| Bacteriocins are ribosomally synthesized peptide with antimicrobial activity produced by Lactic acid bacteria (LAB). It has been reported that a variety of bacteriocins produced by LAB, bacteriocin has been used as a type of natural and safe food antiseptic agent in food industry. Bacillus spp. are a kind of common spoilage or pathogenic bacteria that is often present in dairy food, such as dairy products, canned food, fruit and vegetable,causing severe food spoilage or food-borne diseases. Although a plenty of effective bacteriocins have been identified in previous studies, the bacteriocin with broad spectrum anti-Bacillus spp. activity has not been reported. Currently, the possible harmful health effects from chemical additives in daily foods have been gradually realized, more effective bacteriocins which have potential use to natural and safe food preservatives will need to be explored.The aim of the present paper was to isolate the LAB strain from Suan-Tsai (traditional fermented vegetables from Jilin province, China) with a broad inhibitory spectrum against Bacillus spp. and identified the structure and properties of novel bacteriocin produced by LAB. Then the inhibitory mechanisms of Bacillus cereus spores was explored and the antibacterial mechanism of Bacillus cereus on molecular level was studied by transcriptome method. The aim of the research was to provide a solid groundwork for developing new biological preservatives. The main result in this work were as follows:1. Screening and identification of LAB producing bacteriocin with a broad inhibitory spectrum against Bacillus spp. from Suan-TsaiA total of 580 Gram-positive acid-producing strains isolated from Suan-Tsai, a traditionally fermented cabbage collected from Jilin province of China were cultured in MRS medium, of which the cell-free supernatants of 49 strains exhibited the antibacterial activity against Bacillus cereus and Bacillus pumilus by primary screening. After secondary screening, cell-free supernatants of five strains showed stronger antibacterial activity against Bacillus spp. indicators including Bacillus megaterium, Bacillus coagulans,Bacillus subtilis, Clostridium difficile ,Clostridium perfringens and so on Among them,strain JLA-9 showed the highest antibacterial activity against Bacillus spp. It was identified by physiological, biochemical tests, and the homology comparison of 16SrDNA was analyzed in the NCBI web site and the phylogenetic tree was drawn using the MEGA 5.05 software. According to the analysis result of the bacterial phylogenetic systematics, the JLA-9 was identified as Lactobacillus plantarum.2. Purification, structure identification and properties of plantaricin JLA-9The acids were constantly produced by Lactobacillus. plantarum JLA-9 during 48 h of fermentation and pH remained unchanged after stationary phase. Lactobacillus. plantarum JLA-9 started to produce bacteriocin at the early logarithmic phase and the production reached to its maximum at the late stationary phase. After cultuerd for 30 h, bacteria entered the decline phase, the amount of antimicrobial substances remained the same.Active substance was purified using butanol extraction, Sephadex LH-20 and Sephadex G-25gel filtration, and reverse-phase high-performance liquid chromatography. Active substance showed better antibacterial activity against Bacillus cereus. The molecular mass of bacteriocin was showed to be 1044 Da by MALDI-TOF-MS analyses and the entire amino acid sequence further revealed Phe-Trp-Gln-Lys-Met-Ser-Phe-Ala by Edman degradation. The bacteriocin was identified as a new bacteriocin by BLAST comparison and named plantaricin JLA-9. This bacteriocin exhibited broad-spectrum anti-Bacillus spp.activity, highly thermal stability and pH stability, but it was sensitive to common protease.3. Optimization of MRS medium producing bacteriocin by Lacotobacillus plantarum JLA-9Screening the MRS medium producing bacteriocin by Lacotobacillus plantarum JLA-9 by single factor experiment determined that the best carbon source was fructose (30 g/L); the best nitrogen source was the proportion of fishmeal tryptone: beef extract: yeast extract (15:15:7.5 g/L); the best phosphate was KH2PO4 (3 g/L). The optimization of MRS medium producing bacteriocin by Lacotobacillus plantarum JLA-9 by response surface analysis determined that the best formula were fructose 35.03 g/L; fishmeal tryptone: beef extract: yeast extract 14.5:14.5:7.3 g/L; KH2PO4 2.95 g/L, under this condition, the relative antibacterial titer was 2.3 folds than previous medium.4. Inhibitory mechanisms of Bacillus cereus spores by plantaricin JLA-9The MIC and OIC of plantaricin JLA-9 against Bacillus cereus were determined to be 16 μg/mL and 32 μg/mL respectively using the method of doubling dilution. By studying the changes of germinal refractive index, thermostabily and DPA release of Bacillus cereus,we found that the germinal refractive index, thermostabily was reduced and DPA was released from Bacillus cereus, thus, the plantaricin JLA-9 was not inhibited the initiation of spore germination. The experiment of spore germination proved that germination was the precondition of plantaricin JLA-9 acting on the spore, plantaricin JLA-9 restrained the germinated spore from growing into bacteria. Using the methods of MTT, we studied the effects of Plantaricin JLA-9 on the metabolic activity of spores growth, the results showed that the formation of formazan was not detected by treated with 1×OIC and 2xOIC (32 pg/mL and 64 μg/mL),so we could found that plantaricin JLA-9 inhibited the metabolic activity of spores growth. Flow cytometry was used to study the effect of plantaricin JLA-9 to the integrity of the membrane, the results showed that fluorescence intensity of spores was increased, treated by plantaricin JLA-9 at the concentration of 1×OIC and 2xOIC (32 pg/mL and 64 μg/mL),we could learn that plantaricin JLA-9 destroied the integrity of the membrane. Using DiOC2 as fluorescent dyes,the fluorescent microplate was applied to determined the fluorescence intensity of spores growth, treated by plantaricin JLA-9, and the membrane potential was studied. The membrane potential of spores which was treated with 1×OIC and 2xOIC (32 μg/mL and 64 μg/mL) reduced, the results indicated that plantaricin JLA-9 could disrupted the membrane potential, then inhibited spores becoming bacteria.5. The antibacterial mechanism of Bacillus cereus by plantaricin JLA-9 on molecular level by transcriptome methodPlantaricin JLA-9 could affect the metabolism of carbon and nitrogen of Bacillus cereus. After treated by plantaricin JLA-9 at the concentration of 1/2 MIC (8 μg/mL), the glycolysis pathway genes fbp, fbaA and gapB, the genes encoding TCA cycle-related enzymes sucC, sdhC and mdh, the genes involved in fatty acid β-oxidation such as mmgC and atoB, and the a-oxoglutarate pathway genes proC, rocF and glsA were significantly upregulated. These results indicate that after plantaricin JLA-9 treatment, the glycolysis pathway was promoted to reverse to gluconeogenesis, glucose decomposition was repressed, the TCA cycle of B. cereus was enhanced, the acetyl-CoA produced by the degradation of fatty acids could enter into the TCA cycle to increase the energy yield,redundant amino acids in microorganisms can be converted into common metabolic.After treated by plantaricin JLA-9, the expression of a number of important regulatory genes in the spore-forming process, including sigE, sigF, sigG and sigK, was strongly decreased, and the expression of numerous spore formation related genes in different spore-forming stages was also significantly reduced. These data therefore suggest that spore formation was inhibited by plantaricin JLA-9 treatment. The genes fabG and fabZ were upregulated, it indicated that B. cereus could improve the fluidity of its cell membrane,and repair damaged membrane, by increasing branched fatty acid biosynthesis.The cation transport in B. cereus would be changed distinctly after treatment with plantaricin JLA-9. We found that the expression of the genes kcH and cor A were significantly enhanced by plantaricin JLA-9. The remarkable upregulation of kcH indicates that the bacteria need uptake more K+ to maintain osmotic pressure. nhaC, encoding a Na+/H+ antiporter, is associated with the equilibrium of phosphate and the regulation of pH balance in Bacillus, The upregulation of nhaC suggested that since the cell membrane might be disrupted leading to the loss of K+, B. cereus must also discharge Na+ from the interior of cells to the extracellular environment to maintain the osmotic pressure.The expression of genes purL, purQ and purC, which encode various enzymes for the synthesis of inosine 5-monophosphate (IMP), was repressed by plantaricin JLA-9 treatment.Hence, the synthesis of inosine 5-monophosphate (IMP) which is the precursor of other purine nucleotides was restricted, and we can learn that the plantaricin JLA-9 likely led to inhibition of the synthesis pthway of nucleotide in B. cereus. The expression of genes which involved in the catabolic pathway for adenine and uracil were upregulated, it showed that nucleic acid precursors was degraded after plantaricin JLA-9 treatment. |
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