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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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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] 16 17
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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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
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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
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Edman degradation. This bacteriocin exhibited broad-spectrum antibacterial activity
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against Gram-positive and Gram-negative bacteria, especially Bacillus spp., high
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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
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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
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activity produced by numerous bacteria. Bacteriocins inhibit the growth of other
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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
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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.
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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.
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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.
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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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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.
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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.
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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
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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.
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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)
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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
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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
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excitation and emission at 488 nm and 525 nm, respectively45.
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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
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cell-free supernatants of 49 strains exhibited antibacterial activity against B. cereus
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and C. sporogenes in primary screening (data not shown). After secondary screening,
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cell-free supernatants of five strains showed strong antibacterial activity against
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indicators including B. pumilus, B. megaterium, B. coagulans, B. subtilis, C. difficile
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and C. perfringens. Among these, strain JLA-9, which had the highest antibacterial
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activity against Bacillus spp. (data not shown), was selected for the following studies.
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Identification of bacteriocin-producing strain JLA-9. Strain JLA-9 was a
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Gram-positive, nonflagellated, catalase negative and rod-shaped bacillus without
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spore formation that did not produce gas. It could grow at 15°C but not at 45°C, and
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in 3% but not in 6% or 9% NaCl. Moreover, it was able to produce acid from glucose.
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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,
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mannitol, sorbitol, melezitose, melibiose, raffinose, saligenin, sucrose, trehalose,
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xylose, inulin, and maltose, but not rhamnose. Regarding 16S rDNA sequence
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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),
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which showed 100% similarity to L. plantarum WCFS1 (NR075041.1). Hence, on the
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basis of biochemical and morphological characteristics, we concluded that the strain
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JLA-9 belongs to L. plantarum.
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Fermented foods and vegetables are good sources for isolating L. plantarum.
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Many bacteriocin-producing L. plantarum stains have been isolated, such as L.
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plantarum C1923, L. plantarum LPCO1024, L. plantarum ZJ00815, L. plantarum 1634,
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L. plantarum A-18 and L. plantarum ZJ513. Similar to many previous studies of LAB,
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the strain L. plantarum JLA-9 is capable of producing bacteriocin that, in this case,
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exhibits high antibacterial activity against Bacillus spp. as found in foods.
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Lactobacillus plantarum are often used for food fermentation, for instance, in the
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production of yogurts, pickles and fermented meats; L. plantarum use not only
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improves the flavors, nitrogen content and food texture, but also extends the shelf-life
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of the foods. In the present study, strain L. plantarum JLA-9 was capable of secreting
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high concentrations of lactic acid and bacteriocin. Therefore, it has potential for
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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.
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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
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absorbance wavelength at 259 nm. The active fractions collected by gel
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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
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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
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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.
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plantarum JLA-9 did not result in the collection of more antibacterial substances
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compared with n-butyl alcohol extraction (data not shown). In recent years, the
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pollution of food by Bacillus spp. has increasingly been reported26-28. For instance,
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partly dairy products often suffer from Bacillus spp. contamination because of their
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low temperature sterilization. Spores are not killed completely, leading to the spoilage
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of the food products.
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Molecular mass determination and structure identification of plantaricin JLA-9.
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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
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bacteriocins are >2.0 kDa, for example, plantaricin C19 (3.8 kDa) from L. plantarum
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C1929, plantaricin 423 (3.5 kDa) from L. plantarum 42323, plantaricin Y from L.
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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
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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.
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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.
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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
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and bactericidal action of plantaricin UG1. Int. J. Food Microbiol. 1996, 30, 189-215.
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(36) Toit, D. Plantaricin D, a bacteriocin produced by Lactobacillus plantarum BFE
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905 from ready‐to‐eat salad. Lett. Appli. Microbiol. 1998, 26, 231-235.
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(37) Rojo-Bezares, B.; Sáenz, Y.; Navarro, L.; Zarazaga, M.; Ruiz-Larrea, F.; Torres,
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C. Coculture-inducible bacteriocin activity of Lactobacillus plantarum strain J23
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isolated from grape . Food Microbiol. 2007, 24, 482-491.
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(38) Hernández, D.; Cardell, E.; Zarate, V. Antimicrobial activity of lactic acid
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bacteria isolated from Tenerife cheese: initial characterization of plantaricin TF711, a
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bacteriocin ‐ like substance produced by Lactobacillus plantarum TF711. J.
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Appl.Microbiol. 2005, 99, 77-84.
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(39) Delves-Broughton, J.; Blackburn, P.; Evans, R.; Hugenholtz, J. Applications of
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the bacteriocin, nisin. Anton Leeuw.1996, 69, 193-202.
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(40) Rajkovic, A.; Uyttendaele, M.; Courtens, T.; Debevere, J. Antimicrobial effect of
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nisin and carvacrol and competition between Bacillus cereus and Bacillus circulans in
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vacuum-packed potato puree. Food Microbiol. 2005, 22, 189-197.
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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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(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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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.
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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