Isolation, Characterization, and Application of Bacteriophages Against Several Food Spoilages Bacteria


 Objective: This study was conducted to isolate bacteriophages from soil sample, retail food, and wastewater from fish and then the bacteriophages will be characterized for their activity against several food spoilage bacteria, such as Bacillus cereus, Bacillus subtilis, and Shewanella putrefaciens and will be further investigated for application as food preservation.Results: Total of four bacteriophages were isolated with B. cereus, B. subtilis, and S. putrefaciens as host bacteria. Bacteriophage titers observed around 109 PFU mL-1. Bacteriophages that isolated with B. cereus and B.subtilis as host bacteria tend to have high EOP with the same species bacteria. All the Bacillus phages (S1-BC, S2-BC, and S1-BS) can reduce the Bacillus species bacteria concentration for more than 90%. Refers to their activity, the isolated bacteriophages in this study have a great potential as biocontrol against several food spoilage bacteria.


Introduction
Food spoilage is a metabolic process that causes changes in sensory characteristics, such as off-odor, textural changes, and visible growth of the colony in food. This spoilage is mediated by microbes that use food as their carbon and energy source. Some microbes are commonly found in many spoiled foods such as Bacillus cereus, Bacillus subtilis, and Shewanella putrefaciens [1].
Food preservation is purposed to prevent all of those events usually done by physical or chemical method. However, these methods have been known to reduce the sensory or organoleptic properties of the food products. Preservation using an excessive amount of antibiotic can increase the antibiotic resistance capability in bacteria which give a negative impact to human antibacterial treatment [2].
Bacteriophages as the natural alternative natural method to prevent food contamination is very promising to be used. They are viruses that can targeting bacteria speci cally to disrupt bacterial metabolism and can cause bacteria to lyse without disrupting human, animal, or plant cell. It can be isolated from a wide range of foods, such as rice, sh products, meat products, dairy products, and fermented foods [3]. The objectives of this research were to isolate and characterize lytic bacteriophages for Bacillus cereus, Bacillus subtilis, and Shewanella putrefaciens from soil sample, retail food, and wastewater from sh and analyze their capability as food biocontrol.

Methods
Inoculum Preparation. Bacillus cereus from Atma Jaya culture collections, Bacillus subtilis ATCC 6633, and Shewanella putrefaciens ATCC 8071 were used in this study. Bacterial cultures were inoculated onto luria agar (LA) and incubated at 37 °C and 28 °C (for S. putrefaciens) overnight. Sample Collection. Different types of food samples (rice, pasta, tofu, tempeh), soil samples (black soil and laterite soil), and wastewater sample of freshwater sh and seawater sh were collected in Jakarta,

Indonesia.
Bacteriophage Isolation for B. cereus and B. subtilis. The bacterial host strain was grown by incubation at 37 °C overnight at 120 rpm. Six grams of sample and 300 µL of bacteria culture were added into 30 mL of luria broth (LB) and then incubated at 37 °C overnight at 150 rpm. The medium was centrifuged at 6300 x g for 15 minutes, and then the supernatant was ltered using a 0.2 µm syringe lter. The ltrate was centrifuged again at 6300 x g for 10 minutes and tested for the presence of bacteriophages using agar overlay assay [4,5].
Bacteriophage Isolation for S. putrefaciens. The bacterial host strain was grown to mid-log phase by incubation at 28 °C overnight at 120 rpm. Then, 30 mL of each wastewater sample was centrifuged at 6300 x g for 10 minutes and then ltered using a 0.45 µm syringe lter. Subsequently, 5 mL of ltrate was mixed with 100 µL of host bacteria and added into 5 mL of 2X LB then incubated at 28 °C overnight. The culture was centrifuged at 6300 x g for 10 minutes, and the supernatant will be ltered using a 0.2 µm syringe lter. After that, 10 µL of the ltrate was mixed with 100 µL of host bacteria and then mixed it in 3 mL of soft agar and poured onto LA plate according to agar overlay assay [4].
Bacteriophage Puri cation and Enrichment. Phages were puri ed by removing a single plaque using a sterile tip onto micropipette. The plaque was transferred into 500 µL of SM buffer (50 mM Trishydrochloride (Tris-HCl) [pH 7.5], 0.1 M NaCl, 8 mM magnesium sulphate heptahydrate (MgSO4•7H2O), and 0.01% (w/v) gelatine) and then vortexed. The plaque was then diluted with a 10-fold serial dilution (100 µL of phages: 900 µL of SM buffer). After that, 400 µL of bacteria culture was mixed with 100 µL of the respective phage dilutions. The phage-host mixture was incubated at 37 °C and 28 °C (for S. putrefaciens) to ensure phage adsorption to the host bacteria. The 10 − 4 until 10 − 8 dilution were plated according to the agar overlay assay [4,6].
Bacteriophage Titer Determination. The bacteriophage titer determination was done using agar overlay assay [4]. A series of 10-fold dilutions with SM buffer were made of bacteriophage lysate solution up to 10 − 8 . 100 µL of diluted bacteriophage solution was mixed with 100 µL of mid-log phase bacteria culture in 3 mL of soft agar. The soft agar mixture then poured onto the agar and incubated at 37 C and 28 °C (for S. putrefaciens) overnight. The number of visible plaques were counted between 30 to 300 plaques and calculated as plaque forming unit (PFU) mL −1 [6].
Host Range Determination. This method was done using different species of the host bacteria, two Escherichia coli pathotype (EHEC and EPEC) and Vibrio cholerae. They were tested for the susceptibility towards isolated bacteriophage. Brie y, 100 µL of bacteriophage solution was mixed with 100 µL of tested bacteria in 3 mL of soft agar. The mixture then poured on top of agar and incubated at 37 C and Page 4/10 E ciency of Plating (EOP). Bacteriophages that can lyse the tested bacteria in host range determination method were continued to the EOP. The EOP was calculated by dividing the average PFU of target bacteria with the average PFU of host bacteria [8].
Bacteriophage Application. Rice and pasta sample were sterilized for 15 minutes at 121 °C. Tested bacteria (B. cereus, B. subtilis, V. cholerae) were inoculated 10 4 colony forming unit (CFU) cm − 2 onto the surface of the food surface followed by phage application at concentration 10 9 PFU mL − 1 (S1-BC, S2-BC, and S1-BS). Samples were incubated at 25 °C overnight. For the control, same volume of SM buffer was used instead of phage suspension [9]. Samples were suspended with 9 mL of SM buffer. A 10-fold serial dilution was made then spreaded onto LA plate and incubated at 37 °C overnight. The appropriate dilutions giving number colony between 30 to 300 colonies were expressed as CFU mL − 1 [9].

Results
Bacteriophage Isolation. Three bacteriophages could be isolated from two types of soil, which is black soil and laterite soil (S1-BC, S2-BC, S1-BS). One bacteriophage can be isolated from wastewater of seawater sh (SW-SP). All positive results were classi ed as lytic bacteriophages due to the clear zone plaques on top of agar.

Discussion
In this study, phages that were isolated from soil samples can invade B. cereus and B. subtilis; also, bacteriophage that was isolated from wastewater of seawater sh can invade S. putrefaciens as their host bacteria. These isolated phages could be classi ed as lytic type phages based on the clear zone plaques that appeared on top of the agar [7]. However, no phages could be obtained from food samples (tofu, tempeh, pasta, rice) and also wastewater of freshwater sh. This might be due to the food samples used in this study are processed foods so the processing can affect the presence of host bacteria and due to different environment and interaction of biotic and abiotic components [5,10].
Bacteriophage titers were observed around 10 9 PFU mL − 1 . Other study also showed a similar result with bacteriophage titers observed > 10 8 PFU mL − 1 which can be used for further analysis of the bacteriophage [9]. Bacteriophage titer determination is needed to prevent higher bacteria concentration than the phage concentration for further analysis such as bacteriophage application [11].
Phages that were isolated in this study are viruses that are really speci c so they can't attack other bacteria with different species. A host range that limited to a single species is desirable because it prevents the phage from killing other species of bacteria and leaving the rest of host's microbiome intact [12]. Each bacteriophage has different tail bres with receptor binding protein (RBP) that can bind to speci c receptor on host bacteria surface. Each host bacteria cell wall also has different components and additional constraints [12].
The EOP method uses to determine which phage was the most effective in killing other bacteria. In this study, all the phages tested for EOP (S1-BC, S2-BC, and S1-BS) were indicated to be highly effective and work speci cally for the other species of Bacillus with EOP ≥ 0.5. EOP between any phage pair may vary with different host bacteria [12]. High results of EOP bacteriophage might be useful to further research to make a bacteriophage cocktail [8].
All the Bacillus phages (S1-BC, S2-BC, S1-BS) can reduce B. cereus and B. subtilis e ciently. Higher concentration of phages have a correlation with the e ciency of the bacteriophage application process to lyse targeted bacteria [13]. However, different results were found when the Bacillus phages were applied against V. cholerae. The reason why V. cholerae was added in this method because it is known that V. cholerae also can survive on cooked rice and pasta for up to 5 days and can multiply rapidly at ambient temperature [14,15]. This result was also related to the result of host range determination method where phages can't lyse other species bacteria. The reduction of bacteria can be affected by the food texture and matrix to absorb the phages' suspension and the distribution of phages in food samples [16]. Phages' growth also depends on their ability to diffuse and contact with the host bacteria. The effectivity will be lower due to limited diffusion and contact with the host bacteria. This is also in uenced by differences in receptors between phages and host bacteria [12].

Conclusion
Four type of lytic phages were successfully isolated from black soil, laterite soil, and wastewater of seawater sh with B. cereus, B. subtilis, and S. putrefaciens as the host bacteria. All of the isolated phages showed high titer concentrations for more than 1 × 109 PFU mL-1. Phages isolated with B. cereus and B. subtilis as host bacteria tend to have high EOP and also can reduce the bacteria concentration effectively. All the isolated phages might have a great prospect to be used as food biocontrol and can be further tested to make phage cocktail.

Limitations
This study only carried out until bacteriophage application with Bacillus phages, thereby further research needs to be conducted to determine the application of S. putrefaciens phage, the minimum inhibitory multiplicity of infection, and also the morphology of isolated phages.

Declarations
Ethics approval and consent to participate. Not applicable.

Consent for publication. Not applicable
Availability of data and material. All data generated or analysed during this study are included in this published article.
Competing interests. The authors declare that they have no competing interests Funding. This study was funded by Ristekdikti 2019 and UNIKA Atma Jaya 2019. The funder has no contribution in this study.
Authors' contributions. DEW and YG made substantial contributions in proposal conception and design; FRS contribute in acquisition of data and analysis, and manuscript preparation under advisory of DEW.
All authors have read and approved the manuscript.