eun Hye Ryu a, Eun Ju Yang a, Eun Rhan Woo b, Hae Choon Chang a, * a b

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

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 February 2013 Received in revised form 15 January 2014 Accepted 19 January 2014 Available online 25 January 2014

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 filamentous fungi and yeasts in a spot-on-the-lawn assay. Antifungal activity of Lb. plantarum HD1 was stronger against filamentous fungi than yeast. Antifungal compounds were purified using solid phase extraction (SPE) and recycling preparative-HPLC. Structures of the antifungal compounds were elucidated by electrospray 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 antifungal compounds 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 supernatant of Lb. plantarum HD1 (equivalent to 2.5% addition of culture supernatant) until 27 days of incubation. The addition of antifungal compounds to Korean draft rice wine extended shelf-life up to 27 days at 10  C without any sterilization process. Therefore, the antifungal activity of Lb. plantarum HD1 may lead to the development of powerful biopreservative systems capable of preventing food- and feed-borne fungal spoilage. Ó 2014 Published by Elsevier Ltd.

Keywords: Lactobacillus plantarum Antifungal activity 3-Hydroxy fatty acids 5-Oxododecanoic acid Kimchi

1. Introduction Molds and yeasts are able to grow on most foods, including natural foods, processed foods, and fermented foods. Molds and yeasts play a central role in the spoilage of food products and feed systems (Loureiro and Malfeito-Ferreira, 2003; Filtenborg et al., 1996). Such spoilage can cause considerable economic loss, and further contamination by mycotoxins can cause public health problems (Schnürer and Magnusson, 2005). The current need for biopreservation has spurred the search for foodcompatible antimicrobials produced by microorganisms. Lactic acid bacteria (LAB) are promising alternatives to chemical preservatives, as antimicrobial compounds from LAB show potential in suppressing food-borne yeasts and molds (De Muynck et al., 2004). Additionally, LAB have a long history of use as biopreservatives for food and feed storage (Stiles, 1996). Antifungal

* Corresponding author. Tel.: þ82 62 230 7345; fax: þ82 62 222 8086. E-mail address: [email protected] (H.C. Chang). 0740-0020/$ e see front matter Ó 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.fm.2014.01.011

compounds from LAB include metabolites containing proteinaceous (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 their derivatives, fatty acids and their derivatives, cyclic dipeptides, and nucleosides (Talarico et al., 1988; Corsetti et al., 1998; NikuPaavola 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; Wang et al., 2012; Li et al., 2012). The number of reports characterizing of antifungal compounds from LAB is still low, whereas there have been numerous investigations on antibacterial compounds from LAB (Reis et al., 2012). An antifungal compounds from LAB have been shown to be applicable to the control of food-borne yeasts and molds. However, to date, the antifungal activity of LAB remains poorly understood. Thus, new food-grade antifungal compounds should be continuously identified along with the development of commercial formulations. Korean draft rice wine (makgeolli) is a traditional Korean fermented rice wine using cooked rice as a main material and a

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mixture of molds and yeasts (nuruk) as a fermentation starter. After the fermentation process, its alcohol content reaches 6e8% (Kang et al., 2012; Lee et al., 2012). Rice wine is bottled without any sterilization process, even though this has changed in recent years due to heat-sterilization. This paper reports the isolation and identification of antifungal activity of Lactobacillus plantarum HD1 from kimchi, a traditional Korean fermented vegetable product. Furthermore, the present study describes the purification and characterization of the low molecular mass antifungal compounds obtained from this isolated strain. In addition, it evaluates the antifungal capacity of Lb. plantarum HD1 as a biopresevative in a food model of Korean draft rice wine.

(Hanil, Seoul, Korea) for 2 min. The obtained kimchi juice was filtered through a sterile thin cloth, after which the filtrate was serially diluted with sterile-distilled water and then spread onto MRSþ2% CaCO3 agar. The plates were incubated at 30  C for 2 days, and the tentatively considered LAB strains were selected. Among the selected strains, rod-type LAB were selected. Their antifungal activities against a food-borne spoilage fungus, Aspergillus fumigatus 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 supernatants of LAB were concentrated 5-fold and their antifungal activities assayed using paper disc assay.

2.3. Identification of the isolate

2. Materials and methods 2.1. Cultures and media The microbial strains used in this study and their culture media with culture conditions are listed in Table 1. LAB were grown in de Man Rogosa Sharpe (MRS) broth (Difco, Detroit, MI, USA) at 30  C for 24 h. Yeast strains were grown on yeast extractepeptonee dextrose agar (YPD; Difco) or yeast mold agar (YM; Difco). Molds were grown on malt extract agar (MEA; Difco) or potato dextrose agar (PDA; Difco).

The isolate was identified based on its morphological characteristics under a microscope, biochemical properties using an API 50 CHL (BioMérieux, Marcy-I’Etoile, France), and determination of 16S rRNA gene sequences using an ABI prism 3730 DNA analyzer (Applied Biosystems, Foster city, CA, USA) according to the method described by Yang and Chang (2008). The determined 16S rRNA gene sequences were compared with sequences available in the GenBank database (http://blast. ncbi.nlm.nih.gov/Blast.cgi) using the BLASTN program.

2.2. Isolation of antifungal activity of LAB

2.4. Antifungal activity assays

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

The paper disc assay (Yang and Chang, 2008) and spot-on-thelawn assay (Hoover and Harlander, 1993) were used to detect antifungal activities. Plates were prepared by adding the mold (106 spores per 20 mL of MEA) to 1.5% (w/v) bactoagar (Duchefa, Harlem, The Netherlands) or by spreading the yeast (106 CFU/mL) onto YPD agar, as listed in Table 2. A spore solution was prepared as previously reported (Yang and Chang, 2010). For the paper disc assay, paper discs (diameter 8 mm; Advantec, Tokyo, Japan) on MEA plates were spotted with 100 mL of sample. The plates were incubated at 30  C for 48 h and examined for inhibition zones. For the spot-on-the-lawn assay, 10e25 mL of sample was spotted onto the sensitive mold and yeast plates. Antifungal activity, expressed as arbitrary units (AU) per milliliter, was defined as the reciprocal of the highest dilution at which fungal growth was inhibited. The antifungal titer was calculated as (1000/d) D, where D is the dilution factor and d is the dose (amount of antifungal samples pipetted onto each spot). The above experiment was done in triplicate.

Table 1 Microbial strains used in this study. Strain LAB Lactobacillus plantarum HD1 Yeasts Pichia kudriavzevii GY1 Saccharomyces servazzii GY2 Saccharomyces bulderi HY Kazachstania exigua WY3 Candida albicans ATCC 11006 Molds Aspergillus flavus ATCC 22546 Aspergillus fumigatus ATCC 96918 Aspergillus petrakii PF-1 Aspergillus ochraceus PF-2 Aspergillus nidulans PF-3 Cladosporium gossypiicola KF-2 Penicillium roqueforti ATCC 10110

Mediuma

Culture condition

Reference

MRS, 30  C, 24 h This study MRS þ 2% CaCO3 YPD

30  C, 24 h Chang and Yang, 2012

YPD

30  C, 24 h Chang and Yang, 2012

YPD

30  C, 24 h Chang and Yang, 2012

YPD

30  C, 24 h Chang and Yang, 2012

YM

25  C, 48 h Rudek, 1978 Table 2 Antifungal activity of Lb. plantarum HD1 against yeasts and molds.

MEA

30  C, 48 h Richard et al., 1969 

MEA

30 C, 48 h Pettit et al., 1998

PDA

30  C, 48 h Yang and Chang, 2008

PDA

30  C, 48 h Yang and Chang, 2008

MEA

30  C, 48 h Yang and Chang, 2008

PDA

25  C, 72 h Yang and Chang, 2008

PDA

25  C, 72 h Pillai and Weete, 1975

Microorganism

Indicator species

Activitya (AU/mL)

Yeasts

Pichia kudriavzevii GY1 Saccharomyces servazzii GY2 Saccharomyces bulderi HY Kazachstania exigua WY3 Candida albicans ATCC 11006 Aspergillus flavus ATCC 22546 Aspergillus fumigatus ATCC 96918 Aspergillus petrakii PF-1 Aspergillus ochraceus PF-2 Aspergillus nidulans PF-3 Cladosporium gossypiicola KF-2 Penicillium roqueforti ATCC 10110

32 16 8 128 8 640 640 320 320 320 160 160

Molds

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

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 at least two times using three independent Lb. plantarum HD1 culture preparations.

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

2.5. Purification of antifungal compounds from Lb. plantarum HD1 Solid phase extraction (SPE) of the culture supernatant was carried out according to a previously described method (Yang and Chang, 2010). Briefly, a 24 h culture of Lb. plantarum HD1 grown in MRS broth at 30  C was centrifuged (9500  g, 15 min) and then filter-sterilized through a 0.45 mm pore filter (Advantec). Supernatant (2.5 L) of Lb. plantarum HD1 was 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) aqueous acetonitrile and eluted with 30 mL of 95% (v/v) aqueous acetonitrile. The eluted sample was then concentrated by vacuum evaporation in a Speed Vac apparatus (VS-802, Vison, Daejeon, Korea). The SPE-prepared sample containing the active substance was further separated using a HPLC. We used a recycling preparativeHPLC system (LC9104; Japan Analytical Industry, Tokyo, Japan) fitted with a 3702 UV detector (Japan Analytical Industry). The column used was a JAIGEL-W252 gel permeation chromatography column (20  500 mm, Japan Analytical Industry). The first and second HPLC elution solvent was 50% (v/v) aqueous acetonitrile while the third and fourth HPLC elution solvent was 40% (v/v) aqueous acetonitrile applied at a flow rate of 3 mL/min. For the fourth HPLC, active fractions obtained from the third HPLC were reseparated by recycling preparative-HPLC until a peak with antifungal activity was obtained. Elution was monitored using UV detector at 210 nm. All peaks were measured for antifungal activity using the spot-on-the-lawn method (Hoover and Harlander, 1993).

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activity of Lb. plantarum HD1 was compared to those of three commonly 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, as set by the Food and Drug Administration (FDA). The three preservatives were dissolved in 20 mM sodium acetate (pH 4.0). Prepared cell-free supernatant of Lb. plantarum HD1 was concentrated (5-fold) in 20 mM sodium acetate (pH 4.0). A. fumigatus ATCC 96918 and P. kudriavzevii GY1 were used as sensitive strains. Antifungal activities were examined by paper disc assay. The antifungal compounds from Lb. plantarum HD1 as biopreservatives were added to Korean draft rice wine. An SPE sample containing active substances prepared as in Materials and methods Section 2.5 was dissolved in 20 mM sodium acetate (pH 4.0), after which 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 not sterilized after fermentation. Korean draft rice wine was incubated at 10  C for 30 days, and growth of film-forming yeasts, which induce decay of Korean draft rice wine, was observed. 2.8. Replication of experiments Antifungal activity assays as well as application of antifungal compounds from Lb. plantarum HD1 to food were carried out in duplicate, with three independent sample preparations each. 3. Results and discussion

2.6. Identification of antifungal compounds 3.1. Isolation and identification of antifungal activity of LAB Structures of the antifungal compounds were determined using nuclear magnetic resonance (NMR) spectroscopy, gas chromatography-mass spectrometry (GCeMS), and electrospray ionization-mass spectrometry (ESI-MS). NMR spectra were recorded on samples in CD3CN and D2O on a Bruker Advance-500 NMR spectrometer (Bruker Biospin GmBH, Rheinstetten, Germany). All spectra were recorded at 24  C. Onedimensional 1H and 13C NMR experiments in combination with DEPT (Distortionless Enhancement by Polarization Transfer), twodimensional 1He13C heteronuclear multiple quantum coherence (HMQC), 1He13C heteronuclear multiple bond correlation (HMBC), and 1He1H correlation spectroscopy (COSY) experiments were performed. 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 to 280  C at a rate of 15  C/min, and then held for 15 min. The injector and GC transfer line temperatures were 250  C and 260  C, respectively. The mass detector was operated in electron impact mode at an ionization energy of 70 eV. Compound identifications were made by comparison of the GC-retention index (Sadtler Research Laboratories, 1986) with the NIST mass spectral search program (NIST/EPA/NIH mass spectral library ver. 2.0 f). To acquire the mass spectra in positive-ion and negative-ion modes, ESI-MS was performed using a quadrupole orthogonal time-of-flight (QTOF) mass spectrometer (synapt G2, Waters, Manchester, UK). 2.7. Application of antifungal compounds as biopreservatives in food Application of antifungal compounds from Lb. plantarum HD1 as biopreservatives in food was carried out. First, the antifungal

Ninety-one rod-type LAB were isolated from approximately 80 collected kimchi samples. Among them, strain HD1 showed the strongest antifungal (anti-mold and anti-yeast) activity. Strain HD1 was gram-positive and catalase-negative. Assessment of biochemical characteristics using the API CHL system found that strain HD1 belonged to Lb. plantarum (data not shown). When the 16S rRNA gene sequences (1368 bp) of isolate HD1 were determined (GenBank accession No. JQ343914) and compared with those of type strains of LAB in GenBank, the sequences of strain HD1 showed 99.9% 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 yeasts and molds were determined (Table 2). Lb. plantarum HD1 showed stronger activities against molds (160e640 AU/mL) than yeasts (8e 128 AU/mL). The strongest activities (640 AU/mL) of Lb. plantarum HD1 were against Aspergillus flavus and A. fumigatus, whereas the weakest activities (8 AU/mL) were against Saccharomyces bulderi and Candida albicans. 3.3. Purification of antifungal compounds Twenty-one fractions were acquired in the first injection into preparative-HPLC (Fig. 1A), and their antifungal activities (antimold activity against A. fumigatus; anti-yeast activity against P. kudriavzevii) were assayed using the spot-on-the-lawn method (Fig. 1a). Among the 21 fractions in Fig. 1, active fractions 11 to 15 were collected, combined together, and injected into the second preparative-HPLC. The injected sample was then separated into 11 fractions (Fig. 1B), with fractions 9 to 11 showing the highest activities (Fig. 1b). These three fractions were then combined and injected 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 fractions 9e11 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 their antifungal 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 preparativeHPLC 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 concentrated by 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.

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

fractions, of which three were selected displaying both high antimold 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 into the fourth HPLC and then reseparated by recycling preparativeHPLC. Fraction 7 was reseparated into 13 fractions after six runs of recycling preparative-HPLC (Fig. 2A), with fraction 7e7 showing the highest activities against A. fumigatus and P. kudriavzevii (Fig. 2a). Fraction 11 was reseparated two times by recycling preparative-HPLC, whereas fraction 11 showed one neat peak and could not be further separated (Fig. 2B). Fraction 12 was reseparated into seven fractions by four runs of recycling preparativeHPLC (Fig. 2C), with fraction 12e5 showing the highest activities against both mold and yeast (Fig. 2c). Recovery of the antifungal compounds and determination of their activities according to the purification steps are shown in Table 3. Purified fractions 11, 7e7, and 12e5 showed total activities of 6.4  103 AU, 3.8  103 AU, and 8.0  102 AU, and their total activities were 0.02%, 0.012%, and 0.003% 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 was detected with retention time of 18.06, 16.70, and 17.90 min, respectively. However, compound identification using the NIST mass spectral search program failed; we could only obtain information 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 of fractions 11, 7e7, and 12e5 were determined to be ESI-MS(m/z); 214, 188, and 214, respectively. According to the NMR spectra, the 1 H NMR spectrum of fraction 11 showed one primary methyl group at d 0.89 (3H, t, J ¼ 7.2 Hz) as well as nine methylene groups at d 1.30 (8H, m), 1.53 (2H, qui, J ¼ 7.2 Hz), 1.81 (2H, qui, J ¼ 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, including two carbonyl groups at d 213.5 and 178.8, one methyl group at d 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 7e7 showed one primary methyl group at d 0.89 (3H, t, J ¼ 7.2 Hz), one oxymethine proton at d 3.97 (1H, br s), and seven methylene groups at d 1.32 (10H, br s), 1.47 (2H, s), and 2.39 (2H, m). In addition, the 13 C NMR and HSQC spectral data revealed 10 carbons, including one carboxylic group at d 175.9, one oxygenated carbon at d 69.0, one

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 fraction 12e5 showed one primary methyl group at d 0.89 (3H, t, J ¼ 7.2 Hz), one oxymethine proton at d 4.00 (1H, qui, J ¼ 6.5 Hz), two olefinic protons at d 5.49 (1H, m), 5.43 (1H, m), and seven methylene groups at 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 revealed 12 carbons, including one carboxylic group at d 176.3, one oxygenated 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-MS and NMR analyses, the active compounds in fractions 11, 7e7, and 12e5 were elucidated as 5-oxododecanoic acid, 3-hydroxy decanoic acid, and 3-hydroxy-5-dodecenoic acid, respectively. 3-Hydroxy fatty acid compounds, such as 3-hydroxy decanoic acid and 3-hydroxy-5-dodecenoic acid in this study, have already been 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 3hydroxytetradecanoic acid, from Lb. plantarum MiLAB 14. They further reviewed that the mechanisms behind the antifungal effects of 3-hydroxy fatty acids are due to detergent-like properties of the compounds that alter cellular membrane structure in the target organisms. However, to date, 5-oxododecanoic acid has never been reported as an antifungal compound of LAB. 5-Oxododecanoic acid is a peach- and cream-like aromatic compound accepted as a generally recognized as safe (GRAS) flavoring substance by the FDA (Smith et al., 2009). It can be chemically synthesized and used in imitation dairy and milk products (http://www.fao.org/ag/agn/ jecfa-flav/details.html?printable¼true&flavId¼6964). We believe that our study is the first report regarding the natural production of 5-oxododecanoic acid by LAB Lb. plantarum HD1 with anti-yeast as well as anti-mold activities.

3.5. Application of antifungal compounds from Lb. plantarum HD1 to food Fig. 3 shows the antifungal activities of Lb. plantarum HD1 culture supernatant along with those of other widely used antifungal preservatives, sodium benzoate, potassium sorbate, and pimaricin. Sodium benzoate is approved by the FDA as a food preservative and was 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), whereas pimaricin is used at less than 20 ppm (Davidson, 2001). In this

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

Vol. (mL)

Culture supernatant 50000 Solid phase extraction 40 1st HPLC fractions 11e15 40 2nd HPLC fractions 9e11 25 3rd HPLC Fraction 7 3.5 Fraction 11 2.0 Fraction 12 1.5 4th HPLC (recycling process) Fraction 7e7 1.2 Fraction 12e5 1.0

Activitya (AU/mL)

Total activityb (AU)

640 51200 6400 3200

3.2 2.1 2.6 8.0

107 106 105 104

100.000 6.400 0.800 0.250

6400 3200 1600

2.2  104 6.4  103 2.4  103

0.070 0.020 0.008

3200 800

3.8  103 8.0  102

0.012 0.003

   

Recovery (%)

a Activity was determined against A. fumigatus ATCC 96918 and was calculated in AU/mL, as described in the text. b Total activity was calculated as the total AU within the volume of the sample (mL).

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

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

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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 control Korean draft rice wine (B) during incubation at 10  C for 30 days.

study, the filamentous fungus A. fumigatus was much more sensitive to all antifungal agents compared to the film-forming yeast P. kudriavzevii. Antifungal activity of the 5-fold-concentrated culture supernatant of Lb. plantarum HD1 (equivalent to 0.5 mL of culture broth) was significantly higher compared to those of other food preservatives, even at their maximum approved concentrations. Anti-yeast activity of the 5-fold-concentrated culture supernatant of Lb. plantarum HD1 was clearly observed, whereas those of other food preservatives could hardly be detected. Five-foldconcentrated MRS broth itself as a control did not show any antimold or anti-yeast activity (Fig. 3). The ability to prevent or to retard the growth of food spoilage fungi on foods considered to be the most important regarding biopreservation 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 Korean draft rice wine. To prepare the partially purified antifungal compounds from Lb. plantarum HD1, the acetonitrile-eluted culture supernatant of Lb. plantarum HD1 from the SPE column (C18column) was vacuum evaporated, after which the dried sample was dissolved in 20 mM sodium acetate buffer (pH 4.0). The SPEprepared sample contained the three identified antifungal compounds as well as other undefined antifungal compounds produced by Lb. plantarum HD1, as we observed antifungal activities in other fractions (Figs. 1 and 2). The shelf-life of draft rice wine was 10 days below 10  C due to the growth of film-forming yeasts such as P. kudriavzevii or P. membranifaciens. Further, growth of film-forming yeasts in Korean rice wine is associated with offflavors, which compromises beverage quality. When we treated the partially purified antifungal compounds mixture (equivalent to 2.5% addition of culture supernatant) to Korean draft rice wine, as shown in Fig. 4, film-forming yeasts were not observed in rice wine treated with the SPE-prepared culture supernatant of Lb. plantarum HD1 until 27 days, after which thin film spots were observed from 28 to 29 days. On the other hand, film-forming yeasts were observed in control rice wine after 11 days of incubation at 10  C, and they completely covered the surface of control rice wine by 27 days. These results show that Lb. plantarum HD1

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Purification and characterization of antifungal compounds from Lactobacillus plantarum HD1 isolated from kimchi.

Strain HD1 with antifungal activity was isolated from kimchi and identified as Lactobacillus plantarum. Antifungal compounds from Lb. plantarum HD1 we...
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