Abstract: Background and Objective: The Bacillus cereus strain HVR22 bacteria was obtained from isolated budu which produces bacteriocins. The aim of this study was to analyze the effect of crude bacteriocins on pathogen bacteria to observe the effect on cellular morphology of Escherichia coli using electron microscopy and to evaluate its effect on fish fillets. Materials and Methods: A diffusion method was used to measure the width of the clear zone made by Escherichia coli. Antimicrobial solutions of the Bacillus cereus strain HVR22 microbes were used to soak the snapper fillets, bacterial counts were performed on day 0 and 7. Results: Changes in E. coli membrane cell structure were observed under Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) after 48 h. Membranes were exposed to antimicrobial compounds from the Bacillus cereus strain HVR22. Antimicrobials produced by the Bacillus cereus strain HVR22 inhibited E. coli and Salmonella sp., growth on day 0 and 7. Conclusion: The bacteriocin-producing Bacillus cereus strain HVR22 could be used as a natural preservative for fish products.
INTRODUCTION
The Bacillus cereus strain HVR22 is an isolate from budu which has antimicrobial activity toward Escherichia coli, Staphylococcus aureus, Salmonella typhi, Bacillus subtilis and Listeria monocytogenes bacteria. Budu is a traditional fish-fermented product from West Sumatra that is usually made from pelagis fish tenggiri (Scomberomorus sp.) and talang-talang (Chorinemus sp.)1. The fermentation product is made without additional starter (called spontaneous fermentation) over 30 h and then gutted, salted and dried. Lactic Acid Bacteria (LAB) is usually produced from the fish fermented product.
The LAB is a fermented bacteria that acts as biopreservative because it can produce antimicrobial compounds, such as lactic acid, hydrogen peroxide (H2O2), diacetyl, carbon dioxide (CO2) and bacteriocins2,3. Bacteriocins are polypeptide compounds that act as antimicrobial compounds4. Bacteriocins can be used as a starter culture that is produced through the fermentation process as a component of the food probiotic and as biopreservative, extending the shelf life of different food products5,6. Using LAB in food processing can reduce the use of chemical preservatives and can improve food palatability and nutritional quality by increasing protein and vitamins. The LAB also produces detoxifying agents and inhibitory compounds, such as CO2, organic acids, ethanol, hydrogen peroxides and bacteriocins7-10.
Bacteriocins are produced by prokaryotic and eukaryotic organisms11, proteinaceous compounds which have antagonistic effects against related species and against spoilage and food-borne pathogens12-14. These peptides are very important for food and fodder, since a sufficient amount of peptides can inhibitor kill pathogenic microorganisms that compete for the same nutritive demands6. Bacteriocins can be produced by gram-positive and gram-negative bacteria15,16. Bacillus sp., B. subtilis, B. thuringiensis, B. stearothermophilus, B. licheniformis, B. megaterium, B. thermoleovorans, B. cereus and B. coagulans can also produce bacteriocins. Colicin, warnerin and coagulin are bacteriocins produced by Escherichia coli, Staphylococcus warneri (FM10, FM20 and FM30) and Bacillus coagulan, respectively16. Bacteriocins from LAB have drawn considerable attention recently because they are "Generally Recognized as Safe (GRAS)" and may be used as biopreservatives in food processing17,18. Different types of bacteriocins are widely used in the food industry, including niacin from Lactococcus lactis subsp. lactis and pediocin from Pediococcus pentosaceus19,20. Different strains of enterococci can produce active enterocins against Listeria monocytogenes, Staphylococcus aureus and Clostridium spp.21,22.
Bacteriocin producing LABs can be isolated from different types of food products such as dairy products, sausages, fish and vegetables23-27. Many Sudanese products are rich in LAB and can be used as biopreservatives. Different strains of LAB have been recorded as part of the process of producing traditional fermented milk products but the knowledge of their health benefits and species properties require further study28. Sudanese dairy products may be a rich source of LAB29. One study reported that the dominant species of LAB include Lactobacillus xylosus, Lactococcus lactis sub. sp. cremoris, Lactobacillus delbrueckii and Pediococcus cerevisiae. Several other studies on different Sudanese products (meat, fish, dairy, vegetables and cereal products) reported on their microbial load, however these products require additional research. This study was conducted to evaluate the effect of partial purification of bacteriocins on pathogen bacteria and to observe of its effect on the cellular morphology of Escherichia coli with applications to fish fillets.
MATERIALS AND METHODS
Bacterial strains: The Bacillus cereus strain HVR22 was provided by Yusra1. The indicator strain used in this study was Escherichia coli, which was provided by the Laboratory of Clinical Microbiology Research, Faculty of Medicine and Microbiology, University of Indonesia.
Antagonistic activity of bacteriocins: Diffusion method on sterile paper tests were used to evaluate the antimicrobial activity of the extracted bacteriocins. This method uses a paper disc. The culture of the Bacillus cereus strain HVR22 was refreshed in MRSA medium for 48 h, then 1 oz of bacteria was added to 10 mL of MRS broth and was incubated in 37°C for 24 h. Following, the culture was inserted into eppendorf and was centrifuged at 6000 rpm for 10 min. A 0.1 mL smear of Escherichia coli was removed using sterile cotton. Then, 20 mL of extracted bacteriocin preparation (CBP) was placed in each paper disc and the plates were incubated in 37°C for 24 h to measure the diameter of the microbe free zone30.
Scanning electron microscopy: The treated Escherichia coli cells exposed to bacteriocins of the Bacillus cereus strain HVR22 after incubation at 37°C for 48 h were examined by SEM to visualize any morphological changes that occurred in the cell. The cell suspensions were fixed with 3% glutaraldehyde in Na-cacodylate buffer (100 mM, pH 7.1). The cells were pelleted, washed to remove glutaraldehyde and resuspended in the same buffer. A drop from each suspension was transferred to poly-L-lysine-treated silicon wafer chips, which were kept for 30 min in a hydrated chamber for the cells to adhere. The attached cells were post fixed by immersing the chips in 1% osmium tetroxide (OSO4) in cacodylate buffer for 30 min, rinsed in the same buffer and dehydrated in ethanol in ascending concentrations (%) of: 50, 70, 95 (2x) and 100 (2x), for 10 min each. The chips were mounted on aluminium stubs and coated with gold-palladium in a sputter coater (Emitech K550, Ashford, Kent, England). The chips were viewed at 3 kV accelerating voltage in a Hitachi S-4000 field emission scanning electron microscope (JEM-JEOL JSM-5310LV type) and secondary electron images of cells for topography contrast were taken at several magnifications31.
Transmission electron microscopy: The cell suspension of pathogenic bacteria exposed to bacteriocins from the Bacillus cereus strain HVR22 which was incubated in 37°C for 48 h, where harvested by centrifugation and washed twice with 0.1 M phosphate buffer (pH 7.3). The cells were fixed with 2.5% (v/v) glutaraldehyde and 2.0% (v/v) formaldehyde in 0.12 M phosphate buffer for 10 days and then post-fixed in 2% (w/v) osmium tetroxide in the same buffer for 45 min. The samples were dehydrated in a graded acetone series (30-100%) and embedding in Araldite-Durcupan for 72 h at 60°C. Thin sections (microtome UPC-20, Leica) were mounted on grids, covered with collodion film and post-stained with 2% uranyl acetate in Reynold's lead citrate. All preparations were observed with a tipe JEOL-101032.
Application of bacteriocins to fresh fish fillets: Crude bacteriocins were used to soak the sample. The bacteriocins were exposed by cutting the snapper fish fillet into approximately 10 g and soaking it in a bacteriocins for 30 min (the control was not soaked). After soaking the fillet was removed and drained. The fish fillet was packed in sterile HDPE plastic and labeled. All samples were stored between -2 and 0°C. Observations were made on day 0 and 7. Inspection of fillet quality was done by Total Plate Count (TPC) to determine the total number of Coliform and Salmonella bacteria in each sample.
RESULTS AND DISCUSSION
Antagonistic activity of bacteriocins: The Bacillus cereus strain HVR22 isolates had the highest inhibitory effect against the Escherichia coli in 14 mm as shown in Fig. 11. Antimicrobial activity can be determined by the width of the clear zone around the paper disc after incubation for 48 h. The emergence of the clear zone is caused by acid metabolism from lactic acid that prevented growth of the indicator bacteria, the same effect was also found in Moroccan sardines (Sardina pilchardus) fermented fish. The width of the inhibitory zone of LAB was 20.6 mm and the bakasang fermentation inhibition zone diameter against Escherichia coli was 15 mm33,34.
Scanning electron microscopy: The SEM revealed damage to the cells and cellular material loss. Cell morphological damage from Escherichia coli was used as a representative of gram negative cells, after treatment with the antimicrobial compounds from the Bacillus cereus strain HRV22. As shown in Fig. 2, damage to the Escherichia coli cell membrane resulted from interaction with the Bacillus cereus strain HRV22 from budu fish fermented product. The cell surface was wrinkled and rough and formed irregular shapes and there was a notch in a few cells with septa that had not yet split. Antibacterial bacteriocins disrupted the cell membrane target, such that the cells lost their function35.
| Fig. 1: | Antimicrobial activity of isolate Bacillus cereus strain HVR22 on inhibiting the growth of Escherichia coli bacteria |
| Fig. 2: | Electron micrograph of Escherichia coli bacteria exposed to antibacterial of the Bacillus cereus strain HVR22 using Scanning Electronic Microscopy (SEM) |
| Fig. 3: | Electron micrograph of Escherichia coli bacteria exposed to antibacterial of the Bacillus cereus strain HVR22 using Transmission Electronic Microscopy (TEM) |
Some bacteriocins cause bacteriolysis (lysis of bacterial cells) due to the deregulation of autolysis systems and damage to the peptidoglycan layer. Bacteriocins work at very low nanomolar concentrations36.
Transmission electron microscopy: The TEM observation of ultra-structure pathogenic bacteria of the bacteriocins of Bacillus cereus strain HRV22 is shown in Fig. 3. The addition of bacteriocins to Escherichia coli pathogenic bacteria produced a few visible changes, including irregular cytoplasm, formation of a space between cytoplasm and cell membrane, wrinkled cells (collapse) and cell lysis. Based on microstructure, some Escherichia coli cells were broken and irregularly-shaped. Antimicrobial compounds produced by the Bacillus cereus strain HVR22 can damage the cytoplasm membrane and affect the integrity of Escherichia coli cell surface. This damage can cause increased permeability and intercellular discharge.
Cerein-8A activity analyzed against Bacillus cereus and Listeria monocytogenes bacteria using TEM showed that cerein-8A killed bacterial cells in 60 min37. Bacteriocins also inhibited some gram-positive bacteria, such as Bacillus spp., bacteria but these bacteriocins were not able to kill Escherichia coli and Salmonella enteritidis cells. Bacteriocins derived from Bacillus cereus SBS02 isolated from sea shells can inhibit the growth of pathogenic bacteria and bacteria that cause decay of food, such as Candida albicans, Listeria monocytogenes, Pseudomonas aeruginosa, Bacillus cereus and Bacillus subtilis38.
Electron microscopy showed cell lysis following treatment with antimicrobial compounds of the Bacillus cereus strain HVR22. Cell damage caused by antimicrobial compounds resemble that observed with crude bacteriocins39. Type-A1antibiotics, to which nisin, pediocin and apidermin and many other amphiphilic antimicrobial peptides belong, exert their activity by disrupting the functional barrier of microbial cytoplasmic membranes40. In addition to nisin and apidermin, lipid I and II binding has been observed in subtilin and mersacidin produced by Bacillus spp.41. The hydrophobic bacteriocin cerein 7, produced by B. cereus Bc7 has also been characterized as a membrane-active compound42. The antimicrobial compound from the Bacillus cereus strain HVR22 has a bactericidal effect caused from disrupting the membrane function of target organisms.
Application of bacteriocins to fresh fish fillets: Antibacterial compounds from the bacteria Bacillus cereus strain HVR22 can reduce the number of pathogenic bacteria contained in snapper fillets, including Salmonella sp. and Escherichia sp. bacteria, using PCA, SSA and EMBA medium as shown in Table 1. On observation day 0 using a PCA medium, the average colonies of bacteria were 6.1x108 CFU g–1 colonies. On observation day 7, bacteria were growing in the PCA but there were no Salmonella sp. and Escherichia sp. bacteria. Bacteriocins except in selective SSA and EMBA medium, as a result of the submersion of snapper fish fillet in crude bacteriocins from the Bacillus cereus strain HVR22 that contained antimicrobial compounds. Generally, bacteriocins oppose other bacteria through a bactericidal effect.
Red tilapia fillet submerged in 108 CFU g–1 liquid for 5, 10 and 15 min achieved a 9 day shelf life43. The addition of lactic acid bacteria (Lactobacillus acidophilus) can decrease the pH of tilapia (Oreochromis niloticus)44. Lactobacillus fermentum UN01 can extend the shelf life of fish in cold temperatures (refrigeration) up to 9 days45.
| Table 1: | Colonies of bacteria in treatment concentrations of crude bacteriocin in snapper fish fillet |
Supernatant from cultivation media of L. plantarum (SK5) can be used as a biopreservative for snapper fillets at chilling temperatures and can replace formaldehyde46.
CONCLUSION
Antimicrobial compounds produced by the Bacillus cereus strain HVR22 can damage the cell membrane and cellular structures inside Escherichia coli cells. Antimicrobial compounds of the Bacillus cereus strain HVR22 can inhibit the growth of Salmonella sp. and Escherichia sp. in snapper fish fillet until 7 days of storage and can be used as natural preservatives.
SIGNIFICANCE STATEMENT
This study evaluated the effect of bacteriocins produced from the Bacillus cereus strain HVR22 and its application to fish fillet preservation. The researchers certify that they have no affiliations with or involvement in any organization or entity with any financial or non-financial interest in the subject matter or materials discussed in this study.
ACKNOWLEDGMENTS
This study was supported under study grant (Hibah Bersaing) Directorate of Research and Community Service, Ministry of Research Technology and Higher Education Republic of Indonesia [No. 045-33/SP/HATTA-1/LPPM/IV-2015].