Weissellicin 110, a Newly Discovered Bacteriocin from Weissella cibaria 110, Isolated from Plaa-Som, a Fermented Fish Product from Thailand

Weissella cibaria 110, isolated from the Thai fermented fishproduct plaa-som, was found to produce a bacteriocin activeagainst some gram-positive bacteria. Bacteriocin activity wasnot eliminated by exposure to high temperatures or catalasebut was destroyed by exposure to the proteolytic enzymes proteinaseK and trypsin. The bacteriocin from W. cibaria 110 was purified,and sodium dodecyl sulfate-polyacrylamide gel electrophoresisshowed that the purified bacteriocin contained one protein bandthat was approximately 2.5 kDa in size. Mass spectrometry analysisshowed the mass of the peptide to be approximately 3,487.8 Da.N-terminal amino acid sequence analysis was performed, and 27amino acids were identified. Because it has no similarity toother known bacteriocins, this bacteriocin was defined as anew bacteriocin and termed weissellicin 110.

INTRODUCTION

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Introduction

Lactic acid bacteria (LAB) have long played important rolesin food technology. The LAB include a wide variety of cell typeswith various physiological and biochemical characteristics.The isolation of LAB from milk products, fermented foods, andplants has frequently been reported. The phylogeny of the bacteriaclassified currently in the genus Weissella was clarified in1990 (16), and the taxonomy of Weissella species was furtherassessed in 1993 (5). Weissella species have been isolated froma variety of sources, and some of them play important rolesin fermentation (1). Weissella cibaria was first described byBjörkroth et al. (1) and later found in various kinds offermented foods (6, 19).

Bacteriocins are peptides produced by bacteria that kill orinhibit the growth of closely related bacteria. Bacteriocinsproduced by LAB have attracted special interest as potentialsafe, alternative food preservatives (4, 8, 15). Many bacteriocinsassociated with Lactobacillus, Enterococcus, and Leuconostocspecies have been described previously (4). However, bacteriocinsfrom Weissella species remain rare, and to our knowledge, nobacteriocins from W. cibaria (1, 9) have been reported previously.

W. cibaria 110 (AB261010/DDBJ; DNA Data Bank of Japan [http://www.ddbj.nig.ac.jp/])isolated from plaa-som (17), a fermented fish product from Thailand,was found to produce a bacteriocin active against some gram-positivebacteria. The present paper describes the purification and analysisof this bacteriocin and discusses its similarities to otherknown peptides. This is the first study to clarify the characteristicsof a W. cibaria bacteriocin.

MATERIALS AND METHODS

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Materials and Methods

W. cibaria 110.
Ten strains of W. cibaria were isolated from plaa-som samplescollected from Bangkok, Thailand, and their activities againstthe indicator strain Lactobacillus sakei JCM 1157^T were determined.Only strain 110 showed activity; this strain was identifiedusing the API 50CHL kit, and the identification was confirmedusing 16S rRNA sequence analysis. W. cibaria 110 was thereforeused as the bacteriocin-producing strain in this study. Inhibitoryactivity was determined using the agar well diffusion assaydescribed by Yanagida et al. (24).

Other bacterial strains and their culture conditions.
The culture conditions for strains used for the determinationof the antibacterial spectrum are listed in Table 1.

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TABLE 1. Inhibition spectrum of the bacteriocin produced by W. cibaria 110

Optimum temperature for growth and bacteriocin production.
To study the optimum temperature for growth and bacteriocinproduction, 100 µl of the overnight bacterial culturewas inoculated into 5 ml of lactobacillus Man-Rogosa-Sharpe(MRS) broth (Difco, Sparks, MD) and then incubated at 15, 20,25, 30, 37, or 45°C for 14 h. After incubation, the opticaldensity at 660 nm was determined and bacteriocin activity waschecked by determining the size of the zone of inhibition aroundthe well (8 mm in diameter) in the agar well diffusion assay.

Effects of enzymes and heat on bacteriocin activity.
To evaluate heat stability, samples of neutralized supernatantfluid from the W. cibaria 110 culture were incubated at 80°Cfor 30 min, 90°C for 30 min, 100°C for 30 min, 110°Cfor 15 min, or 121°C for 15 min. To analyze sensitivityto various enzymes, neutralized supernatant fluid was treatedwith proteinase K (Merck, Darmstadt, Germany), trypsin (Wako,Osaka, Japan), or catalase (Wako, Osaka, Japan) at 30 IU mg^–1and 37°C for 5 h. After the treatments described above,bacteriocin activity was determined using the agar well diffusionassay.

Production of the bacteriocin by W. cibaria 110.
To study bacteriocin production, 5 ml of the overnight bacterialculture was inoculated into 1 liter of MRS medium. At specifictime intervals, 1-ml samples were removed and the optical densityat 660 nm of the culture and the arbitrary activity units (AU)ml^–1 (reciprocal of the highest dilution at which activitywas still obtained) of the bacteriocin were determined accordingto the method of Henderson et al. (13). L. sakei JCM 1157^T wasused as the indicator strain. The incubation temperature wasset based on the results obtained from the optimum growth temperatureanalysis.

Purification of the bacteriocin.
Cell-free culture supernatant (2.5 liters) was prepared andthen purified by ammonium sulfate precipitation (40%) and columnchromatography with a hydrophobic column of phenyl-650M TOYOPEARL(Tosoh, Tokyo, Japan) and Sep-Pak C₁₈ cartridges (Waters, Milford,MA) (24). Eluted bacteriocin fractions from Sep-Pak C₁₈ cartridgeswere freeze-dried and then stored at 4°C.

Molecular size approximation.
The molecular size of the purified bacteriocin was analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) by following the method described by Yanagida etal. (24). Bacteriocin size was estimated using rainbow-coloredprotein molecular mass markers (Amersham Biosciences, Piscataway,NJ).

Mass spectrometry.
The molecular mass of the purified bacteriocin was determinedby matrix-assisted laser desorption ionization-time of flight(MALDI-TOF) mass spectrometry (MS) using a mass spectrometer(Microflex; Bruker, Bremen, Germany) (14).

The molecular mass of the purified bacteriocin was also determinedby liquid chromatography with an ion trap mass spectrometer(LC/MSD Trap XCT; Agilent, CA).

N-terminal amino acid sequence analyses.
The activity of the purified bacteriocin was confirmed on theSDS-PAGE gel, and the gel was then blotted onto polyvinylidenedifluoride membranes and stained with CBB R-250 (Wako, Osaka,Japan). The objective bands were cut out and analyzed, and theN-terminal amino acid sequence was determined by Edman degradationon a protein sequencer (model 491; Applied Biosystems, FosterCity, CA).

RESULTS

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Results

Maximum cell numbers and activity against the indicator strainL. sakei JCM 1157^T were observed at 30°C (Table 2). Thehighest bacteriocin titers (5,120 AU ml^–1) were obtainedafter 10 h of incubation at 30°C, and the highest cell density,based on the optical density at 660 nm, was observed after 10to12 h of incubation (Fig. 1).

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TABLE 2. Effects of various factors on bacteriocin produced by W. cibaria 110

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FIG. 1. Production of bacteriocin during the growth of W. cibaria 110. OD 660 nm, optical density at 660 nm.

The effects of enzymes and heat on the inhibitory agent fromW. cibaria 110 are shown in Table 2. The bacteriocin was inactivatedby proteinase K or trypsin but not affected by treatment withcatalase. The bacteriocin was considered to be heat stable,as activity remained after heating at 121°C for 15 min.

The neutralized supernatant from W. cibaria 110 showed activityagainst some gram-positive bacteria, as listed in Table 1, buthad no activity against Listeria monocytogenes.

The molecular mass of the purified bacteriocin was approximately2.5 kDa, according to SDS-PAGE (Fig. 2).

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FIG. 2. SDS-PAGE analysis of purified bacteriocin from W. cibaria 110. (A) CBB-stained gel. (B) Gel placed onto MRS agar surface overlaid with L. sakei JCM 1157^T. Lanes M, low-molecular-mass standards; lanes 1 and 2, purified bacteriocin from W. cibaria 110.

The purified bacteriocin was analyzed by MALDI-TOF MS, and theresult revealed a major peak at 3,487.86 Da (Fig. 3). The samesample subjected to liquid chromatography electrospray ionizationMS revealed one peak with an identified molecular mass of 3,490.8Da; this result coincided with the expected molecular mass.N-terminal amino acid analysis of this band revealed the followingpartial sequence: NH₂-SDKNNVFFQIGKRYVAPVLYXFGKXAE, where X representsunidentified amino acids. MALDI-TOF MS analysis of the proteinafter tryptic digestion confirmed the identities of amino acids1 to 12 and 14 to 24 and revealed that the 21st amino acid wastryptophan (W) and the 25th amino acid was glycine (G) (datanot shown). No corresponding protein sequence was found in thedatabase (DNA Data Bank of Japan [http://www.ddbj.nig.ac.jp/])or NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). Based onthe results described above, we strongly suggest that W. cibaria110 produces a novel, natural bacteriocin and have termed thisbacteriocin weissellicin 110.

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FIG. 3. MALDI MS-determined mass spectrum of weissellicin 110 (mass, 3,487.86 Da).

DISCUSSION

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Discussion

Bacteriocins produced by LAB such as Lactobacillus (12, 25),Enterococcus (2, 3, 11, 24, 25), Leuconostoc (7, 10), Streptococcus(22), and Carnobacterium (20, 23) species have been frequentlyreported in previous studies. However, studies of bacteriocinsfrom Weissella sp. remain scarce (18). To our knowledge, thisis the first study of a bacteriocin from W. cibaria.

The optimum growth temperature always has an influence on theproduction of bacteriocins (7, 21). The results of our investigationsof growth temperature and bacteriocin production indicated thatW. cibaria 110 had higher bacteriocin production when incubatedat 30°C for 10 to 12 h than when incubated at other temperatures(Fig. 1). The sensitivity of the substance to proteinase K andtrypsin is proof of its proteinaceous nature. In addition, noeffect was observed after treatment with catalase; this findingprovided evidence that the active agent did not originate fromH₂O₂. The same treatments were later performed with the purifiedbacteriocin, and the results were confirmed (data not shown).

Weissellicin 110 showed a narrow spectrum of inhibition of otherLAB. Unlike most class II bacteriocins produced by LAB, weissellicin110 had no activity against Listeria monocytogenes. This characteristicmay limit its potential application in fermented foods.

The molecular mass of the 27-amino-acid peptide was calculatedusing the tool Compute pl/M_w from the ExPASy proteomics server(http://ca.expasy.org), and a result of 3,205.71 Da was obtained.However, results from MALDI-TOF MS and liquid chromatographyelectrospray ionization MS indicated that the true molecularmass of the bacteriocin was approximately 3,488 Da (Fig. 3).We therefore believe that two or three of the amino acids remainunknown in this study. To clarify this result, an advanced analysissuch as PCR DNA sequencing analysis will be conducted in thefuture.

In conclusion, the evidence presented in this report indicatesthat W. cibaria 110 isolated from plaa-som produces a novelbacteriocin, which we have named weissellicin 110. Weissellicin110 is stable after high-temperature treatment but has a narrowspectrum of inhibition of other LAB and does not inhibit Listeriamonocytogenes. Future work in our laboratory will focus on theclarification of the full amino acid sequence of weissellicin110 and the possibility of applying weissellicin 110 as a biopreservative.

ACKNOWLEDGMENTS

We thank Tsutomu Takayanagi for his technical assistance duringthe N-terminal amino acid sequence analyses. We also thank YanKwok of the Vaccine Research and Development Center at the NationalHealth Research Institutes for his assistance and patience inhelping us understand the analysis of mass spectrometry andfor discussions about protein purification.

FOOTNOTES

* Corresponding author. Mailing address: Institute of Enology and Viticulture, University of Yamanashi, 1-13-1, Kitashin, Kofu, Yamanashi 400-0005, Japan. Phone and fax: 81-55-220-8605. E-mail: yishen{at}yamanashi.ac.jp.

Published ahead of print on 9 February 2007.

REFERENCES

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