Characterization of a Bacteriocin-Like Substance Produced from a Novel Isolated Strain of Bacillus subtilis SLYY-3

LI Junfeng, LI Hongfang, ZHANG Yuanyuan, DUAN Xiaohui, and LIU Jie

1) College of Chemical Engineering, Qingdao University of Science & Technology, Qingdao 266042, P. R. China

2) Yantai Entry-Exit Inspection and Quarantine Bureau, Yantai 264000, P. R. China

Characterization of a Bacteriocin-Like Substance Produced from a Novel Isolated Strain of Bacillus subtilis SLYY-3

LI Junfeng1),*, LI Hongfang1), ZHANG Yuanyuan1), DUAN Xiaohui2), and LIU Jie1)

1) College of Chemical Engineering, Qingdao University of Science & Technology, Qingdao 266042, P. R. China

2) Yantai Entry-Exit Inspection and Quarantine Bureau, Yantai 264000, P. R. China

In the present research, the strain SLYY-3 was isolated from sediments of Jiaozhou Bay, Qingdao, China. The strain SLYY-3, which produced a bacteriocin-like substance (BLS), was characterized to be a strain of Bacillus subtillis by biochemical profiling and 16S rDNA sequence analysis. It is the first time to report that Bacillus subtilis from Jiaozhou Bay sediments could produce a BLS. The BLS of B. subtillis SLYY-3 exhibited strong inhibitory activity against gram-positive bacteria (including Staphylococcus aureus and B. subtillis) and some fungi (including Penicillium glaucum, Aspergillus niger and Aspergillus flavus). The antimicrobial activity was detected from culture in the exponential growth phase and reached its maximum when culture entered into stationary growth phase. It was thermo-tolerant even when being kept at 100℃ for 60 min without losing any activity and stable over a wide pH range from 1.0 to 12.0 while being inactivated by proteolytic enzyme and trypsin, indicating the proteinaceous nature of the BLS. The BLS was purified by precipitation with hydrochloric acid (HCl) and gel filteration (Sephadex G-100). SDS-PAGE analysis of the extracellular peptides of SLYY-3 revealed a bacteriocin-like protein with a molecular mass of 66 kDa. Altogether, these characteristics indicate the potential of the BLS for food industry as a protection against pathogenic and spoilage microorganisms.

bacteriocin-like substance; Bacillus subtillis SLYY-3; antimicrobial activity; food protection; purification

1 Introduction

Antimicrobial substances are widespread among bacteria. Bacteriocins and bacteriocin-like substances (BLS) are antimicrobial peptides produced by a number of different bacteria that are usually effective against closely related species (Baugher and Klaenhammer, 2011). Bacteriocins have received increasing attention due to their potential use as natural preservatives in food industry, as probiotics in the human health, and as therapeutic agents against pathogenic microorganisms (Riley and Wertz, 2002). Although most research efforts were mainly focused on bacteriocins produced by lactic acid bacteria, bacteriocins from other various species have also been characterized (Turgis et al., 2012; McAulife et al., 2001).

Bacillus is a genus that has been investigated for antimicrobial activity since Bacillus species produce a large number of peptide antibiotics representing several different basic chemical structures (von D?hren, 1995). The production of bacteriocins or bacteriocin-like substances has been already described for B. thuringiensis, B. subtilis, B. amyloliquefaciens, B. licheniformis, B. megaterium and B. cereus (Balciunas, 2013; Gray et al., 2006; Liu et al., 2012; He et al., 2006; Senbagam et al., 2013).

The objective of this study is to evaluate the potential antimicrobial activity of a bacteriocin-like substance produced by a Bacillus subtilis SLYY-3 isolated from sediments of Jiaozhou Bay, Qingdao, China. The antimicrobial spectrum and some properties of this bacteriocin-like substance are investigated.

2 Materials and Methods

2.1 Isolation of Microorganisms

The samples (5 g moist weight) collected from Jiaozhou Bay sediments were mixed with sterile water (1:1 w/v), homogenized for 5 min, and 1 mL of this suspension was inoculated into 50 mL of nutrient medium. After microbial growth was observed by turbidity, aliquots were inoculated onto nutrient agar plates incubated at 28℃, and single colonies were isolated and screened for antimicrobial activity.

2.2 Indicator Bacterial Strains

The indicator strains Enterobacter aerogenes, Proteus vulgaris and Pseudomonas aeruginosa were kindly offered by UNESCO Chinese Center of Marine Biotechnology. Bacillus subtilis, Escherichia coli, Staphylococcus aureus ATCC 6538, Penicillium glaucum, Aspergil-lus niger, and Aspergillus flavus were the collections of our laboratory.

2.3 Taxonomical Studies

Strain SLYY-3 was identified based on 16S rDNA sequence analyses and the characterization of bacteria recorded in Bergey’s Manual of Determinative Bacteriology. Genomic DNA of strain was isolated as described by Edwards et al. (1989), 16S rDNA gene was amplified via PCR and then amplicon was sequenced. The primers used for amplification were: F (5’-AGAGTTTGATCCTG GCTCAG-3’) and R (5’-ACGGCTACCTTGTTACG ACT-3’). Alignment of different 16S rDNA nucleotide sequences was carried out by CLUSTAL W program (Thompson et al., 1994). Phylogenetic trees for 16S rRNA genes were constructed by the NJ method (Saitou and Nei, 1987) using the MEGA4.0 program (Tamura et al., 2007).

2.4 Activity Assay

To determine the activity spectrum of BLS, strain SLYY-3 was cultured in LB broth for 24 h at 28℃ in a rotary shaker at 150 r min-1. The cells were harvested (10000 r min-1, 15 min, 4℃), and the cell-free supernatant (CFS) was obtained by filtering through a Milipore filtre with 0.22 μm pore size. Pre-poured agar media plates were spread with 107CFU of the respective indicator microorganism and allowed to dry. The sterile Oxford-cups (8 mm×10 mm) were placed on the plates. 200 μL of CFS was added to each cup and incubated at optimal temperature of the test organism for 24 h and the diameter of the inhibition zone was determined (Li et al., 2008).

2.5 Characterization of Bacteriocin-Like Substances

To determine the thermal stability, the BLS samples were heated at 100℃ for 0 (control), 10, 20, 30, 40, 50 and 60 min, cooled and assayed for activity. The effect of trypsin on activity of BLS was also tested by the following method: 0.2 mL phosphate buffer as Control I (C1); 0.1 mL CFS containing BLS + 0.1 mL phosphate buffer as Control II (C2); 1 mg of enzyme-Trypsin (Sigma Chemicals) was dissolved in 1 mL of 0.1 molL-1phosphate buffer, pH 7.0 and then added to CFS of B. subtilis in the ratio of 1:1 as Enzyme reaction (ER). The activities of enzyme reaction and control I and II were assayed on the indicator plates. To test the sensitivity of the BLS to pH, each of aliquots was adjusted to 1.0-12.0 with 0.1 mol L-1HCl or 0.1 mol L-1NaOH and incubated for 30 min at 37℃. Then each sample was adjusted back to pH 7.0 and assayed for the residual activity. After each treatment, the samples were tested for antibacterial activity against S. aureus ATCC 6538 using diffusion method.

2.6 Purification of BLS and Molecular Weight Determination by SDS-PAGE

Precipitation of the BLS was induced by acidification using 6 mol L-1hydrochloric acid (HCl). The BLS was extracted from the pellet with 100 mL methanol. After evaporation, the light brown viscous extract was resuspended in 20 mL of 10 mol L-1sodium phosphate. This extract was loaded on a Sephadex G-100 column (2.6 cm × 80 cm, Pharmacia, Uppsala Sweden), equilibrated with 10 mmol L-1sodium phosphate, pH 7.2 and eluted with the same phosphate buffer. The elution with bactericidal activity was used to determine the molecular size of BLS by SDS-PAGE according to the method described by Laemmli (1970). The apparent molecular masses of proteins were estimated by co-electrophoresis of marker proteins (Biorad, Hercules, CA, USA) with masses ranging from 14.4 to 116 kDa. One half of the gel was stained with Coomassie Blue R250, and the position of the active bacteriocin was determined on the other unstained gel. S. aureus ATCC 6538 (107CFU mL-1) suspended in 1% nutrient agar was used to overlay the gel and cleared zone due to inhibition was examined after overnight incubation at 37℃.

3 Results and Discussion

3.1 Isolation and Identification of BLS-Producing Strain

In this study strain SLYY-3 was isolated from sediments which produced the highest inhibition zones using B. subtilis and S. aureus as indicator strains. The microorganism is Gram-positive, aerobic, endospore forming and strongly catalase positive. The morphological and physiological characteristics (data not shown) and the phylogenetic analysis of strain SLYY-3 confirmed that the strain belonged to B. subtillis. The 16S rDNA sequence of SLYY-3 showed a high similarity (99%) to B. subtillis. The cluster formed by SLYY-3 and B. subtillis was supported by high bootstrap values (Fig.1).

Fig.1 Phylogenetic tree of the SLYY-3 and related type species based on the 16S rDNA domain sequences.

3.2 Bacteriocin-Like Substances Production

SLYY-3 was grown in flasks with 50 mL LB medium at 28℃ on a rotary shaker. The optical density (OD) of the culture was determined at 600 nm at an interval of 2 h with a Hitachi U-1100 spectrophotometer (Hitachi, Tokyo, Japan). Cells reached the stationary phase after 12 h ofcultivation (Fig.2). Kinetics of BLS production showed that its synthesis and ? or secretion started at the early exponential phase, and reached to its maximum antibacterial activity at the stationary phase. Afterward, the inhibitory activity slowly decreased (Fig.2). Similar results have been reported with other bacteriocins (Samy et al., 2010; Cladera-Olivera et al., 2004), the antibacteria activity was detected at the middle exponential growth phase and the maximum activity was obtained at the early stage of the stationary growth phase.

Fig.2 Growth and BLS of SLYY-3: (◆) OD600 and (■) inhibitory zone diameter.

The cell-free supernatant of SLYY-3 exhibited a broad spectrum of antagonistic activities against all indicator strains of Gram-positive bacteria and some fungal pathogens, but not against the strains of Gram-negative bacteria (Table 1). These findings are consistent with bacteriocins or BLS by other Bacillus species reported. Although some bacteriocin are active against a narrow spectrum of bacteria (Lee et al., 2001), several strains produce bacteriocins with a broad range of activity against important pathogens (Khochamit et al., 2013, Cherif et al., 2001). The BLS produced by SLYY-3 was able to inhibit the growth of A. flavus, a very important pathogen in food safety. Therefore, the BLS may be useful for controlling several important pathogenic and spoilage microorganisms.

3.3 Characterization of Bacteriocin-Like Substances

3.3.1 Effect of temperature on BLS activity

Cell-free supernatant of SLYY-3 was assayed for the thermal stability. The activity of BLS produced from SLYY-3 showed 100% activity even after exposure to 100℃ for 60 min (Fig.3), the same as reported for the low-molecular-weight bacteriocin from B. licheniformis MKU3 (Kayalvizhi and Gunasekaran, 2008). The results are characteristic of other bacteriocins reported, such as thuricin 7, being stable after exposure to 90℃ for 30 min, and losing all activity after exposure to 121℃ for 20 min (Cherif et al., 2001). The bacteriocin produced from Bacillus sp. strain 8 A was reported to be heat-stable only up to 80℃ and the activity disappeared dramatically after incubation at 100℃ only for 15 min (Bizani and Brandelli, 2002). Therefore, this superior thermostability of BLS from B. subtillis SLYY-3 is a remarkable property for biopreservation of food.

Fig.3 Effect of temperature on activity of BLS of SLYY-3.

3.3.2 Effect of pH on BLS activity

Taking S. aureus as indicator strain, BLS produced from B. subtillis SLYY-3 retained its activity between pH 1.0 to 12.0. There was a very small difference in the zone of inhibitions formed after interaction of indicators with different pH treated BLS (Fig.4). Similar studies have been reported for bacteriocin of Bacillus sp., such as thuricin 7, which was stable between pH 3.0 and 9.0 (Cherif et al., 2001). The bacteriocin from strain 8A remained active between pH 5.0 and 8.0 (Bizani and Brandelli, 2002). When the pH was higher than 9.25, the biological activity of thuricin 17 disappeared (Gray et al., 2006). The activity of low molecular weight bacteriocin from the strain MKU3 was found to be stable under a pH range of 3.0-10.0 (Kayalvizhi and Gunasekaran, 2008). As a result, this wide range pH property of BLS in our study further recommends its application in biopreservation of acidic and alkaline food.

Fig.4 Effect of pH on activity of BLS of SLYY-3.

3.3.3 Effect of Proteolytic Enzyme-Trypsin on BLS Activity

Cell free supernatant containing the BLS from SLYY-3 pretreated with trypsin (ER) did not show any zone of inhibition against the S. aureus, with the sample as similar as the negative control by using phosphate bufferalone (C1), whereas the CFS mixed with phosphate buffer (C2) resulted in an inhibition zone at a diameter of 22 mm (Fig.5). This result showed that enzyme trypsin had completely inactivated the BLS of B. subtillis SLYY-3. This sensitivity to proteolytic enzyme trypsin reveals its proteinaceous nature and further supports its use as food biopreservative since it can be easily degraded in the digestive system of human beings.

Fig.5 Effect of trypsin on activity of bacteriocin-like substances of SLYY-3.

3.3.4 Partial Purification and Molecular Weight Determination of BLS

The inhibitory antibacterial component was isolated from the cell free culture supernatant by a combination of acid precipitation and gel filtration chromatography as shown by the results presented in Fig.6. Gel filtration resulted in fractions exhibiting antibacterial activity corresponding to peak II. Since antibacterial activity was present over a wide range of elution tube (Nos.15-19) and proteins in these fractions were not well resolved, it was difficult to determine precisely the elution tube for proteins having antibacterial activity. However, as the maximum zone of inhibition (23 mm) was observed at No.18 tube of elution, this point was considered arbitrary for determination of molecular weight of the antibacterial protein. SDS-PAGE followed by Coomassie blue R250 staining indicated that the peak consisted of a single peptide with an estimated molecular mass of 66 kDa (Fig.7) that exhibited antibacterial activity against S. aureus ATCC 6538. Some other bacteriocins with high (>10 kDa) molecular weight produced by Bacillus spp. had been previously studied in detail, such as bacteriocins (150 and 20 kDa) from B. licheniformis P40 (Cladera-Olivera et al., 2004); entomocin 9 (12.4 kDa) from B. thuringiensis ssp. entomocidus HD9 (Cherif et al., 2003); and thuricin 7 (11.6 kDa) from B. Thuringiensis BMG17 (Cherif et al., 2001). However, no bacteriocins with the same characteristics as the peptide described here have been reported from B. subtilis SLYY-3.

Fig.6 Elution profile of BLS from gel-filtration column.

Fig.7 Molecular weight of BLS estimated by SDS-PAGE.

4 Conclusion

In this study, we have successfully isolated a strain Bacillus subtilis SLYY-3 from sediments of Jiaozhou Bay, Qingdao, China. It is the first time to report the production of bacteriocin-like substance of Bacillus subtilis from this source. The bacteriocin-like substance (66 kDa) from B. subtilis SLYY-3 shows strong antimicrobial activity against most challenging and serious food pathogens such as Aspergillus flavus and S. Aureus. It is active over a wide range of temperatures and pH, which is a common characteristic of a number of bacteriocins produced by Lactobacilli (Anacarso et al., 2014). In addition, this BLS is more heat-stable when compared with other antimicrobial proteins produced by different species of Bacillus and Lactobacillus. As B. subtilis SLYY-3 produces a higher activity of BLS with a broad spectrum of activity and stability, this BLS can effectively be used as a biopreservative to prevent the growth of spoilage bacteria. It could also be proposed as a potential product used as medicine, natural biopreservative in the food processing industry, and pesticide for plant diseases control.

Acknowledgements

This work was supported by the National Science and Technology Support Program (No. 2011BAD14B04), Project of Shandong Province Higher Educational Science and Technology Program (J14LE59), Applied & Basic Research Foundation of Qingdao (No. 12-1-4-3-(3)-jch), and Science & Technology Project of AQSIQ (No. 2012IK176).

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(Edited by Ji Dechun)

(Received December 4, 2013; revised March 28, 2014; accepted April 11, 2014)

? Ocean University of China, Science Press and Springer-Verlag Berlin Heidelberg 2014

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E-mail: lijf1999@qust.edu.cn