Bioresource Technology 111 (2012) 504–506

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Bioresource Technology

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Short Communication

Tostadin, a novel antibacterial peptide from an antagonistic microorganismBrevibacillus brevis XDH

Zhen Song a, Qingxin Liu b, Hui Guo a,c, Ruicheng Ju a, Yuhua Zhao a, Jinyu Li b, Xunli Liu b,⇑a College of Life Science, Shandong Agricultural University, Taian, Chinab College of Forestry, Shandong Agricultural University, Taian, Chinac Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences, Beijing, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 27 December 2011Received in revised form 5 February 2012Accepted 11 February 2012Available online 18 February 2012

Keywords:Brevibacillus brevisAntibacterial peptidePurificationCharacterization

0960-8524/$ - see front matter � 2012 Elsevier Ltd. Adoi:10.1016/j.biortech.2012.02.051

⇑ Corresponding author. Postal address: DepartmeForestry, Shandong Agricultural University, 61 DaizProvince 271018, China. Tel.: +86 538 8249131; fax:

E-mail address: xunliliu@gmail.com (X. Liu).

A novel small antibacterial peptide was obtained from the liquid culture of Brevibacillus brevis XDH,which is a broad-spectrum antagonistic bacterium isolated from the soil of Mountain Tai, China. This pep-tide was purified from the fermentation medium of strain XDH via ammonium sulfate precipitation, cat-ion exchange chromatography, and reversed-phase high-performance liquid chromatography (HPLC),successively. The structure of the active linear peptide was elucidated using mass spectra (MS) andnuclear magnetic resonance (NMR) analyses that consisted of nine amino acids. This peptide was easilysoluble in water, thermally stable and strongly inhibited the growth of Escherichia coli and Staphylococcusaureus in vitro. The present data support the identification of a novel antibacterial peptide, which wasnamed Tostadin.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Escherichia coli and Staphylococcus aureus, which are commonand damaging pathogens of humans and animals, have a widerange of hosts and are a major threat to public health and foodsafety (Eaton et al., 2008). Chemical control currently remains asthe primary means for preventing diseases caused by E. coli andS. aureus. Numerous synthetic chemicals comprise the major mar-ket share of antibiotics (Cambau et al., 2009). However, the de-mand for more effective and safer compounds with novel modesof action has been increasing because of the rapid emergence ofdrug-resistant pathogens. Consequently, the selection of naturalantibiotics from antagonistic microorganisms is considered as analternative method for the disease control. Antimicrobial peptidesare the most important and effective antibiotics for combating theincrease of drug-resistant bacteria (Wang et al., 2010).

In the past few years, biologically based drugs have aroused awide public concern because of its environmental protection andsecurity features. Scientists have focused on the use of antagonisticbacteria and their active substances (Viscardi et al., 2008). Severalclasses of proteins and peptides that inhibit the growth of fungiand bacteria in vitro assays have been identified in the last decade.In 1996, the genus Brevibacillus, which is a well-known rod-shaped

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nt of Sericulture, College ofong Street, Taian, Shandong+86 538 8249164.

Gram-positive bacterium, was established as an independentgenus from the reclassification of Bacillus brevis (Shida et al.,1996). Brevibacillus is omnipresent in agricultural soils, and it cansecrete structurally diverse secondary metabolites with broad anti-biotic spectra. Some of these metabolites, such as chitinase andgramicidin S, have been extensively studied, and numerousBrevibacillus species, which have the potential as antimicrobialagents, have become research hotspots in the recent years (Mogiand Kita, 2009).

Brevibacillus brevis XDH (Genbank accession number: DQ279738), which can inhibit many pathogens both in vivo and in vitro,was isolated from the soil of Mountain Tai, China. In the presentstudy, a novel antibacterial peptide, which was named as Tostadin,was isolated and purified from the liquid culture of strain XDH.Moreover, the structure of Tostadin was elucidated and some ofits properties were analyzed.

2. Methods

2.1. Microorganism and culture conditions

Brevibacillus brevis XDH was isolated from the soil of MountainTai and was stored in the College of Forestry, Shandong Agricul-tural University. It was maintained at 4 �C on potato dextrose agar(PDA: 200 g potato, 20 g glucose, 5 g beef extract, 5 g sodium chlo-ride, and 20 g agar in 1 L distilled water) plate and was subculturedevery month prior to use.

Z. Song et al. / Bioresource Technology 111 (2012) 504–506 505

The liquid medium used in the present study was composed of20 g glucose, 30 g soybean meal, 6 g starch, 2 g calcium carbonate,4 g magnesium sulfate, and 1 L distilled water. The seed liquid in a250 mL Erlenmeyer flask with 50 mL medium was cultured in a ro-tary shaker under 30 �C at 200 rpm for 18 h. Fermentation wasthen performed in 250 mL flasks containing 50 mL medium witha 2% aseptic inocula under similar conditions described above.

2.2. Determination of the antibacterial activity

The mixed-germs-plate method was used to determine the anti-bacterial activity of the antibacterial substances using the Oxfordplate assay system. An E. coli strain, which was kindly providedby the College of Animal Sciences and Technology, Shandong Agri-cultural University, was used as the indicating microorganism withthe concentration controlled at 2–6 � 104 CFU/mL in the medium.The fermentation broth was heated at 100 �C for 10 min and wascentrifuged at 9500g for 20 min. Up to 250 lL supernatant was theninjected onto the Oxford cup, and the diameters of the inhibitionzones were measured after being incubated at 30 �C for 6 h. The fit-ting equation for antibacterial activity (titer) counting is given byY = 10[(X � 2.6475)/5.9311] � N, where Y represents the titer (using con-centration unit lg/mL), X represents the diameter of the inhibitionspot (11.3 mm < X < 14.5 mm), and N represents the dilution timesof the fermentation broth.

2.3. Isolation and purification of the antibacterial substance

The fermentation broth of B. brevis XDH was sterilized at 100 �Cfor 10 min and was centrifuged at 4200 rpm for 30 min. The super-natant, which was mixed with ammonium sulfate to the saturationof 45%, was kept at 25 �C overnight and was centrifuged at4200 rpm for 20 min. The salting-out sediment was dissolved usingdistilled water and was condensed using a rotary evaporator. TheCM Sepharose Fast Flow gel was rinsed with NaCl (1 mol/L) anddistilled water successively for 2–3 times before being loaded ontothe column (1.6 � 30 cm). It was then equilibrated with deionizedwater for 12 h. Approximately 15 mL of the salting-out sampleswas added onto the surface of the gel, and was rinsed absolutelywith plenty of distilled water to clean the substances that werenot adsorbed. Linear gradient elution of NaCl (0–1.0 mol/L) wasthen performed at a flow rate of 60 mL/h. The eluate was moni-tored using a UV detector, and a bioassay was used to examinethe antibacterial active peaks of the elution. The effective compo-nents were collected and desalinated using Sephadex G-25 chro-matography, based on the same method as the CM Sepharose FFin loading, monitoring, and collecting, except that it was onlyrinsed with distilled water. Ultimately, the antibacterial substanceswere condensed and lyophilized.

The crude material was then prepared using reversed-phaseHPLC. The condition was as follows: Sinochrom ODS-BP column(300 � 10 mm, 10 lm), injection volume of 200 lL, temperatureof 30 �C, flow rate of 2.0 mL/min, water and acetonitrile containing0.1% trifluoroacetic acid (v/v: 68/32) as the mobile phase, andabsorption wavelength of 210 nm. The active component was col-lected for property analysis.

2.4. Property analysis of the antibacterial substance

The antibacterial component was sent to Shandong Analysisand Test Center, China, and the structure was elucidated via elec-trospray ionization mass spectrometry (ESI-MS) and experimentsof TOCSY, NOESY, ROESY, 13C HSQC 2D NMR. After obtaining thedetailed structure, China Peptides Co., Ltd. in Shanghai synthesized100 mg of the pure sample. The modified 100 mg sample (acetyla-tion at N-terminal and amidation at C-terminal) was also produced

by the company to investigate its antibacterial ability. The mini-mum inhibitory concentration (MIC) and minimum bactericidalconcentration (MBC) values of the natural product and the twokinds of synthetic peptides were determined according to litera-ture (Gauri et al., 2011).

The active peptides, both natural and synthetic, were preparedto 100 lg/mL with distilled water and were incubated at 100 �C for0, 10, and 20 min, respectively, to determine the thermal stability.After cooling to room temperature, the residual antibacterial activ-ities were measured as described above.

3. Results and discussion

3.1. Isolation and purification of antibacterial component

Brevibacillus species generally exist in many environments. As agroup, this genus offers several advantages for protection againstpathogens, because of the broad-spectrum activity of their antibi-otics (Song et al., 2011). In general, the mechanisms of pathogencontrol are classified as competition, parasitism or predation, andantibiosis. B. brevis XDH is a potent antimicrobial agent isolatedfrom the soil of Mount Tai, and it has a strong antibacterial activityagainst many pathogens of humans and animals (Song et al., 2012).B. brevis XDH showed great potentials in application, and thus itwas chosen for further investigation.

The crude antibacterial substances were precipitated usingammonium sulfate from the supernatant of fermentation broth.The extracts were then used for ion exchange chromatographyon a CM FF column, which produced two unadsorbed fractions(P1 and P2) and an adsorbed fraction (P3), to enrich the active com-ponents from the culture of B. brevis XDH. The three fractions wereassayed for growth inhibition on the test pathogens (E. coli and S.aureus). No antibacterial activity was detected in P1 and P2,whereas, it was observed in P3, resulting from the linear NaCl gra-dient elution at the concentration of around 0.3 mol/L. Furtherpurification for the P3 was conducted using the reversed-phaseHPLC, and four main peaks (A, B, C, and D) were obtained. Aftereach peak was assayed for the antibacterial activities of E. coliand S. aureus, the third fraction (C) was found to show strong inhib-itory activities. This compound was purified and collected usingpreparative HPLC on a Venusil XBP C18 preparative column underthe same conditions mentioned above. About 35 mg purified com-ponent C was obtained from the 5 L liquid culture of B. brevis XDH.

3.2. Property analysis for the antibacterial compound

The experiments of ESI-MS and NMR were performed, and theexperts from the Shandong Analysis and Test Center analyzed theresults. The molecular mass of the active peptide was determinedto be about 1102.35 Da. Structure elucidation revealed that the ac-tive component was a linear peptide that consisted of nine aminoacids. The detailed sequence was illustrated as Ser1-Leu2-Tyr3-Lys4-Leu5-Thr6-Cys7-Lys8-Phe9 (SLYKLTCKF). Further unscram-bling of the NMR spectra showed that residues 4 and 9 are of D-configuration, and others are of L-configuration. No same or similarsequences were found in the protein databases online. The presentdata support the identification of a novel antibacterial peptide,which was named Tostadin.

Tostadin demonstrated significant antibacterial activitiesagainst E. coli and S. aureus. Pure Tostadin samples, both unmodi-fied and modified, were synthesized in China Peptides Co., Ltd. tofurther analyze some of its properties. The MIC and MBC valuesof the unmodified peptide were 16 lg/mL and 32 lg/mL, respec-tively, with E. coli as the indicating microorganism. S. aureus wasless sensitive to Tostadin with the MIC and MBC values of 32 lg/

506 Z. Song et al. / Bioresource Technology 111 (2012) 504–506

mL and 64 lg/mL, respectively. However, the modified peptide(AC-SLYKLTCKF-NH2) showed a stronger antibacterial activity, ofwhich the MIC and MBC values against both of the two pathogenswere 8 lg/mL and 16 lg/mL, respectively, basically the same withthose of the natural product. This demonstrated that the syntheticpeptides have the similar antibacterial abilities with the naturalTostadin.

The results of the thermal stability tests suggest that both thenatural and synthetic Tostadin are considerably thermally stable.The antibacterial activity was almost unchanged (remained morethan 95% activity) even after being maintained at 100 �C for 20 min.

Hundreds of antimicrobial peptides have been isolated andidentified from a great number of microorganisms (Dale andFredericks, 2005). Their modes of actions, including disruptingmembranes, interfering with metabolism, and targeting cytoplas-mic components have been well studied (Saikia et al., 2010).Brevibacillus is a well-known antibiotic-producing species thatcan also produce a wide variety of metabolites with antimicrobialactivities (Zhang et al., 2008). A number of the active metaboliteswere bactericidal or bacteriostatic peptides, which were non-ribos-omally synthesized using the multi-enzyme-catalyzed systems.Most of these peptides are very stable because of their low molec-ular weight and specific structure (Frueh et al., 2008). Gramicidin Sis such a cyclic decapeptide with wide antibiotic spectra, which is awell-studied antimicrobial compound produced by B. brevis (Elmaret al., 1999). Moreover, these compounds have become researchhotspots in the recent years because of their broad antibacterialspectrum, high potency, and no-crossing drug resistance withother antibacterial drugs (Zhao et al., 2010).

In the present study, the antibacterial peptide Tostadin was iso-lated using a three-step isolation procedure, including ammoniumsulfate precipitation, cation exchange chromatography on CM-Sepharose Fast Flow column, and the reversed-phase HPLC. Thispurification process was successfully used for this antibacterialpeptide from strain XDH. The pure active component, namely Tost-adin, which showed strong inhibition against E. coli and S. aureus,was purified and identified. Moreover, the modifications at bothends of Tostadin were found to display higher activities than thoseof the unmodified compounds. This phenomenon may be causedby the stable structure of the modified peptide since it is not easilyresisted or decomposed. Besides, both the natural and syntheticTostadin exhibited thermal stability, which can be found in othernon-ribosomal peptides. In summary, this novel compound is a sol-uble peptide with great antibiotic effects and high stability, and itis easily synthesized because of its low molecular weight and lin-ear structure. Thus, Tostadin may have great potential applicationsas a novel antibacterial agent.

4. Conclusions

In this work, a linear peptide namely Tostadin was isolated andidentified from a B. brevis strain XDH, which showed strong anti-bacterial activities against E. coli and S. aureus. Thirty five milligram

purified Tostadin was obtained by ammonium sulfate precipita-tion, ion exchange chromatography, and reversed-phase high-per-formance liquid chromatography. MS and NMR analysisdemonstrated that this compound consisted of nine amino acids,of which two amino acids residues were of D-configuration. Fur-ther studies illustrated that both the natural and syntheticTostadins were thermally stable. When modified at both ends, itexhibited higher activity and stability. These findings supportedthe characterization of a novel antibacterial peptide.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.biortech.2012.02.051.

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