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 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 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 . 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 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 subtilis could induce compounds, such as bacteriocins and antifungal peptides. In production of Paracin1.7 in the culture supernatant of particular, bacteriocins from 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. In order to observe the effects tate as controls. Also, the eluted material was mixed with gel- of Paracin1.7 on the growth of other microbes, we added the loading buffer, boiled for 5 min, and 20-μL samples were supernatant to the different cultures at 10 % (v/v) and mea- applied to a polyacrylamide separating gel. Gel electrophore- sured the growth curves. Sterile modified MRS broth was sis was carried out at a constant voltage of 120 V for 60 min, used as the blank control. Simultaneously, morphological or until the tracking dye reached the end of the gel. The gel changes in the microbes following treatment with the bacteri- was stained with coomassie blue R-250 for 30 min, and then ocin were observed by electron microscopy. After co-culture, fixed in 30 % ethanol: 10 % glacial acetic acid in water for the cell-containing sediments were fixed in 2.5 % (v/v) glu- 30 min at room temperature. In our work, the SD data were taraldehyde, post-fixed with 1 % (v/v) osmium tetroxide for obtained from three independent experiments and averaged. 1 h, and dehydrated with an ethanol concentration series. Cells were coated with gold and then viewed on a scanning electron microscope. The culture supernatants of the co-cultured bacteria and Results yeast at various cell densities were filtered through a 0.25-μm bacterial filter and the cells were washed two times with Defining the threshold concentration of strain HD1-7 sterile, double-distilled water; these were then added to the L. paracasei HD1-7 culture to determine the threshold con- The threshold concentration is defined as the cell density centration. After shaking at 30 °C for 24 h, the culture broth below which L. paracasei HD1-7 could not secrete the bac- was centrifuged at 5,031×g, 4 °C for 15 min and the superna- teriocin Paracin1.7. By adding inoculum at different cell den- tant was concentrated 10-fold. The concentrated solution was sities, we found that the production of Paracin1.7 varied assayed using the methods described previously to measure during culturing (Table 1 and Fig. 1). The inoculum from the change in bacteriocin production. For controls, we de- 107 to 109 CFU/mL gave cultures with final densities of up signed both a blank control and an inactivation control in this to 109 CFU/mL, and the supernatants from these cultures experiment. The blank control, which was used to investigate contained Paracin1.7. Only the 106 CFU/mL group had no whether the increase in bacteriocin production was influenced bacteriocin production, and the cell density showed little by adding exogenous molecules during culture, contained change after culturing for 48 h. Based on these results, we strain HD1-7 at the threshold concentration without any addi- were able to define the threshold concentration for tives. The inactivation control, which contained the superna- L. paracasei HD1-7; beginning with an inoculum at tant and cells of the co-cultured microbes that were sterilized 106 CFU/mL and after culturing for 24 h, the culture super- at 121 °C as negative control, was compared to the test group natant contained no Paracin1.7. This special condition could to determine whether the additives could increase the produc- therefore be used to determine whether culture with other tion of bacteriocin. microbes could promote L. paracasei HD1-7 to produce more Paracin1.7. Purification of the inducer from the Bacillus subtilis culture supernatant Table 1 Changes in cell density in Lactobacillus paracasei HD1-7 starting with inoculum of different densities The overnight Bacillus subtilis culture was added to sterile BP Cell density of inoculuma Culture time Cell density of broth broth and cultured at 37 °C, with shaking at 160 rpm 24 h. The supernatants were then centrifuged at 5,031×g at 4 °C for 106 24 h 1.57±0.03×107 15 min and filtered through a 0.25-μm filter as before. The 36 h 2.64±0.04×107 supernatants were concentrated 10-fold; ammonium sulfate 48 h 3.02±0.01×107 was added to a final concentration of 60 % (m/v), incubated 107 24 h 1.52±0.04×108 at 4 °C for 12 h, and centrifuged at 10,976×g for 15 min. After 36 h 3.41±0.05×108 discarding the supernatant, the remaining liquid and ammoni- 48 h 3.37±0.03×109 um sulfate was removed with filter paper. Following dialysis 108 24 h 5.61±0.01×108 into 0.05 mol/L ammonium acetate, the precipitate was dis- 36 h 5.56±0.03×109 solved in sterilized deionized water and purified by anion 48 h 6.27±0.07×109 exchange chromatography (CM Sepharose Fast Flow). The 109 24 h 5.13±0.04×108 eluted fraction was added to L. paracasei HD1-7 cultures of 36 h 5.69±0.02×109 threshold concentration to observe its effect on the production 48 h 6.07±0.03×109 of Paracin1.7. In this process, we respectively used the strain HD1-7ofthresholdconcentrationwith0.05mol/L a Initial concentration unit is CFU/mL 1738 Ann Microbiol (2014) 64:1735–1743

Fig. 1 Concentration of Paracin1.7 in cultures of Lactobacillus paracasei HD1-7 starting with inoculum at four different cell densities. a 106 CFU/mL; b 107 CFU/mL; c 108 CFU/mL; d 109 CFU/mL. au/mL is units of antibacterial activity

Effect of Paracin1.7 on the co-cultured microbes morphological changes after the addition of Paracin1.7 (Fig. 3d and e); the surface of some bacterial cells appeared After adding the L. paracasei HD1-7 supernatants from cul- rough, and some cells were also swollen. Saccharomyces tures with different cell densities, the growth curves of the cerevisiae showed no morphological changes during culture tested microbes were determined in order to observe the influ- in the presence of Paracin1.7 (Fig. 3c), which confirmed ence of Paracin1.7. As the results show, the supernatants that the bacteriocin had no effect on yeast. This result obtained from L. paracasei HD1-7 cultures at different densi- was very different from the other tested microbes, pos- ties had varying effects on the growth of the tested microbes. sibly because Saccharomyces cerevisiae is a eukaryotic, We found that supernatants from low-cell-density cultures (106 rather than a prokaryotic, microorganism. Investigating and 107 CFU/mL) had a weak effect on the growth of Bacillus the mechanism(s) of action of Paracin1.7 on the growth subtilis, Escherichia coli, Lactabacillus plantarum,and of the tested microbes will be the subject of future Lactococcus lactis. From the growth curves (Fig. 2a and b), research. we found that the logarithmic phase and biomass of Bacillus subtilis and Escherichia coli, which respectively represent Effect of the tested microbes on production of Paracin1.7 Gram-positive and Gram-negative bacterial species, were both slightly affected. Two species of lactic acid bacteria were also In this study, we also examined the supernatants and cells of affected by the L. paracasei HD1-7 culture supernatants the co-cultured bacteria and yeast at various cell densities to (Fig. 2d and e). When the starting cell density of determine whether there were some factors that could induce L. paracasei HD1-7 was high (108 and 109 CFU/mL), the L. paracasei HD1-7 of threshold concentration to secrete added supernatants had a large effect on the growth of the four more bacteriocin. The results of co-culture experiments tested species of bacteria. Under these conditions, both showed that only the supernatants and cells of Bacillus subtilis Bacillus subtilis, Escherichia coli showed very little growth could induce L. paracasei strain HD1-7 to produce bacterio- in culture, and the two species of lactic acid bacteria cin (data for other strains not shown). The production of had prolonged logarithmic phases and reduced biomass. Paracin1.7 was also observed after adding the supernatants During this research, we also found that the growth of and cells of Bacillus subtilis at various concentrations. The the yeast Saccharomyces cerevisiae, one of the five results showed that when the cells were at logarithmic phase tested species of microbes, was not influenced by the (107 and 108 CFU/mL), the co-culture broth gave higher addition of culture supernatants containing Paracin1.7 bacteriocin production than did the blank control, and that (Fig. 2c). the supernatant from cells at a density of 106 CFU/mL induced the highest level of Paracin1.7 production (Fig. 4). These Scanning electron microscopy results strongly suggest that the supernatant of Bacillus subtilis contains a factor that can induce L. paracasei HD1-7 We used scanning electron microscopy to examine the effects to produce more bacteriocin. of Paracin1.7 (obtained from the supernatant of the strain The results of the co-culture experiments showed that both HD1-7 at 109 CFU/mL) on the morphology of the tested the culture supernatants and the cells of Bacillus subtilis could microbes. Cells of Escherichia coli and Bacillus subtilis induce L. paracasei HD1-7 to produce more bacteriocin. showed obvious morphological changes (Fig. 3a and b). Because this could be due to the presence of molecules that Compared to the blank control groups, the bacteria were function as signals for quorum sensing in Gram-positive bac- swollen into rod shapes, and pore formation was observed at teria, we purified the Bacillus subtilis culture supernatants. the surface of the bacteria; some bacterial cells had ruptured. The Bacillus subtilis culture supernatant was preliminarily We also found cell debris during our observations (data not purified by adding ammonium sulfate, and the resulting pre- shown). The two species of lactic acid bacteria (Lactabacillus cipitate was dissolved in sterile deionized water and purified plantarum and Lactococcus lactis) had only slight by anion exchange chromatography (CM Sepharose Fast Ann Microbiol (2014) 64:1735–1743 1739

Fig. 2 The effects of adding L. paracasei HD1-7 supernatants from the Sterile modified MRS broth was used as a blank control: 106 CFU/ cultures of differing cell densities on the growth of microbes. The symbols mL; 107 CFU/mL; 108 CFU/mL; 109 CFU/mL; indicate the starting cell density of the L. paracasei HD1-7 cultures. blank control

Flow) using a UV detector. The eluted peak with the highest Discussion absorbance at a wavelength of 280 nm was collected (Fig. 5). After adding the eluted column fraction, the bacteriocin was Lactobacillus paracasei HD1-7 was isolated from Chinese also detected in the L. paracasei HD1-7 culture (Fig. 6). sauerkraut fermentation broth. It produces a type of bacterio- Denaturing polyacrylamide gel electrophoresis showed cin called Paracin1.7, which is active against many microbes that the eluted fraction contains several polypeptides such as Staphylococcus aureus, Escherichia coli, Bacillus between ∼15and45kDa(Fig.7). Therefore, the cereus, Bacillus subtilis, Bacillus megaterium,andProteus Bacillus subtilis culture supernatant must contain one vulgaris Houser (Ge et al. 2009). Similar to the bacteriocin or more factors that can promote the production of nisin produced by Lactococcus lactis, Paracin1.7 is an antibi- bacteriocin in L. paracasei HD1-7. otic peptide that is effective against a broad spectrum of 1740 Ann Microbiol (2014) 64:1735–1743 Ann Microbiol (2014) 64:1735–1743 1741

R Fig. 3 Morphological changes in the co-cultured microbes. Scanning electron microscopy of five co-cultured microbes treated with Paracin1.7 that came from L. paracasei HD1-7 culture supernatant at a cell density of 109 CFU/mL. The group treated with Paracin1.7 (left column) is indicated with a (+), and the control group (right column) is indicated with a (−). a Escherichia coli; b Bacillus subtilis; c Saccharomyces cerevisiae; d Lactabacillus plantarum; e Lactococcus lactis

bacteria, and is widely used as a safe food antimicrobial agent (Delves-Broughton et al. 1996; Kaur et al. 2011). In our previous research, we found that the production of Paracin1.7 was related to the cell density of strain HD1-7 (Ge et al. 2011). In this study, we demonstrated that this strain can produce higher levels of bacteriocin in co-cultures with other bacterial species. Among the strains that we included in the test, Bacillus subtilis had a higher ability to induce strain Fig. 5 Purification of the Bacillus subtilis culture supernatant precipitate HD 1.7 to produce more Paracin1.7. by CM Sepharose Fast Flow column chromatography. The eluted peaks Some studies have examined the effect of certain factors on were detected by absorption at a wavelength of 280 nm the production of bacteriocins in culture such as pH, temper- ature, nutrition, and growth conditions (Mandal et al. 2008; as quorum sensing, or cell-to-cell communication in bacteria, Altuntas et al. 2010). Our data shows that the concentration of which is achieved through the production and subsequent bacteriocin in the culture supernatant is influenced by the cell response to the accumulation of some specific extra-cellular density of L. paracasei HD1-7. When the primary cell density molecules (Carbonell et al. 2002). of the inoculum is lower than 107 CFU/mL, Paracin1.7 will Hasslöf et al. (2010) have shown that some not be detected in the culture broth. However, when the lactobacilli strains, which are readily found in daily life, can primary inoculum cell density is >107 CFU/mL, the bacterio- inhibit the growth of certain pathogenic bacteria such as cin will be produced under the same conditions. Hence, high mutans streptococci and Candida albicans. In these studies, cell densities favor a high level of bacteriocin production. the effects on the tested bacteria vary greatly. Some Indeed, an increased nutrient supply in batch fermentation lactobacilli can inhibit the tested bacteria at low and high results in an increased biomass and volumetric bacteriocin concentrations, and others only at high concentrations. production. However, some specific bacteriocin production Furthermore, the effects on the tested bacterium have certain strains have been found. In Lactobacillus amylovorus DCE high levels of specificity. In our research, we also observe 471, a rapid decline in bacteriocin activity is observed after the these types of phenomena. The effects of L. paracasei strain growth-associated bacteriocin production phase (Callewaert HD1-7 on the growth of Bacillus subtilis, Escherichia coli, and De Vuyst 2000). Similarly, enterocin production by Lactabacillus plantarum,andLactococcus lactis all begin at Enterococcus faecium RZS C5 seems to be limited to the very low cell densities. The range of organisms inhibited by each early growth phase (Leroy and De Vuyst 2002). Thus, the bacteriocin varies greatly. In general, the bacteriocins pro- relationship with cell concentration suggests that bacteriocin duced by Gram-positive bacteria are directed primarily against production may be controlled by a regulatory mechanism such other Gram-positive species and do not have a bactericidal

Fig. 4 Changes in the level of Paracin1.7 produced by L. paracasei strain HD1-7 with the addition of cells and supernatants of Bacillus subtilis. The antibacterial activity of Paracin1.7 was detected after adding cells and supernatants from Bacillus subtilis cultures of different cell densities. — Fig. 6 The effects of CM Sepharose Fast Flow purification of Bacillus addition of Bacillus subtilis cells; □—addition of Bacillus subtilis culture subtilis culture supernatant on the production of Paracin1.7 by supernatants. (1) Blank control; (2)106 CFU/mL; (3)107 CFU/mL; (4) L. paracasei strain HD1-7. (1) Blank control; (2) addition of 0.05 mol/L 108 CFU/mL; (5)109 CFU/mL; (6) the supernatants and cells of the co- ammonium acetate as the control; (3) addition of CM Sepharose Fast cultured microbes which were sterilized at 121 °C as negative control Flow column fraction 1742 Ann Microbiol (2014) 64:1735–1743

Through our co-culture experiments, we found that bacte- riocin production by L. paracasei HD1-7 can be affected by growth in the presence of other species of bacteria. Furthermore, one of the most common results of co- culturing bacteria is a shift in the enzymatic activity profiles (Di Cagno et al. 2007). In a previous study, Ulrich and Hughes (2001) determined that the inducing factor for lactacin B production by Lactobacillus acidophilus N2 in the presence of Lactobacillus delbrueckii ATCC 4797 could be a cell wall- associated protein of approximately 58 kDa. This observation has been also reported by Rojo-Bezares et al. (2007), who found that the bacteriocin of Lactobacillus plantarum strain J23, which was recovered from a grape must sample in Spain, can be detected in co-cultures during the exponential phase. Interestingly, Ruiz-Barba et al. (2010)alsoshowedthat Lactobacillus plantarum NC8 is greatly enhanced in its growth and survival when co-inoculated with two specific bacteriocin-inducing strains, Enterococcus faecium 6T1a-20 and Pediococcus pentosaceus FBB63, as compared to singly- inoculated . From our data, production of the bacteriocin Paracin1.7 is only induced by Bacillus subtilis of the group of microbes tested in this study. The Bacillus subtilis culture supernatant could contain some special signal mole- cules that can initiate quorum sensing in Gram-positive Fig. 7 SDS-PAGE of the purified inducer fraction. M—protein size marker (10–200 kDa) to estimate the molecular weight of the signal bacteria. molecules; Lane 1—purified fraction from the Bacillus subtilis culture supernatant following precipitation with 60 % (w/v) ammonium sulfate and CM Sepharose Fast Flow chromatography Conclusions

Lactobacillus paracasei HD1-7, which produces a bacteriocin effect on Gram-negative species (McAuliffe et al. 2001). called Paracin1.7, can effect the growth and morphology of However, in some cases, activity against Gram-negatives other microbes in mixed cultures. Through co-culture exper- can be observed on disruption of the outer membrane, as iments, we found that a strain of Bacillus subtilis, a Gram- reported for nisin. Nisin has a broad antibacterial spectrum positive bacterial species, can induce the production of that includes strains of Lactococcus, Streptococcus, Paracin1.7 by L. paracasei HD1-7. Also, we performed a Staphylococcus, Listeria,andMycobacterium, as well as preliminary fractionation of the culture supernatant of some pathogenic bacteria found in the food industry Bacillus subtilis, and purified a protein-containing fraction (Lubelski et al. 2008;Ruizetal.2010;Tongetal.2010). by ion-exchange chromatography. We hypothesize that some Wang et al. (2010) also found that five Lactobacillus strains specific signal molecules, possibly one of the proteins purified isolated from the feces of breast-fed infants and Taiwanese from the Bacillus subtilis culture, may initiate quorum sensing pickled cabbage elicited antibacterial activity against both in Gram-positive bacteria. Future research will focus on elu- Gram-positive (Bacillus cereus, Listeria monocytogenes,and cidating the mechanism of quorum sensing and bacteriocin Staphylococcus aureus) and Gram-negative (Escherichia coli production in L. paracasei HD1-7. and ) bacterial pathogens. This bactericid- al effect was observed in this study by scanning electron microscopy. The bacteriocin produced by L. paracasei HD1- 7 has an effect on the growth of both Gram-positive and Funding This work was supported by grants from the National Natural Science Foundation of China (31070446), the National Natural Science Gram-negative bacteria; however, there appeared to be no Foundation of China (31270534), the National Natural Science effect on a eukaryotic microorganism (Saccharomyces Foundation of China (31270143), the National Science Foundation for cerevisiae). Similar to other bacterioicins, Paracin1.7 may Distinguished Young Scholars of China (31300355), the High-level change the shape of the bacterium and disrupt the outer Talents (Innovation Team) Projects of Heilongjiang University (Hdtd2010-17) and the Innovation Team in Science and Technology of membrane (Wal et al. 1995; Yoneyama et al. 2009, 2011; Heilongjiang Province (the Fermentation Technology of Agricultural Stevens et al. 1991). Microbiology, 2012td009). Ann Microbiol (2014) 64:1735–1743 1743

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