Purification and Characterization of Plantaricin JLA-9: A Novel

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Purification and characterization of plantaricin JLA-9: a novel bacteriocin against Bacillus spp. produced by Lactobacillus plantarum JLA-9 from Suan-Tsai, a traditional Chinese fermented cabbage Shengming Zhao, Jinzhi Han, Xiaomei Bie, Zhaoxin Lu, Chong Zhang, and Fengxia Lv J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05717 • Publication Date (Web): 17 Mar 2016 Downloaded from http://pubs.acs.org on March 19, 2016

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Journal of Agricultural and Food Chemistry

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Purification and characterization of plantaricin JLA-9: a novel bacteriocin

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against Bacillus spp. produced by Lactobacillus plantarum JLA-9 from Suan-Tsai,

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a traditional Chinese fermented cabbage

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Shengming Zhao, Jinzhi Han, Xiaomei Bie*, Zhaoxin Lu, Chong Zhang, Fengxia Lv

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College of Food Science and Technology, Nanjing Agricultural University, Key

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Laboratory of Food Processing and Quality Control, Ministry of Agriculture of China,

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No.1 Weigang Nanjing 210095, P.R. China

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Corresponding author: Xiaomei Bie. Email: [email protected]

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Address: College of Food Science and Technology, Nanjing Agricultural University,

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Key Laboratory of Food Processing and Quality Control, Ministry of Agriculture of

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China, Nanjing, P.R. China

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Tel: 0086-25-84396570;

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Fax: 0086-25-84396431;

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ACS Paragon Plus Environment


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ABSTRACT

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Bacteriocins are ribosomally synthesized peptides with antimicrobial activity

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produced by numerous bacteria. A novel bacteriocin-producing strain, Lactobacillus

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plantarum JLA-9, isolated from Suan-Tsai, a traditional Chinese fermented cabbage,

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was screened and identified by its physiobiochemical characteristics and 16S rDNA

28

sequence analysis. A new bacteriocin, designated plantaricin JLA-9, was purified

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using butanol extraction, gel filtration, and reverse-phase high-performance liquid

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chromatography. The molecular mass of plantaricin JLA-9 was shown to be 1044 Da

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by MALDI-TOF-MS analyses. The amino acid sequence of plantaricin JLA-9 was

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predicted to be FWQKMSFA by MALDI-TOF-MS/MS, which was confirmed by

33

Edman degradation. This bacteriocin exhibited broad-spectrum antibacterial activity

34

against Gram-positive and Gram-negative bacteria, especially Bacillus spp., high

35

thermal stability (20 min, 121°C) and narrow pH stability (pH 2.0-7.0). It was

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sensitive to α-chymotrypsin, pepsin, alkaline protease and papain. The mode of action

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of this bacteriocin responsible for outgrowth inhibition of B. cereus spores was

38

studied. Plantaricin JLA-9 had no detectable effects on germination initiation over 1 h

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on monitoring the hydration, heat resistance and 2,6-pyridinedicarboxylic acid (DPA)

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release of spores. Rather, germination initiation is a prerequisite for the action of

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plantaricin JLA-9. Plantaricin JLA-9 inhibited growth by preventing the establishment

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of oxidative metabolism and disrupting membrane integrity in germinating spores

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within 2 h. The results suggest that plantaricin JLA-9 has potential applications in the

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control of Bacillus spp. in the food industry.


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Keywords: Lactobacillus plantarum; Bacteriocin; Purification; Characterization;

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Bacillus spp.

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INTRODUCTION

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Bacteriocins are a class of ribosomally synthesized peptides with antimicrobial

51

activity produced by numerous bacteria. Bacteriocins inhibit the growth of other

52

bacteria, sometimes of similar or closely related species. It is well known that lactic

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acid bacteria (LAB) produce a variety of bacteriocins. The LAB bacteriocins, which

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have been widely applied in many countries for food preservation, are defined as safe

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and non-toxic 1.

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The bacteriocins from LAB have wide ability to inhibit the growth of bacteria and

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fungi such as Bacillus cereus, Salmonella enteritidis, Escherichia coli, and

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Penicillium notatum, preventing food spoilage and extending the shelf-life of food

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products

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discovered5, has been used as a natural food antiseptic agent in the food industry in

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dozens of countries6, 7.

2-4

. For instance, nisin, produced by Lactococcus lactis, the first bacteriocin

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Although many bacteriocins have been studied, relevant reports concerning their

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application in food preservation are relatively rare8. Accordingly, more novel

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bacteriocins need to be explored for applications in the food industry9.

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Bacteriocin-producing bacteria exist widely in Chinese fermented vegetables.

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Meanwhile, LAB are found to be the dominant microorganisms in fermented food4, 10.

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Suan-Tsai is the most popular traditional fermented vegetable in the north-east region

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of China. LAB responsible for the fermentation, especially L. plantarum, are derived

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from the air, raw Chinese cabbages and the fermentation containers. A variety of

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bacteriocins produced by L. plantarum species isolated from fermented vegetables


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have been reported, including plantaricin 1634, plantaricin A-18, plantaricin 35d11,

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plantaricin MG12, and plantaricin ZJ513.

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Bacillus spp. are common bacteria that are often present in dairy foods, canned

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food, fruit, vegetables and bread, causing severe food spoilage or food-borne diseases.

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Toxins produced by these bacteria, especially botulinum toxin, cause diarrhea,

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vomiting and even death14. Hence, there is an urgent need to prevent or control

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Bacillus spp. in food. Although many bacteriocins have been identified in previous

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studies, no bacteriocin with broad spectrum anti-Bacillus spp. activity has been

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reported. As the possible harmful health effects from chemical additives in foods are

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gradually realized, more effective bacteriocins, which have potential for use as natural

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and safe food preservatives, need to be explored.

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Therefore, the aim of the present paper was to isolate an LAB strain from Suan-Tsai

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with a broad inhibitory spectrum against Bacillus spp., then to identify the structure

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and properties of a novel bacteriocin, and to preliminarily reveal its mechanism of

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action against B. cereus spores, in order to develop a natural and highly efficient food

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preservative to prevent contamination by Bacillus spp. and to extend the shelf-life of

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food.

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MATERIALS AND METHODS

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Samples, Bacterial Strains and Culture Conditions. Samples of Suan-Tsai were

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collected from Jilin Province, north-east China. All LAB stains were grown in MRS

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medium at 37°C. Other bacteria were mainly grown in LB broth, LB medium was



93

also used for antibacterial activity tests. FT medium was used for the culture of

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Clostridium. All bacteria used as indicator organisms in this study were grown at

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37°C unless stated. All bacteria were stored as frozen cultures at −80°C in appropriate

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culture medium with 30% (v/v) glycerol.

97 98

Screening for LAB strains with bacteriocin activity. Lactobacillus isolated from

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samples were spread directly onto the surface of MRS agar and then incubated for 24

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h at 37°C15. Single bacterial colonies were inoculated at random into 2 ml of MRS

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medium for bacteriocin screening and incubated for 48 h at 37°C. Cultures were

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centrifuged at 9000 × g for 20 min at 4°C and the supernatants obtained were filtered

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through 0.22-µm filters to produce cell-free supernatants (CFSs) for subsequent

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experiments12.

105 106

Primary screening for bacteriocin activity. The antibacterial activities of CFSs of

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all selected Lactobacillus strains were screened using agar well diffusion assays. The

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106 CFU/ml Bacillus cereus AS 1.1846 or Clostridium sporogenes CICC 10385 were

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used as the indicator strain. Then, the antibacterial activities of the CFSs were

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determined by measuring the diameters of inhibition zones with a vernier caliper. The

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CFSs of strains with a relatively large inhibition zone were used in subsequent

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studies.

113 114

Secondary screening for bacteriocin activity. Positive strains identified in the


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primary screening assay were further screened for antibacterial activity in agar well

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diffusion assays using other pathogenic and food-spoiling Bacillus spp. (B. pumilus

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CMCC 63202, B. megaterium CICC10448, B. coagulans CICC20138, B. subtilis

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ATCC9943, Clostridium difficile CICC22951, C. sporogenes CICC 10385 and C.

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perfringens CICC22949) as indicator strains. After 12 h, the diameters of inhibition

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zones formed by tested CFSs were measured and the diameters of inhibition zones >9

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mm recorded. A strain with broad-spectrum antibacterial activity and relatively large

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inhibition zones was selected for the following studies.

123 124

Identification of the bacteriocin-producing strain. The identification of one

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bacteriocin-producing strain, JLA-9, from Chinese fermented cabbage was based

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initially on phenotypical, biochemical and physiological characteristics, including

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Gram staining, the catalase reaction, cell morphology, and carbohydrate fermentation

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patterns16. Genotypic identification was carried out by 16S rDNA sequence analysis.

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The species identity was confirmed by PCR using universal primers: 16SAF

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(5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ)

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(5ʹ-TACGGYTACCTTGTTACGACTT-3ʹ). The PCR conditions were used as

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described by Hu et al.4. The PCR products were purified using Omega Gel Extraction

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Kits (Omega, USA) and sequenced by Genscript (Nanjing, China). Sequence

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homologies were examined by blasting the obtained sequences against the GenBank

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database (http://www.ncbi.nlm.nih.gov/BLAST).

and

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16SAR


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Purification of Plantaricin JLA-9. Strain L. plantarum JLA-9 was cultured in 100 ml

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MRS medium with agitation to an initial OD600 of 0.5, and then 2% (v/v) of the liquid

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culture was inoculated into 1 l MRS medium with agitation and grown to

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post-stationary phase. The fermentation broth was centrifuged at 9000 × g for 20 min

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at 4°C in order to remove bacterial cells. Then the cell free supernatant was extracted

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with n-butyl alcohol for 10 h using a separating funnel. The extracted liquid was

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vacuum concentrated to 20 ml to eliminate the n-butyl alcohol and the antimicrobial

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crude extracts obtained were further purified.

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A Sephadex G25 column (1.6 cm × 80 cm) was equilibrated with phosphate

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buffer (pH 6.0). After the equilibration, 1 ml of extracted sample filtered through a

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0.22-µm filter membrane was loaded onto the column and eluted at a flow rate of 0.3

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ml/min with phosphate buffer (pH 6.0). Each 3 ml fraction was collected and the

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antibacterial activity against B. cereus AS1.1846 was evaluated by the agar diffusion

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method. The active fraction obtained from gel filtration was freeze-dried and

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resuspended in 1 ml of phosphate buffer (pH 6.0).

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A Sephadex LH-20 column (1.6 cm × 120 cm) chromatography step4 was then

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performed in a similar manner to the Sephadex G25 step, except for the substitution

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of 80% methanol in water for phosphate buffer. 1 ml of freeze-concentrated fraction

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filtered with a 0.22-µm filter membrane was loaded onto the Sephadex LH-20 column

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and then eluted at a flow rate of 0.3 ml/min with 80% methanol over 10 h. The active

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fractions were collected by determining the antibacterial activity using B. cereus

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AS1.1846 as the indicator strain. Then, the active fractions were centrifugally vacuum


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concentrated in a Christ RVC 2-25 CD plus instrument. The fraction with the highest

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antibacterial activity was collected and the absorption spectrum was obtained using an

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ultraviolet-visible spectrophotometer (UV-2450, Shimadzu, Japan); the wavelength

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displaying the highest absorbance was recorded17.

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To further purify the concentrated antibacterial fraction from the Sephadex LH-20

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column, an Acchrom XCharge C18 column (4.6/250 column) incorporated in a

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reversed-phase high performance liquid chromatography (RP-HPLC) system (Agilent

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1100 series, USA), was used. The elution phase used a linear gradient from 95%

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water containing 0.1% TFA to 95% acetonitrile containing 0.1% TFA over 40 min

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with a flow rate of 0.2 ml/min, monitored at 259 nm4. The purified fractions were

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collected, concentrated and screened for antibacterial activity using B. cereus

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AS1.1846. The purified active fraction was finally vacuum freeze-dried in a Christ

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ALPHA 1-4 LD plus for structural identification of the bacteriocin.

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Molecular mass and amino acid sequence determination of plantaricin JLA-9.

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The molecular mass of plantaricin JLA-9 was determined by matrix-assisted laser

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desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) (Bruker

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Daltonic, Bremen, Germany) using α-cyano-4-hydroxycinnamic acid (CHC) as the

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matrix4, 18. The N-terminal amino acid sequence of HPLC-purified plantaricin JLA-9

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was determined via automated Edman degradation. Sequencing was carried out in a

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PPSQ-33A protein sequencer (Shimadzu Corporation, Kyoto, Japan) by Applied

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Protein Technology (Shanghai, China).

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Antibacterial spectrum and MIC values of plantaricin JLA-9. The antibacterial

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spectrum of plantaricin JLA-9 purified by RP-HPLC was assessed using selected

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indicator strains (Table 1). The minimum inhibitory concentration (MIC) of

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plantaricin JLA-9 against different indictor strains was detected by the double broth

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dilution method as previously described19. In brief, a stock solution of 2048 µg/ml

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plantaricin JLA-9 was two-fold serially diluted in LB medium in a 96-well

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round-bottom polystyrene plate. Overnight-cultured different indicator bacteia were

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diluted into LB medium and approximately 106 CFU were added to each well of the

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microtiter plate supplemented with the range of concentrations of plantaricin JLA-9.

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The microplate was incubated at 37°C for 24 h to determine the lowest concentration

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of plantaricin JLA-9 inhibiting visible growth. After incubation, the MIC values were

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measured by adding 10 µl of a 0.5% triphenyl tetrazolium chloride (TTC) aqueous

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solution. The MIC was defined as the lowest concentration of plantaricin JLA-9 that

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inhibited visiblegrowth, as indicated by the color change of TTC.

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Effect of enzymes, pH, and heat treatment on the activity of plantaricin JLA-9.

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Firstly, the sensitivity of partially purified plantaricin JLA-9 collected from the

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Sephadex LH-20 column towards different proteases was evaluated. Plantaricin

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JLA-9 was incubated with different enzymes (Sigma) including pepsin, alkaline

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protease, papain and α-chymotrypsin (each at a final concentration of 5 mg/ml), at

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appropriate pHs, at 37°C, for 3 h. Secondly, pH stability of plantaricin JLA-9 was

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assessed by adjusting the pH value of the plantaricin JLA-9 with NaOH or HCl in a


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range from 2.0 to 10.0 at 37°C for 3 h. Thirdly, the thermal stability of plantaricin

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JLA-9 was determined by exposure to temperatures of 60, 80 and 100°C for 10 and

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30 min respectively, in a thermostatic water bath, as well as to 121°C for 20 min in an

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autoclave.

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After the enzyme, pH, and thermal treatments, the samples were readjusted to pH

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7.0. Then, an agar diffusion assay was performed using B. cereus AS1.1846 as the

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indicator to evaluate the remaining antibacterial activity. The zones of inhibition were

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measured by vernier caliper. All experiments were performed in triplicate.

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Spore preparation. Spores were prepared from B. cereus AS 1.1846 as previously

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described19. The enumeration of spore suspensions was performed by a plate

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colony-counting method. The final spore concentration we obtained was 5.6×109

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spores/ml.

216 217

Determination of outgrowth inhibitory concentration (OIC). Antibacterial

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susceptibility tests were performed by the double broth dilution method as previously

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described19. The outgrowth inhibitory concentration (OIC) was defined as the lowest

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concentration of plantaricin JLA-9 in which no growth of spores was observed after

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24 h at 37°C. The OIC assays was carried out three times to ensure the reproducibility

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of results.

223 224

Spore hydration. Spore hydration was evaluated by detecting the spore refractility



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variation using a Varioskan™ spectral scanning multimode reader (Thermo, Finland).

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The spores at a final concentration of 5.6×106 spores/ml were added into LB broth in

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96-well microtiter plates with various concentration of plantaricin JLA-9. The OD600

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of the spore suspensions was measured before the incubation and after incubation for

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1 h at 37°C and the percentage change of the spore refractility was determined45.

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Spore thermostability. Ten millimolar D-histidine and D-alanine were added to

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spores to prevent further germination initiation45. Then, the spore suspensions were

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incubated in LB broth supplemented with various concentrations (2-fold dilutions) of

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plantaricin JLA-9, or 0.1 M PBS (pH 6.8) as negative control. Samples were

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incubated for 1 h at 37°C, and then in a 65°C water bath or in an ice bath for 30 min.

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The samples were serially diluted and plated onto LB agar to quantify viable B. cereus.

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The percentage of heat-resistant spores was evaluated by comparing the samples

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incubated at 65°C with those treated in the ice bath.

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Detection of 2,6-pyridinedicarboxylic acid (DPA) release. Germination of spores

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was monitored by measuring the release of 2,6-pyridinedicarboxylic acid (DPA) by

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the fluorescence resonance energy transfer (FRET) between terbium and DPA using a

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Varioskan™ spectral scanning multimode reader. The spores were incubated with

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different concentrations (2-fold dilutions) of plantaricin JLA-9 as described in the

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section “spore hydration”, except for further incubation with TbCl3 (200 µM) in an ice

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bath for 15 min. The DPA-Tb complex was assessed by FRET with excitation and


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emission at 280 nm and 546 nm, respectively20.

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Detection of oxidative metabolism.

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The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-h-terazolium bromide) assay

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was used to evaluate the change of oxidative metabolism of spore suspensions

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incubated in LB broth at 37°C in 96-well microtiter plates supplemented with various

253

concentrations (2-fold dilutions) of plantaricin JLA-9, or with 0.1 M PBS (pH 6.8) as

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negative control21. D-Histidine and D-alanine (10 mM) were added to the collected

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spores to prevent further germination initiation before the MTT (5 mg/ml) assay. The

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conversion of tetrazolium to formazan was determined at 540 nm using a Varioskan™

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spectral scanning multimode reader.

258 259

The integrity of spore membranes. Spore membrane integrity was evaluated using

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BD Accuri™ C6 flow cytometry (Ann Arbor, MI, USA) with propidium iodide (PI)

261

by measuring the uptake of PI21, 22. The spore concentration was adjusted to 5.6×106

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spores/ml by dilution in LB broth supplemented with different concentrations (2-fold

263

dilutions) of plantaricin JLA-9, or with 0.1 M PBS (pH 6.8) as a negative control.

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After different incubation times, plantaricin JLA-9-treated spores were washed twice

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and then cultured with PI (100 µM) in PBS in an ice bath for 15 min. In flow

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cytometry, 1000 events were collected for each sample at the medium flow rate, with

267

excitation and emission at 488 nm and 525 nm, respectively45.

268



269

Data Analysis. Results are presented as mean values ± standard deviation (SD).

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Origin 9.0 and SPSS 16.0 statistical software were used for data analyses.

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RESULTS AND DISCUSSION

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Screening of bacteriocin-producing strains. A total of 580 Gram-positive

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acid-producing strains isolated from Suan-Tsai were cultured in MRS medium. The

275

cell-free supernatants of 49 strains exhibited antibacterial activity against B. cereus

276

and C. sporogenes in primary screening (data not shown). After secondary screening,

277

cell-free supernatants of five strains showed strong antibacterial activity against

278

indicators including B. pumilus, B. megaterium, B. coagulans, B. subtilis, C. difficile

279

and C. perfringens. Among these, strain JLA-9, which had the highest antibacterial

280

activity against Bacillus spp. (data not shown), was selected for the following studies.

281 282

Identification of bacteriocin-producing strain JLA-9. Strain JLA-9 was a

283

Gram-positive, nonflagellated, catalase negative and rod-shaped bacillus without

284

spore formation that did not produce gas. It could grow at 15°C but not at 45°C, and

285

in 3% but not in 6% or 9% NaCl. Moreover, it was able to produce acid from glucose.

286

To further characterize the strain, sugar fermentation assay tests indicated that it could

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metabolize L-arabinose, cellobiose, aesculin, D-fructose, glucose, lactose, D-mannose,

288

mannitol, sorbitol, melezitose, melibiose, raffinose, saligenin, sucrose, trehalose,

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xylose, inulin, and maltose, but not rhamnose. Regarding 16S rDNA sequence

290

analysis, the nucleotide sequence of a 1544 bp fragment was amplified from strain


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JLA-9 genomic DNA (GenBank accession no. KP406154). A neighbor-joining

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phylogenetic tree was drawn from sequence alignment and comparison (Figure 1),

293

which showed 100% similarity to L. plantarum WCFS1 (NR075041.1). Hence, on the

294

basis of biochemical and morphological characteristics, we concluded that the strain

295

JLA-9 belongs to L. plantarum.

296

Fermented foods and vegetables are good sources for isolating L. plantarum.

297

Many bacteriocin-producing L. plantarum stains have been isolated, such as L.

298

plantarum C1923, L. plantarum LPCO1024, L. plantarum ZJ00815, L. plantarum 1634,

299

L. plantarum A-18 and L. plantarum ZJ513. Similar to many previous studies of LAB,

300

the strain L. plantarum JLA-9 is capable of producing bacteriocin that, in this case,

301

exhibits high antibacterial activity against Bacillus spp. as found in foods.

302

Lactobacillus plantarum are often used for food fermentation, for instance, in the

303

production of yogurts, pickles and fermented meats; L. plantarum use not only

304

improves the flavors, nitrogen content and food texture, but also extends the shelf-life

305

of the foods. In the present study, strain L. plantarum JLA-9 was capable of secreting

306

high concentrations of lactic acid and bacteriocin. Therefore, it has potential for

307

application in the food preservation field as well as in fermented food industries.

308 309

Purification of plantaricin JLA-9. The plantaricin JLA-9 produced by strain L.

310

plantarum JLA-9 was purified from culture supernatant by n-butyl alcohol extraction

311

and two column chromatography steps using Sephadex G-25and Sephadex LH-20.

312

Then, the UV/visible absorption spectra of the purified fraction revealed a maximum



313

absorbance wavelength at 259 nm. The active fractions collected by gel

314

chromatography were applied to RP-HPLC and an active peak with a retention time

315

of 19.576 min was observed (Figure 2a). A series of classical strategies for isolation

316

and purification were applied in our study, including organic solvent extraction, gel

317

filtration and RP-HPLC. A multitude of previous studies reported that ammonium

318

sulfate precipitation was used as the first-step for bacteriocin isolation, including in

319

the purification of plantaricin 1634, plantaricin A-18 and sakacin LSJ61825. However,

320

ammonium sulfate precipitation applied to the culture supernatant of strain L.

321

plantarum JLA-9 did not result in the collection of more antibacterial substances

322

compared with n-butyl alcohol extraction (data not shown). In recent years, the

323

pollution of food by Bacillus spp. has increasingly been reported26-28. For instance,

324

partly dairy products often suffer from Bacillus spp. contamination because of their

325

low temperature sterilization. Spores are not killed completely, leading to the spoilage

326

of the food products.

327

Molecular mass determination and structure identification of plantaricin JLA-9.

328

MALDI-TOF analysis of purified plantaricin JLA-9 revealed that its molecular mass

329

was 1044 Da (Figure 2b), smaller than previously studied plantaricins8, 12. In the past

330

few years, several new bacteriocins produced by L. plantarum have been successfully

331

purified and characterized. A majority of the molecular masses of L. plantarum

332

bacteriocins are >2.0 kDa, for example, plantaricin C19 (3.8 kDa) from L. plantarum

333

C1929, plantaricin 423 (3.5 kDa) from L. plantarum 42323, plantaricin Y from L.

334

plantarum 510 (4.2 kDa)30, plantaricin 163 (3.5 kDa) from L. plantarum 1634 and


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pediocin LB-B1 (2.5 kDa) from L. plantarum LB-B131, but excepting plantaricin

336

ZJ008 (1.3 kDa) produced by L. plantarum ZJ00815. However, to the best of our

337

knowledge, there is no previous report of a molecular mass as low as 1044 Da for a

338

plantaricin produced by any L. plantarum strain.

339

MALDI-TOF-MS/MS of plantaricin JLA-9 was used to predict its amino acid

340

sequence. The principle of MALDI-TOF-MS analysis of molecular structures of

341

polypeptides is that the polypeptide is blended with a specific matrix and forms a

342

co-crystallization film by laser irradiation. Then, energy is absorbed by the matrix

343

from the laser and transferred to the polypeptide to ionize an electron. The parent ion

344

of the polypeptide and a few large fragment ions can be obtained by this technique,

345

and the primary structure of the polypeptide can be deduced according to the intensity

346

of the fragment ions.

347

MS/MS analysis of the precursor ion of plantaricin JLA-9, with m/z 1044.641, is

348

shown in Figure 2c. The product ion m/z 972.543 was interpreted as having lost an

349

alanine from the peptide, a fragment ion at m/z 828.342 was produced by loss of

350

alanine and phenylalanine from the peptide, and a fragment ion at m/z 728.268

351

derived from the peptide without alanine, phenylalanine and serine. Other fragment

352

ions (m/z 608.279, m/z480.933, m/z352.399 and m/z m/z165.776) in Fig. 2c could be

353

used to determine the remaining peptide sequence by a similar calculation method.

354

According to the peptide fragmentation nomenclature proposed by Roepstorff and

355

Fohlman, we deduced that the entire amino acid sequence was FWQKMSFA.

356

The calculated amino acid sequence was verified by Edman degradation of



357

HPLC-purified plantaricin JLA-9 using an automated PPSQ-33A protein sequencer

358

with previously reported protocols 32. The analysis results determined that the amino

359

acid sequence of plantaricin JLA-9 was FWQKMSFA, completely consistent with the

360

conclusions from MALDI-TOF-MS (Figure S1). Compared with previously studied

361

bacteriocins, plantaricin JLA-9 (8 amino acids) is a short peptide. The numbers of

362

amino acids of the bacteriocins produced by L. plantarum species are generally >20,

363

for example plantaricin C19 (36 amino acids) from L. plantarum C1929, plantaricin

364

423 (33 amino acids) from L. plantarum 42323, plantaricin C11 (25 amino acids) from

365

L. plantarum J33, plantaricin 163 (32 amino acids) from L. plantarum 1634 and

366

plantaricin Sα (25 amino acids) from L. plantarum LPC01034. Meanwhile, the

367

sequence of plantaricin JLA-9 showed no homology with other known bacteriocins

368

using protein BLAST against the GenBank database (www.ncbi.nlm.nih.gov/BLAST).

369

Thus, plantaricin JLA-9 may be identified as a novel bacteriocin produced by L.

370

plantarum.

371 372

Antibacterial spectrum and MIC values of plantaricin JLA-9. The antibacterial

373

spectrum and MIC values of plantaricin JLA-9 are presented in Table 1. Plantaricin

374

JLA-9 exhibited notable antibacterial activity and significantly inhibited spoilage and

375

pathogenic Bacillus species, including B. cereus, B. pumilus, B. megaterium, B.

376

coagulans, B. subtilis, G. stearothermophilus, A. acidoterrestris, Paenibacillus

377

polymyxa, C. difficile, C. perfringens and C. sporogenes. A lower MIC was obtained

378

for plantaricin JLA-9 against B. cereus than was previously reported for


Page 18 of 49

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379

nisin40.Plantaricin JLA-9 also had a broad inhibitory activity, including against other

380

Gram-positive bacteria such as Staphylococcus aureus and Micrococcus luteus, as

381

well as Gram-negative bacteria such as Pseudomonas fluorescens, Serratia

382

marcescens, Escherichia coli, Salmonella enteritidis, Salmonella typhimurium,

383

Salmonella paratyphi A, Salmonella paratyphi B, Shigella flexneri and Proteus

384

mirabilis.

385

A great many bacteriocins produced by L. plantarum strains exhibit significant

386

inhibitory activity against food-borne pathogens. For instance, the plantaricin ZJ008

387

produced by L. plantarum ZJ008 showed a broad inhibitory spectrum, especially

388

against Staphylococcus spp., including S. carnosus, S. citreus, and S. epidermidis15.

389

The inhibition spectrum of plantaricin LB-B1 appeared to be relatively wide; it

390

inhibited the growth of Listeria spp. and many other Gram-positive and

391

Gram-negative bacteria31. Plantaricin MG was mainly active against Gram-negative

392

food-borne pathogens12. The plantaricin UG1 was reported to inhibit some food-borne

393

pathogens, including C. perfringens, C. sporogenes and B. cereus35. In the present

394

study, plantaricin JLA-9 had broad inhibitory activity against a large range of food

395

spoilage and pathogenic Bacillus strains; there are no previously known bacteriocins

396

produced by L. plantarum strains with broad-spectrum inhibitory activity against

397

Bacillus strains. Thus, serving as a good preservative candidate, plantaricin JLA-9

398

may have high potential for application in the food industry to extend product

399

shelf-life.

400



401

Effect of enzymes, temperature and pH on the antibacterial activity of

402

plantaricin JLA-9. The effects of proteases, temperature and pH on the stability of

403

plantaricin JLA-9 are shown in Figure 3. After enzymatic treatment with pepsin,

404

alkaline protease, papain or α-chymotrypsin, complete inactivation in the antibacterial

405

activity of partially purified plantaricin JLA-9 was observed (Figure 3A). Thus,

406

plantaricin JLA-9 is a peptide that can be digested by pepsin after eating; it can

407

therefore be used safely in food preservation. Some plantaricins, including plantaricin

408

1634 and pediocin LB-B131, have similar characteristics, in that their antimicrobial

409

activity was lost after such protease treatments. However, other plantaricins like

410

plantaricin D could be degraded by α-chymotrypsin, trypsin, pepsin and proteinase K,

411

but not papain36.

412

In addition, the antibacterial activity of plantaricin JLA-9 was evaluated at

413

different temperatures and under conditions of different pH. Plantaricin JLA-9

414

remained relatively stable after treatment at 60, 80 and 100°C for 30 min. 58%

415

inhibitory activity was retained after treatment at 121°C for 20 min (Figure 3B).

416

Plantaricin JLA-9 was stable after heat treatment, the same as other plantaricins, for

417

example, bacteriocin J2337 and plantaricin TF71138. However, plantaricin JLA-9 had

418

better thermal stability than nisin5. The high thermal stability of plantaricin JLA-9

419

will make it suitable for use in heat processed food.

420

Assessment of pH stability revealed that plantaricin JLA-9 remained stable after

421

incubation for 3 h at pH values ranging from 2.0 to 7.0, but its activity reduced

422

notably at pH 10.0 (Figure 3C). Similar characteristics were previously reported for


Page 20 of 49

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423

some LAB bacteriocins, but other bacteriocins, including nisin, were easily

424

inactivated by neutral and alkaline condition39 which limit their application in the

425

food industry. Moreover, some plantaricins have characteristics of heat susceptibility

426

and narrow pH stability, e.g., plantaricin UG1 only retained stable inhibitory activity

427

at pH 4.5 to 7.0 and showed thermal inactivation on treatment at 100°C for 30 min35.

428

Overall, assessment of the newly discovered plantaricin JLA-9 revealed that it

429

has the merits of being degradable by various proteases, high thermal stability and

430

wide pH stability in comparison with previously reported LAB bacteriocins.

431

OIC determinations. The OIC of plantaricin JLA-9 against B. cereus strain AS

432

1.1846 were determined to be 32 µg/ml. The OIC assay was used to determine

433

whether plantaricin JLA-9 inhibits the outgrowth of spores into vegetative cells.

434

Plantaricin JLA-9 was effective at preventing the outgrowth of spores. On the basis of

435

these results, 32 µg/ml of plantaricin JLA-9 was defined as 1×OIC, which was used in

436

subsequent experiments.

437 438

Germination initiation was not inhibited by plantaricin JLA-9. Whether or not the

439

germination initiation of spores was inhibited by plantaricin JLA-9 was first evaluated

440

by measuring the loss of spore refractility by the change in OD600. Spores were highly

441

refractile. Refractility decreased by >50% over 60 min in incubation broth

442

supplemented with either plantaricin JLA-9 or 0.1 M PBS (Figure 4A). Even high

443

concentrations (64µg/ml) of plantaricin JLA-9 did not exhibit inhibitory effects on the

444

initiation of germination. Thus, plantaricin JLA-9 did not significantly alter the



445

hydration of spores undergoing germination initiation.

446

Loss of spore thermostability is another hallmark of germination initiation, due to

447

rupture of the exosporium. We observed a decrease in thermal stability of spores

448

of >80% after 60 min both in the presence and absence of plantaricin JLA-9 (Figure

449

4B), which again indicated that the germination initiation of spores was not inhibited

450

by incubation with plantaricin JLA-9.

451

DPA is the unique substance in spores which is not found in vegetative cells or

452

other non-spore forming bacteria. The DPA content of the spore cell weight is almost

453

17%41. Once spores begin to germinate, all DPA is released to the external

454

environment from the spore structure. Thus, the release of DPA was monitored to

455

further evaluate the possibility that plantaricin JLA-9 inhibited the germination

456

initiation of spores. DPA release was the same in either the presence or absence of

457

plantaricin JLA-9 (Figure 4C).

458

On the basis of these results, we conclude that the germination initiation of

459

spores over 1 h was not altered by supplementation with plantaricin JLA-9. These

460

results are in conformity with previous studies showing that nisin did not inhibit B.

461

cereus growth by impeding germination initiation42, 43. Consequently, that the spores

462

of B. cereus did not grow to be vegetative cells on treatment with plantaricin JLA-9

463

was not because of the inhibition of germination, but may be due to the killing of

464

germinated spores by the plantaricin. In our experiments, plantaricin JLA-9 did not

465

result in any reduction in viability counts, thermostability or DPA release of spores,

466

which is consistent with results obtained for nisin43. A previous study on subtilin


Page 22 of 49

Page 23 of 49


467

showed that it also inhibited spore outgrowth without inhibiting germination

468

initiation44.

469

Germination initiation is a prerequisite for the inhibitory action of plantaricin

470

JLA-9 on spores. Next, we investigated whether initiation of germination is essential

471

for the inhibition of the growth of B. cereus from spores to vegetative cells by

472

plantaricin JLA-9. Spores of B. cereus were incubated in LB medium, in LB medium

473

supplemented with 32 µg/ml plantaricin JLA-9, or in 0.1 M PBS (pH 6.8)

474

supplemented with 32 µg/ml plantaricin JLA-9. After incubation for 2 h the spores

475

were washed with PBS to remove the plantaricin JLA-9. Then, each treated group was

476

incubated in LB medium for 8 h. Spores not treated with plantaricin JLA-9 grew well

477

in LB medium. In contrast, spores treated with plantaricin JLA-9 during germination

478

did not grow in LB medium (Figure 5). These results demonstrate that germination

479

initiation is indispensable for the antibacterial activity of plantaricin JLA-9 against

480

spores. Previous work demonstrated that germinated spores were inactivated by nisin

481

to some extent and germination initiation is also a prerequisite for the action of nisin45.

482

The loss of bacteriocin resistance can be explained by degradation of the spore coat.

483

The cortex and the coat are more rigid than the membrane of vegetative cells, which

484

makes it more difficult for spores to be affected by plantaricin JLA-9 than vegetative

485

cells. Therefore, plantaricin JLA-9 only has the ability to inhibit the outgrowth of B.

486

cereus spores when the germination of spores has taken place.

487 488

Outgrowth of spores was inhibited by plantaricin JLA-9. Spores were incubated



489

for 10 h with different concentrations of plantaricin JLA-9 in LB medium to monitor

490

whether the spores developed into vegetative B. cereus. As expected, each treated

491

group rapidly started hydration, shown by the reduction of OD600 over 1 h, which

492

reflected the complete germination initiation of spores (Figure 6). However, the

493

spores did not grow to vegetative B. cereus in the presence of high concentrations (32

494

µg/ml and 64 µg/ml) of plantaricin JLA-9. In contrast, the spores incubated in the

495

absence of plantaricin JLA-9 demonstrated obvious bacterial growth. Outgrowth also

496

appeared to some extent when spores were treated with a low concentration of

497

plantaricin JLA-9 (Figure 6). Therefore, outgrowth of spores was inhibited by

498

plantaricin JLA-9, and 32 µg/ml was determined as the concentration sufficient to

499

inhibit development of the spores into vegetative B. cereus.

500

Nisin is an FDA-approved natural preservative that has been applied in the food

501

industry for 40 years. It was reported that a concentration of 25 µg/ml was the

502

minimum for inhibiting the outgrowth of B. cereus spores46. The inhibitory activity of

503

plantaricin JLA-9 was about the same as that of nisin, but plantaricin JLA-9 exhibited

504

greater efficiency than bacteriocin AS-48 which was reported to require a

505

concentration of 50 µg/ml to inhibit the outgrowth of B. cereus spores47.

506 507

Detection of metabolic activity. Non-germinating spores have hardly any metabolic

508

activity. Outgrowth of spores was inhibited by plantaricin JLA-9, which could be due

509

to the loss of metabolic activity of germinating spores. To test whether the oxidative

510

metabolism of germinating spores was inhibited by plantaricin JLA-9, we used the


Page 24 of 49

Page 25 of 49


511

MTT assay. The production of formazan was not detected when spores were treated

512

with high concentrations (32 µg/ml or 64 µg/ml) of plantaricin JLA-9. Low

513

production of formazan was detected on treatment with a lower concentration (16

514

µg/ml) of plantaricin JLA-9. However, robust production of formazan was observed

515

during germination in the absence of plantaricin JLA-9 (Figure 7). These results are

516

consistent with the hypothesis that metabolic activity of germinating spores was

517

inhibited by plantaricin JLA-9.

518

The establishment of oxidative metabolism is necessary for the outgrowth of B.

519

cereus spores. Plantaricin JLA-9 had the ability to rapidly inhibit the outgrowth of B.

520

cereus spores by preventing the establishment of metabolism. Once oxidative

521

metabolism was inhibited, cell wall biosynthesis did not initiate48, which contributed

522

to the effect of plantaricin JLA-9 against the outgrowth of B. cereus spores. One study

523

reported that nisin was able to cause a disruption of oxidative metabolism to inhibit

524

the outgrowth of B. anthracis spores45. Our results reveal that the absence of oxidative

525

metabolism was a distinct factor explaining the outgrowth inhibition of B. cereus

526

spores by plantaricin JLA-9.

527 528

Flow cytometry-based detection of membrane integrity. The absence of oxidative

529

metabolism in germinating spores could be because the membrane integrity was

530

destroyed. To evaluate whether membrane integrity was involved in the growth

531

inhibition of spores, the membrane integrity of spores was measured by detecting the

532

PI uptake using flow cytometry. PI could not pass through the integrated cell



533

membrane, but could cross the damaged cell membrane and stained the cell nuclear.

534

After 60 min incubation with plantaricin JLA-9, the amount of PI uptake increased

535

distinctly only in samples treated with a high concentration of plantaricin JLA-9 (64

536

µg/ml). After 120 min incubation, samples treated with plantaricin JLA-9 at both low

537

(16 µg/ml) and high (32 µg/ml or 64 µg/ml) concentrations showed increased PI

538

uptake relative to spores incubated in the absence of plantaricin JLA-9 (Figures 8 and

539

S2). These results demonstrated that the membrane integrity of germinating B. cereus

540

spores was disrupted by plantaricin JLA-9, further supporting the idea that the

541

outgrowth of B. cereus spores was inhibited by plantaricin JLA-9 in Figure 5. The

542

spores did not grow into vegetative B. cereus in the presence of plantaricin JLA-9.

543

The establishment of oxidative metabolism from germinating B. cereus spores was

544

inhibited, which was likely related to the disruption of membrane integrity. According

545

to our studies, once the PI uptake increased significantly, the spore outgrowth was

546

also inhibited and spores were not able to establish an integrated oxidative

547

metabolism. These observations are consistent with a previous study in which nisin

548

prevented outgrowth of germinating B. cereus spores by disrupting membrane

549

integrity45.

550

In summary, a novel anti-Bacillus bacteriocin, plantaricin JLA-9, produced by L.

551

plantarum JLA-9 isolated from Suan-Tsai (traditional fermented vegetables from

552

North-eastern China), was reported for the first time in the present study. After

553

purification, the molecular weight of plantaricin JLA-9 was determined as 1044 Da by

554

MALDI–TOF–MS. The amino acid sequence was determined as FWQKMSFA.


Page 26 of 49

Page 27 of 49


555

Plantaricin JLA-9 showed a broad spectrum of activity against food spoilage and

556

pathogenic Bacillus spp., and excellent thermal and pH stability. The mechanism of

557

inhibition of Bacillus cereus outgrowth by the plantaricin JLA-9 showed that

558

plantaricin JLA-9 prevented the development of germinated spores into vegetative

559

bacilli. Consequently, these results reveal that the plantaricin JLA-9 is an attractive

560

and promising natural biologic preservative for food preservation industry against

561

spore-forming Bacillus or Clostridium pathogens.

562 563

ACKNOWLEDGEMENTS

564

This work was supported by grants from the National Natural Science Foundation of

565

China (grant no. 31271828) and the National Science and Technology Support

566

program (grant no. 2012BAK08807).The authors are grateful to the Laboratory

567

Center of Life Sciences at Nanjing Agriculture University for expert technical

568

assistance with Bruker Ultraflex MALDI–TOF mass Spectrometer and BD Accuri™

569

C6 flow cytometry analyses.

570 571



572 573 574

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(41) Bell, S. E.; Mackle, J. N.; Sirimuthu, N. M. Quantitative surface-enhanced

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Raman spectroscopy of dipicolinic acid—towards rapid anthrax endospore detection.

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Analyst. 2005, 130, 545-549.

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(42) Faille, C.; Membre, J. M.; Kubaczka, M.; Gavini, F. Altered ability of Bacillus

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cereus spores to grow under unfavorable conditions (presence of nisin, low

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temperature, acidic pH, presence of NaCl) following heat treatment during sporulation.

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J. Food. Prot. 2002, 65, 1930-1936.

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(43) Pol, I. E.; van Arendonk, W. G.; Mastwijk, H. C.; Krommer, J.; Smid, E. J.;

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Moezelaar, R. Sensitivities of germinating spores and carvacrol-adapted vegetative



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cells and spores of Bacillus cereus to nisin and pulsed-electric-field treatment. Appl.

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Environ. Microbiol. 2001, 67, 1693-1699.

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(44) Liu, W.; Hansen, J. N. The antimicrobial effect of a structural variant of subtilin

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against outgrowing Bacillus cereus T spores and vegetative cells occurs by different

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mechanisms. Appl. Environ. Microbiol. 1993, 59, 648-651.

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(45) Gut, I. M.; Prouty, A. M.; Ballard, J. D.; van der Donk, W. A.; Blanke, S. R.

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Inhibition of Bacillus anthracis spore outgrowth by nisin. Antimicro Agents

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Chemother. 2008, 52, 4281-4288.

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(46) Penna, T. C. V.; Moraes, D. A. The influence of nisin on the thermal resistance of

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Bacillus cereus. J. Food. Prot. 2002, 65, 415-418.

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(47) Abriouel, H.; Maqueda, M.; Gálvez, A.; Martínez-Bueno, M.; Valdivia, E.

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Inhibition of bacterial growth, enterotoxin production, and spore outgrowth in strains

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of Bacillus cereus by bacteriocin AS-48. Appl. Environ. Microbiol. 2002, 68,

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1473-1477.

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(48) Gut, I. M.; Blanke, S. R.; van der Donk, W. A. Mechanism of inhibition of

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Bacillus anthracis spore outgrowth by the lantibiotic nisin. ACS. Chem. Biol. 2011, 6,

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744-752.

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Page 34 of 49

Page 35 of 49


723

Figure 1. Phylogenetic tree derived from the 16S rDNA sequence of L. plantarum

724

JLA-9. All of the sequences used were from LAB-type strains. Numbers at the nodes

725

indicate the bootstrap values on neighbor-joining analyses of 1000 replicates. Bar:

726

sequence divergence of 0.005.

727 728 729 730 731

Figure 2. (a) RP-HPLC of the active antibacterial peak with a retention time of 19.576

732

min, corresponding to plantaricin JLA-9. (b) MALDI-TOF-MS mass spectrum of

733

plantaricin JLA-9. (c) MALDI-TOF-MS/MS of a fragment of plantaricin JLA-9.

734 735 736 737 738

Figure 3. (a) Effect of temperature on the antibacterial activity of plantaricin JLA-9.

739

(b) Stability of plantaricin JLA-9 in different pH conditions; the initial pH value of

740

partially purified plantaricin JLA-9 preparations was 4.0. (c) Stability of plantaricin

741

JLA-9 after different enzyme treatments. Data are shown as mean ± standard

742

deviations of three independent experiments.

743 744



745

Figure 4. Initiation of spore germination is not inhibited by plantaricin JLA-9. (a)

746

spores treated with different concentrations (0 to 64 µg/ml) of plantaricin JLA-9 in LB

747

medium for 60 min were monitored in terms of OD600 at time zero, and at 60 min

748

relative to the value for each culture at time zero. (b) Samples were analyzed for

749

thermostability by detecting the OD600 at time zero, and at 60 min relative to the value

750

for each culture at time zero. (c) The release of DPA was analyzed at the indicated

751

times. All data are presented as the means of three experiments. Differences between

752

the spore refractility (a) and spore heat resistance (b) at 60 min relative to those at 0

753

min were statistically significant (p < 0.05).

754 755 756

Figure 5. Germination initiation is a prerequisite for the action of plantaricin JLA-9 on

757

spores. The data are presented as the OD600 in LB medium at 8 h following different

758

preincubation treatments, and are the average of three independent experiments.

759

Differences between the OD600 at 8 h and that at 0 h were statistically significant (p

Purification and Characterization of Plantaricin JLA-9: A Novel

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