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Bacillus Subtilis Enhances Production of Paracin1.7, a Bacteriocin Produced by Lactobacillus Paracasei HD1-7, Isolated from Chinese Fermented Cabbage

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Bacillus Subtilis Enhances Production of Paracin1.7, a Bacteriocin Produced by Lactobacillus Paracasei HD1-7, Isolated from Chinese Fermented Cabbage Ann Microbiol (2014) 64:1735–1743 DOI 10.1007/s13213-014-0817-z ORIGINAL ARTICLE Bacillus subtilis enhances production of Paracin1.7, a bacteriocin produced by Lactobacillus paracasei HD1-7, isolated from Chinese fermented cabbage Jingping Ge & Baozhu Fang & Yang Wang & Gang Song & Wenxiang Ping Received: 11 September 2013 /Accepted: 14 January 2014 /Published online: 20 February 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014 Abstract Lactobacillus paracasei HD1-7 (CCTCCM Keywords Lactobacillus paracasei . Bacteriocin . 205015), isolated from Chinese sauerkraut fermentation broth, Co-culture experiments . Quorum sensing was found to contain the bacteriocin Paracin1.7, which pos- sesses broad-spectrum antibacterial activity. We examined the relationship between bacteriocin production by L. paracasei Introduction strain HD1-7 and the cell density of co-cultured bacteria. The cells and supernatants from cultures of different densities of Many bacteria are capable of producing bacteriocins, which five microbes, four bacteria, and a yeast were co-cultured with are ribosomally-synthesized peptides or proteins that have L. paracasei HD1-7, and the change in Paracin1.7 levels in the antimicrobial activity against closely related bacterial strains culture broth was measured by the agar-well diffusion meth- or broad-spectrum inhibitory activity against other microbes od. When the initial cell density of the L. paracasei HD1-7 (Tagg et al. 1976;Boman1995; Hancock and Lehrer 1998). inoculum was 106 CFU/mL, the strain did not produce bacte- The use of lactic acid bacteria (LAB) in fermented foods has a riocin, and this was termed the threshold concentration. The long history due to the benefits of their nutritional, organolep- five species of microbes tested were grown with added culture tic, and antimicrobial activities (Klaenhammer 1993). Lactic supernatant of L. paracasei HD1-7, and the growth of the acid bacteria have a range of antimicrobial activities that are tested bacteria was inhibited to varying degrees. We found that mainly due to the production of organic acids, but also other the supernatants and cells of Bacillus subtilis could induce compounds, such as bacteriocins and antifungal peptides. In production of Paracin1.7 in the culture supernatant of particular, bacteriocins from lactic acid bacteria have attracted L. paracasei HD1-7. Lactobacillus paracasei HD1-7 secretes great interest because of their potential uses as biopreservatives a bacteriocin called Paracin1.7 into the culture supernatant that could replace traditional chemical preservatives (Gálvez that inhibits the growth of the four species of bacteria tested, et al. 2007; De Vuyst and Leroy 2007). Bacteriocin production but not Saccharomyces cerevisiae. We found that Bacillus in many species of LAB is controlled by specific peptides subtilis could increase the concentration of Paracin1.7 in the called autoinducers (AIPs) via a three-component regulatory culture supernatant at a certain threshold cell density. We system that involves, in addition to the AIP, a histidine protein ultimately showed that this inducer can be purified from the kinase and a response regulator as part of a quorum sensing Bacillus subtilis culture supernatant, and that it may be a mechanism (Kleerebezem 2004; Henke and Bassler 2004; protein. This study demonstrated that there was a certain Williams et al. 2007; Nakayama et al. 2003). ecological relationship between the L. paracasei HD1-7 and Lactobacillus paracasei is one kind of LAB that has been other microbes. isolated not only from fermented foods such as Xueo, which is a traditional fermented yak milk in the western Sichuan Plateau of China (Ao et al. 2012), as well as cheese (Kmeť and Drugdová 2012) and cereal-based drinks, but also from : : : : * J. Ge B. Fang Y. Wa ng G. Song W. Ping ( ) healthy humans and newborn infant feces (Liang et al. 2010; Key Laboratory of Microbiology, College of Life Science, Heilongjiang University, Harbin 150080, People’sRepublicofChina Chiang and Pan 2012), and which possesses antibacterial and e-mail: [email protected] antifungal activities against oral pathogens. Lactobacillus 1736 Ann Microbiol (2014) 64:1735–1743 paracasei HD1-7 has been isolated from Chinese fermented 16 h until the cell density reached 107 CFU/mL. This inocu- cabbage, which is one of the traditional fermented vegetables lum culture was then used to inoculate fresh, sterile modified eaten in Northeastern China. Lactobacillus paracasei HD1-7 MRS broth at 5 % (v/v) and incubated as before for 24 h. produces a bacteriocin called Paracin1.7 which, like many Escherichia coli, Lactabacillus plantarum,andLactococcus other bacteriocins produced by LAB, has antimicrobial activ- lactis were grown in sterile LB broth and Bacillus subtilis was ity. The level of production of Paracin1.7 is also related to its grown in sterile BP broth, incubated at 37 °C with shaking own concentration in the culture broth. (160 rpm) for 18 h, and Saccharomyces cerevisiae was grown The aim of this work was to determine whether we could in sterile YPD broth and incubated at 30 °C with shaking enhance the production of Paracin1.7 through co-culture with (160 rpm) for 20 h. These five microbes were used for the co- other species of microbes, therefore suggesting a role for the culture experiments. Cells were harvested by centrifuging the production of the bacteriocin itself. In this study, we inoculat- L. paracasei HD1-7 culture at 5,031×g for 15 min at 4 °C. ed cultures of L. paracasei HD1-7 in combination with several Samples were centrifuged three times prior to the bacteriocin strains of bacteria and a yeast that had been isolated from the assay to obtain cell-free samples, which were then used in the same habitat niche. As we reasoned that the cell density of agar-well diffusion test as follows: the plates were loaded with L. paracasei HD1-7 could interfere with the regulatory mech- 30 mL of soft BP containing the indicator bacteria (Bacillus anism for bacteriocin production, we also investigated ways to subtilis) at 5 % (v/v); the cell density was adjusted to 107 CFU/ define a cell threshold concentration below which strain HD1-7 mL and Oxford plates were placed in the upper agar layer would not produce bacteriocin. above the water agar layer. Wells were filled with the cell-free supernatants and the plates were incubated at 37 °C for 15 h. Materials and methods Cell threshold concentration of L. paracasei strain HD1-7 Bacterial strains and culture media Overnight bacterial cultures of L. paracasei strain HD1-7 that were grown in sterile modified MRS broth at 30 °C with Lactobacillus paracasei HD1-7 was isolated from traditional shaking were centrifuged (two times at 5,031×g for 15 min) Chinese fermented cabbage and tested for antimicrobial activ- to recover the cells. The harvested cells were washed with ity. The following bacterial strains maintained in our own sterile 0.9 % saline and the cell concentrations were adjusted laboratory were used to assess the change in antimicrobial to 106 CFU/mL, 107 CFU/mL, 108 CFU/mL, and 109 CFU/ activity of strain HD1-7 after the co-culture experiments: mL. These inoculum cultures were then added to fresh, sterile Bacillus subtilis (ATCC 11774), Escherichia coli (ATCC modified 1/5 MRS broth at 5 % (v/v) and incubated at 30 °C, 25922), Saccharomyces cerevisiae (ATCC 2601), 180 rpm for 48 h. The changes in cell density and bacteriocin Lactabacillus plantarum (ATCC 8014), and Lactococcus concentration were detected by spectrophotometry and the lactis (ATCC 11454). agar-well diffusion test, respectively, in order to determine The following media were used to culture LAB: (1) modi- the minimum inhibitory threshold concentration, and the min- fied MRS broth (soya peptone, 10 g/L; beef extract, 10 g/L; imum cell density in the supernatant that would not inhibit yeast extract, 5 g/L; glucose, 20 g/L; K2HPO4,2g/L;Na2SO3, other microbes. 0.1 g/L; sodium acetate, 5 g/L; MgSO4 7H2O, 0.2 g/L; MnSO4, 0.05 g/L; ammonium citrate, 0.4 g/L; Tween 80, 1 mL; pH 5.5); Co-culture experiments (2) 1/5MRS medium (soya peptone, 2 g/L; beef extract, 2 g/L; yeast extract, 1 g/L; glucose, 4 g/L; K2HPO4,0.4g/L;Na2SO3, The microbes used for the co-culture tests, Bacillus subtilis, . 0.02 g/L; sodium acetate, 1 g/L; MgSO4 7H2O, 0.04 g/L; Escherichia coli, Saccharomyces cerevisiae, Lactabacillus MnSO4, 0.01 g/L; ammonium citrate, 0.4 g/L; Tween 80, plantarum,andLactococcus lactis, were inoculated into the 0.2 mL; pH 5.5) was used to obtain a cell threshold; (3) LB respective liquid broths from slants and grown to the logarith- (peptone, 10 g/L; yeast extract, 5 g/L; NaCl, 10 g/L; pH 7.0); mic phase. These cultures were then inoculated into fresh (4) BP (beef extract, 3 g/L, peptone, 10 g/L, NaCl, 5 g/L; pH broth for continuous culture. Gradient dilution plate culture 7.0–7.2); (5) YPD (yeast extract, 10 g/L, peptone, 20 g/L, counts and spectrophotometry were both used as detection glucose, 10 g/L); (6) soft BP (beef extract, 3 g/L, peptone, methods. Cell density and OD600 were both investigated, and 10 g/L, NaCl, 5 g/L, agarose, 0.75 g/L; pH 7.0–7.2). the changes in cell density were measured by OD600 for the co-culture experiments. Cell harvest and bacteriocin detection An overnight culture of L. paracasei HD1-7 was inoculat- ed into fresh, sterile modified MRS broth at 1 % (v/v) and Lactobacillus paracasei HD1-7 was grown in sterile modified incubated at 30 °C, with shaking at 180 rpm for 24 h. The MRS broth and incubated at 30 °C with shaking (180 rpm) for culture supernatant was filtered through a 0.25-μmbacterial Ann Microbiol (2014) 64:1735–1743 1737 filter after centrifugation (5,031×g for 15 min) to give a ammonium acetate and without 0.05 mol/L ammonium ace- solution containing Paracin1.7.
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