Isolation and characterization of a proteinaceous antifungal compound ...

Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Isolation and characterization of a proteinaceous antifungal compound from Lactobacillus plantarum YML007 and its application as a food preservative I. Ahmad Rather, B.J. Seo, V.J. Rejish Kumar, U.-H. Choi, K.-H. Choi, J.H. Lim and Y.-H. Park Department of Applied Microbiology and Biotechnology, Yeungnam University, Gyeongsan, Korea

Significance and Impact of the Study: After screening 1400 kimchi bacterial isolates, strain Lactobacillus plantarum YML007 was selected with strong antifungal activity against various foodborne pathogens. From the preliminary studies, it was found that the bioactive compound is a low molecular weight novel protein of 1256617 Da. Biopreservative potential of Lact. plantarum YML007 was demonstrated on soybean grains, and the results point out YML007 as a potent biopreservative having broad antimicrobial activity against various foodborne pathogens.

Keywords antifungal, biopreservative, lactic acid bacteria, Lactobacillus plantarum. Correspondence Yong-Ha Park, Department of Applied Microbiology and Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk 712-749, Korea. E-mail: [email protected] 2013/0018: received 4 January 2013, revised 18 March 2013 and accepted 1 April 2013 doi:10.1111/lam.12077

Abstract Korean kimchi is known for its myriad of lactic acid bacteria (LAB) with diverse bioactive compounds. This study was undertaken to isolate an efficient antifungal LAB strain among the isolated kimchi LABs. One thousand and four hundred LABs isolated from different kimchi samples were initially screened against Aspergillus niger. The strain exhibiting the highest antifungal activity was identified as Lactobacillus plantarum YML007 by 16S rRNA sequencing and biochemical assays using API 50 CHL kit. Lact. plantarum YML007 was further screened against Aspergillus oryzae, Aspergillus flavus, Fusarium oxysporum and other pathogenic bacteria. The morphological changes during the inhibition were assessed by scanning electron microscopy. Preliminary studies on the antifungal compound demonstrated its proteinaceous nature with a molecular weight of 1256617 Da, analysed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF). The biopreservative activity of Lact. plantarum YML007 was evaluated using dried soybeans. Spores of A. niger were observed in the negative control after 15 days of incubation. However, fungal growth was not observed in the soybeans treated with fivefold concentrated cell-free supernatant of Lact. plantarum YML007. The broad activity of Lact. plantarum YML007 against various food spoilage moulds and bacteria suggests its scope as a food preservative.

Introduction Prevention of food and feed from fungi and other food spoilage bacteria has always been a major challenge. The harmful metabolites of these organisms are more alarming to human and animal health. In addition, consumers’ demand for safe food with a long shelf life and a preference for minimally processed products that do not contain chemical preservatives make it more challenging for food processors (Paul et al. 2005). Current approaches to prevent food and feed spoilage mainly depend on chemicals

or treatments that are unaffordable for common man, whereas the use of natural preservatives could be a viable alternative (Malik et al. 1994). The use of micro-organisms to prevent fungal contamination has been gaining interest during the recent years as they can mitigate the adverse effects of chemical preservatives (Prema et al. 2010). It has been suggested that 30–99% of the bacteria and archaea produce at least one bacteriocin (Klaenhammer 1988; Riley 1998). Lactic acid bacteria (LAB)produced bacteriocin particularly offers the possibility of manipulating food microbial communities considerably.

Letters in Applied Microbiology 57, 69--76 © 2013 The Society for Applied Microbiology

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Antifungal activity of Lact. plantarum YML007

I. Ahmad Rather et al.

Lactic acid bacteria as biopreservation organisms are of particular interest; they have been used for centuries as starter cultures in the food industry and are able to produce different kinds of bioactive molecules, such as organic acids, fatty acids, hydrogen peroxide and bacteriocins (Lindgren and Dobrogosz 1990; Dodd and Gasson 1994; Stiles 1996; Schnurer and Magnusson 2005). At present, several such antimicrobials have been discovered; however, it has become imperative to search for more stable antimicrobials with a broad range of activities, especially against various spoilage moulds. There are many recent reports on the antifungal activity of LAB isolated from various sources. Sathe et al. (2007) reported the antifungal activity of Weissella paramessenteroides and Lactobacillus paracollinoides isolated from fresh vegetables against wide range of food spoilage fungi. Lactobacillus paracasei ssp. tolerans isolated from sourdough completely inhibited the growth of Fusarium proliferatum and Fusarium graminearum in a dualculture agar plate assay (Hassan and Bullerman 2008). Delavenne et al. (2012) evaluated the antifungal LAB diversity in raw milk over one-year period and ascertained that majority of the antifungal isolates of raw milk belonged to the Lactobacillus genus. Two probiotic strains Lactobacillus rhamnosus L60 and Lactobacillus fermentum L23 were able to inhibit the growth and aflatoxin B1 production by Aspergillus section Flavi strains (Gerbaldo et al. 2012). Belguesmia et al. (2012) isolated Enterococcus durans A5-11 from Mongolian airag cheese showing activity against various fungi including D. hansenii, Fusarium culmorum and Penicillium expansum. Various antifungal compounds have been reported from LAB includes lactic and acetic acids (Dalie et al. 2010; Yang and Clausen 2005), caproic acid (Corsetti et al. 1998), synergic antifungal metabolite like phenyl lactic acid, 3-hydroxylated fatty acids (Strom et al. 2002, 2005) and cyclic dipeptides (Strom et al. 2002; Li et al. 2012). Kimchi is a well-known lactic acid-fermented vegetable product and is a promising source for bioactive LAB. There exist a few earlier findings on the antifungal activity of kimchi LAB (Kim 2005; Yang and Chang 2010). In the present study, we have isolated a kimchi LAB with strong and broad antifungal activity against various food spoilage moulds. Both the nature of the antifungal compound and the biopreservative activity of the bacteria were analysed. Results and discussion Isolation and identification of Lactobacillus plantarum YML007 After screening the antifungal activity by dual-culture agar plate assay, 55 bacteria among the 1400 were observed 70

Table 1 Antimicrobial activity of cell-free supernatant of Lactobacillus plantarum YML007

Indicator strains

Source

Inhibition zone spectrum (mm)

Aspergillus niger Aspergillus flavus Aspergillus oryzae Fusarium oxysporum Escherichia coli E. coli E. coli Staphylococcus aureus Staphylococcus epidermidis Bacillus subtilis Enterobacter cloacae Klebsiella pneumoniae Listeria monocytogenes Salmonella choleraesuis Salmonella enterica Cronobacter muytjensii Neisseria meningitides

KCTC16683 KCTC16682 KCCM 11371 KCTC6434 KCTC 2441 O157:H7 KCTC 1039 KCTC 1621 KCTC 1917 KCTC 1021 KCTC 2361 KCTC 2208 KCTC 3569 ATCC 10727 ATCC 4931 ATCC 51329 ATCC 13098

1723 1543 1543 135 2466 1433 1583 1316 1775 1875 20 155 21 25 2175 2175 225

005 01 005 015 05 074 148 089 077 078 053 086 070

227 027 165

with inhibition against Aspergillus niger. Comparing the strength of their inhibition, strain YML007 was selected for further studies. After 16S rRNA sequencing, the strain was identified and named Lact. plantarum YML007 (nucleotide accession number JN853603) (Fig. S1). The molecular identification was also supported by the biochemical analysis (Table S1). Antimicrobial activity of Lactobacillus plantarum YML007 Lactobacillus plantarum YML007 was observed with a broad antimicrobial activity against various pathogens tested (Table. 1). Generally, the inhibition was higher against the bacterial pathogens than the fungi. Among the fungi, the inhibition was highest against A. niger followed by A. flavus, A. oryzae and F. oxysporum. Most of the pathogenic bacteria tested were very well inhibited by YML007 with significantly higher inhibition in Salmonella choleraesuis ATCC 10727 (P < 001) followed by Escherichia coli KCTC 2441. Lactic acid analysis As shown in Fig. S2, Lact. plantarum YML007 produced more D-lactic acid (881 gl 1) than L-lactic acid 797 gl 1. Both the Man Rogosa Sharpe (MRS) medium and lactic acid at different concentrations failed to impart antifungal activity against A. niger compared with the strong inhibition (173 mm) by YML007 supernatant (Fig. 1). This fact corroborated the presence of an antifungal compound in the cell-free supernatant of Lact. plantarum YML007.




Scanning electron microscopy Scanning electron microscopy (SEM) demonstrated the activity of Lact. plantarum YML007 against A. niger,

A. oryzae, A. flavus and F. oxysporum (Fig. 2). The mycelial damage was visible in all cases. However, the extended damage was observed to be very high in the A. niger and low in the F. oxysporum, which coincides with the disc diffusion assay. The antifungal compound produced by YML007 was not only effective in restricting mycelial growth of target fungi, but also induced marked morphological changes such as damage to the mycelial cell wall and hyphal size reduction.

Characterization of the antifungal compound

Figure 1 Antifungal activities of Man Rogosa Sharpe (MRS) media, Lactic acid and YML007 against Aspergillus niger. 1. MRS media ( ), 2. 75 mmol l 1 lactic acid ( ), 3. 125 mmol l 1 lactic acid ( ), 4. 250 mmol l 1 lactic acid ( ), 5. 500 mmol l 1 lactic acid ( ), 6. 1000 mmol l 1 lactic acid ( ), 7. Fivefold concentrated cell-free supernatant of YML007 (+++).

The antifungal activity of the Lact. plantarum YML007 was not affected by the addition of enzymes (lipase, alpha amylase and catalase) other than the proteolytic enzymes proteinase K and trypsin. This result indicated the proteinaceous nature of the compound. Partial purification of antifungal compound was obtained by ammonium sulfate precipitation, followed by dialysis with cellulose membrane (cut-off value, 3 kDa), which allowed the recovery of 85% of activity, indicating that the molecular weight of compound is less than 3 kDa. Twenty-eight fractions of dialysed sample were collected from the chromatographic column, and among them, fraction 9 showed significant inhibitory activity. This fraction was completely inactivated by the addition of proteinase K and trypsin. MALDI-TOF analysis detected the molecular weight of the active compound as 1256617 Da (Fig. 3). There was no significant change in activity observed after

Figure 2 Scanning electron micrographs of mycelia after 48 h of cultivation at 30°C. Test strains were exposed to 100 ll fivefold cell free supernatant of Lactobacillus plantarum YML007 (a) Aspergillus niger Control, (b) A. niger Test, (c) Aspergillus oryzae Control, (d) A. oryzae Test (e) Aspergillus flavus Control, (f) A. flavus Test, (g) Fusarium oxysporum Control, (h) F. oxysporum Test.


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Figure 3 MALDI-TOF spectra of the purified active fraction from Lactobacillus plantarum YML007 cell free supernatant showing antifungal activity.

adjusting the pH of cell-free supernatant from pH 6 to pH 11. However, there was 20% decrease in activity after incubating cell-free supernatant (pH 9 and 11) at 60°C for 20 mins, and about 50% of the activity was completely lost after autoclaving the sample at 121°C for 20 mins. Lactobacillus plantarum YML007 as a food biopreservative The preservative effect of Lact. plantarum YML007 was demonstrated on soybean grains. The spores of A. niger were visible in the negative control soybeans after 15 days of incubation, whereas fungal growth was not observed in the soybeans treated with fivefold concentrated cell-free supernatant of Lact. plantarum YML007 (Fig. 4). Similarly, the mould count from grinded soybeans (n = 3) was highest in ( ) CON (761E+07), followed by (+) CON (145E+07), and only three colonies were observed in test group (30E+05), and no colony was observed in (+) CON with YML007 (00E+00). This confirms the scope of Lact. plantarum YML007 as biopreservative. In recent years, there has been a growing interest in biopreservation (Muriana 1996; Stiles 1996; Gyu-Sung et al. 2010). The use of LAB in food and feed and its health benefits supports it as a promising alternative to chemical preservatives. Lactobacillus plantarum YML007 isolated from kimchi showed broad antifungal activity against different food spoilage moulds and inhibited a number of pathogenic bacteria. There has been much interest in Lact. plantarum strains because of its potential application as a starter culture for the fermentation of 72

meat, fish products and vegetables (MacKay and Baldwin 1990). Morphological changes in the fungal mycelia were well observed in SEM analysis. Inhibition was visible, for example, the disappearance of surface ornamentation and the depression or flat ribbon-like appearance of cell walls. These manifestations may be associated with the inhibition of chitin synthesis as reported previously (Masahiro et al. 2011). However, this requires further investigation. The antifungal activity of cell-free supernatant and purified compound was completely destroyed by the treatment with proteinase K and trypsin. There was no significant change in activity after changing the pH of cell-free supernatant from pH 6 to pH 11, showing stability at a broad range of pH. The cell-free supernatant of YML007 retained its stability even after 3 months of storage at room temperature (15–20°C). There are several previous reports on the antifungal compounds produced by the Lact. plantarum such as 3-phenyllactic acid (Prema et al. 2010), 3-hydroxy fatty acids (Sjogren et al. 2003), 3,6-bis(2-methylpropyl)-2,5-piperazinedione (Yang and Chang 2010) and benzeneacetic acid, 2-propenyl ester (Wang et al. 2012). The low molecular weight (1256617 Da) peptide produced by YML007 indicates that it is a novel antifungal compound. Lactobacillus plantarum YML007 exhibited a broad spectrum of activity against both fungus and bacteria compared with pediocin-like bacteriocins, which have a narrow spectrum of activity against the food pathogen Listeria monocytogenes (Montville and Chen 1998; Hechard and Sahl 2002). Our soybean model experiment demonstrated the biopreservative potential of a fivefold concentrated cell-free extract of Lact. plantarum YML007. Even after 15 days of




Figure 4 Control and treatment groups of soybean sample after 15 days of incubation at ambient temperature. (a) (+) CON, positive control, without fungi treatment: (b) (+) CON with YML007, without fungi treatment; (c) ( ) CON, negative control treated with Aspergillus niger spores; (d) ( ) TEST, treated with A. niger spores and YML007.

incubation, the grains treated with YML007 extract were without any fungal growth. Lavermicocca et al. (2000) have used a 10-fold concentrated culture filtrate of Lact. plantarum to obtain an efficient antifungal activity against various food spoilage moulds. Our study further underpins the scope and potential of Lact. plantarum YML007 as a biopreservative against various food spoilage moulds. The antifungal compounds produced by food grade Lact. plantarum YML007 will have immediate potential in food applications, as they are more likely to meet with regulatory approval owing to their origin. This is the primary report on the proteinaceous antifungal compound having a molecular weight of 125661 Da from Lact. plantarum YML007, and like nisin and pediocin PA1, it has the potential for large-scale use in food and feed applications. Materials and methods Screening and isolation of the bacteria The one thousand and four hundred bacteria isolated from different kimchi samples and cultured on MRS agar were screened for antifungal activity. Aspergillus niger KCTC1683 was selected as the indicator fungus for the preliminary screening considering its sensitivity to LAB antifungal compounds. Aspergillus niger was grown on potato dextrose agar (PDA) at 30°C for 5–7 days, stored at 4°C and stocked in a medium containing 15% glycerol at 70°C. Spore inoculum was prepared by growing the moulds on PDA slants until the occurrence of

sporulation. Spores were then collected by vigorous shaking with sterile water containing 01% Tween 80. The spore concentration was determined using a haemocytometer (Paul Mariendeld GmbH & Co.KG, LaudaKonigshofen, Germany) and adjusted to 106 spores ml 1. LABs were screened for antifungal activity by a dualculture agar plate assay on MRS agar plates. LABs were grown in two lines on the plate under anaerobic conditions. After incubation, the plate was overlaid with 15 ml PDA soft agar containing fungal spores at concentration of 106 spores ml 1, and the plates were incubated at 30°C and subsequently examined after 24 and 48 h of incubation. The presence of clear zones was considered as antifungal activity. The selected strains were stored at 80°C in MRS broth with 25% (v/v) glycerol. 16S rRNA gene sequencing and biochemical characterization LAB showing the highest antifungal activity after the screening was identified by 16S rRNA sequencing after amplification using the universal primers 27f and 1492r as described by Lane (1991). The sequences were compared with those in GenBank at the National Centre for Biotechnology Information (NCBI) using the BLAST program. The same bacterium was also characterized biochemically using the API 50 CH strip and API CHL medium system according to the manufacturer’s instructions (API bioMerieux, Durham, NC, USA). A freshly grown colony of LAB was harvested and resuspended in sterile water to achieve a cell density of 1010 CFU ml 1.


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An aliquot of the cell suspension (200 ll) was inoculated into a 10 ml API 50 CHL medium and mixed gently by inversion. 120 ll of this suspension was inoculated into API 50 CH strips overlaid with paraffin to maintain anaerobic conditions. The incubation was carried out at 37°C, and all test preparations were incubated for 48 h before reading colour changes. Antimicrobial activity analysis Antimicrobial activity of the selected LAB Lact. plantarum YML007 against various moulds and foodborne pathogens was carried out using agar well diffusion assay. Lactobacillus plantarum YML007 cultured in MRS broth at 30°C for 24 h was centrifuged at 10 000 g for 10 min, and the cellfree supernatant was taken after filtering it through a 045lm-pore-size filter (Sartorius Stedim Biotech, Goettingen, Germany). The pH of the supernatant was adjusted to 6 with 1 mol l 1 NaOH, and 100 ll of this was loaded in each well against F. oxysporum KCTC6434, A. flavus KCTC16682 and A. oryzae KCCM 11371 and foodborne pathogenic bacteria E. coli KCTC2441, E. coli o157: H7, E. coli KCTC1039, Staphylococcus aureus KCTC1621, Staphylococcus epidermidis KCTC1917, Bacillus subtilis KCTC1021, Enterobacter cloacae KCTC2361, Klebsiella pneumoniae KCTC2208, L. monocytogenes KCTC3569, Salm. choleraesuis ATCC10727, Salmonella enterica ATCC4931, Cronobacter muytjensii ATCC51329 and Neisseria meningitides ATCC13098. The plates were incubated at 30°C and examined after 24 and 48 h for inhibition zones. The diameter of each zone of inhibition was measured, and the diameter of the well (8 mm) was subtracted from the total diameter of the inhibited zone. The significantly higher activities above 20 mm diameter were analysed statistically by one-way analysis of variance (ANOVA). Lactic acid assay Lactobacillus plantarum YML007 was tested for its production of D- and L-lactic acid using the D-/L-lactic acid Megazyme kit (Megazyme International Ireland Ltd., Co., Wicklow, Ireland). An overnight culture of YML007 was centrifuged at 10 000 g for 10 min; the supernatant was collected after filtering through 045-lm filter and assayed with the kit according to manufacturer’s instructions. Involvement of lactic acid and the culture media in contributing to the antifungal activity was assessed by agar well diffusion assay. One hundred microlitres of standard lactic acid and MRS media was checked for antifungal activity against A. niger. The zone of inhibition was compared with 100 ll fivefold concentrated cell-free supernatant of YML007. 74

Scanning electron microscopy analysis The morphological changes during the inhibition were assessed by SEM analysis. Two hundred microlitres of the fungal inoculum containing 106 spores ml 1 was spread over a PDA plate. A filter paper (Hyundai Micro Co., Ltd., Junggu, Seoul, Korea) of 1 cm diameter was then placed on the centre of the plate. Adjacent to the filter paper, a paper disc (8 mm diameter) was placed, and 100 ll of fivefold concentrated cell-free supernatant of Lact. plantarum YML007 was spotted on it. After 48 h of incubation at 30°C, the filter paper was slowly removed and then incubated in a solution containing 25% glutaraldehyde for 4 h at 4°C. The fixed samples were washed with 02 mol l 1 sodium phosphate buffer for 20 min, repeated six times and incubated in 1% osmic acid solution at 4°C overnight. The samples were then dehydrated with 50% ethanol for 20 min with 1~2 repeats, followed by a 20-min dehydration each with 70, 80, 90, 95 and 100% ethanol. The samples were soaked in 100% isoamyl acetate twice for 20 mins each. After the removal of isoamyl acetate, the samples were dried by critical point drying (Hitachi, HCP-2, Japan) with carbon dioxide as a transition fluid. The specimens were mounted on aluminium stubs with a double-stick carbon tape and sputter coated with gold (Au) and observed using scanning electron microscope (Hitachi, JEOL, Japan). Control plates containing filter paper devoid of supernatant were also processed for comparison. Characterization of the antifungal compound The preliminary nature of the antifungal compound was assessed by treating the culture filtrate of Lact. plantarum YML007 with proteinase K (Sigma-Aldrich, St. Louis, MO, USA), trypsin (Sigma-Aldrich), lipase (SigmaAldrich), alpha amylase (Sigma-Aldrich) and catalase (Sigma-Aldrich), each at a final concentration of 1 mg ml 1, and the solution was incubated at 37°C for 3 h. The mixtures were then cooled to inactivate the enzyme activity and subsequently analysed for antifungal activity against A. niger KCTC16683 by the paper disc method. To further investigate the nature of the antifungal compound, 1 l of the cell-free supernatant of Lact. plantarum YML007 was subjected to ammonium sulfate precipitation (85% w/v) followed by dialysis. The dialysed sample was purified by gel filtration chromatography using sephadex G-25 column (35 9 2 cm) with fraction ranges 1000–5000 kDa (Sigma Aldrich). Twenty millimolar phosphate buffer (pH 7) was used as an elution buffer at a flow rate of 40 ll min 1. A total of 28 fractions were collected and checked for antifungal activity. The most active fraction was analysed by MALDITOF (Ultraflex III; Bruker Daltonics, Bremen, Germany)




with a-cyano-4-hydroxy-cinnamic acid (HCCA) as a matrix. A model VSL-337 ND S-nitrogen laser (337 nm) was used. The ion acceleration voltage and laser intensity were set to 20 kV and 35%, respectively. A five microlitre suspension of the purified sample was carefully mixed with an equal volume of HCCA and spotted onto a MALDI plate and left to dry at room temperature for 15 min before analysis. pH and temperature stability of the compound were analysed. Lactobacillus plantarum YML007 as a food preservative The biopreservative potential of Lact. plantarum YML007 was assessed on soybean grains. Freshly dried soybean purchased from market was sterilized by following the method of Yang and Chang (2010). The sterilized soybeans were divided into four groups each containing 10 g of soybeans. The test group was treated with 1 ml of spore suspension (106 spores ml 1) prepared in distilled water followed by fivefold concentrated cell-free supernatant of Lact. plantarum YML007. Whereas the control group was without the YML007 supernatant, a negative control group containing only fivefold concentrated cell-free supernatant and a positive control group devoid of both spores and supernatant were kept for comparison. All samples were incubated at 30°C for 15 days for the observation of fungal growth. After 15 days of incubation at 30°C, each sample was grinded in sterilized grinder and assessed the mould population on PDA plates (n = 3). Acknowledgements Irfan Ahmad Rather acknowledges Yeungnam University for its generous financial assistance. The authors have no conflict of interest to declare. Authors acknowledge the anonymous reviewers for their valuable comments. References Belguesmia, Y., Choiset, Y., Rabesona, H., Baudy, F.M., Le Blay, G., Haertle, T. and Chobert, J.M. (2012) Antifungal properties of durancins isolated from Enterococcus durans A5–11 and of its synthetic fragments. Lett Appl Microbiol 56, 237–244. Corsetti, A., Gobetti, M., Rossi, J. and Damiani, P. (1998) Anti mould activity of sourdough lactic acid bacteria: identification of a mixture of organic acids produced by Lactobacillus sanfrancisco CB1. Appl Microbiol Biotechnol 50, 253–256. Dalie, D.K.D., Deschamps, A.M. and Richard-Forget, F. (2010) Lactic acid bacteria – potential for control of mould growth and mycotoxins: a review. Food Control 21, 370–380.

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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Biochemical characterization of Lactobacillus plantarum YML007, using API 50 CHL. Figure S1. Neighbour-joining phylogenetic tree showing the position of strain YML007 among the different Lactobacillus plantarum strains based on 16S RNA sequences. Scale bar represents 0001 substitutions per nucleotide position. Figure S2. D-Lactic acid /L-Lactic acid assay of Lactobacillus plantarum YML007.
