KR102346551B1 - Mass production method for cronobacter sakazakii bacteriophage - Google Patents

Abstract

The present invention relates to a method for mass production of bacteriophages, specifically Cronobacter sakazakii bacteriophages, and specifically, the present invention consists of a two-stage self-cycling (TSSC) process, By adding a nutrient medium necessary for the culture of Chronobacter sakazaki and measuring the pH change of the culture medium, the optimal bacteriophage inoculation time and harvest time can be set. Continuous mass production is possible.

Description

Mass production method of Chronobacter sakazaki bacteriophage {MASS PRODUCTION METHOD FOR CRONOBACTER SAKAZAKII BACTERIOPHAGE}

The present invention relates to a method for mass production of Cronobacter sakazakii bacteriophages.

10 years ago, Enterobacter Chrono bakteo sakajaki was named sakajaki (Enterobacter sakazakii) (Cronobacter sakazakii) is defined as a single species. Chronobacter sakazaki is an opportunistic gram-negative, rod, pathogenic bacterium that can live even in very dry places, such as powered infant formula (PIF). These species can be isolated from environments such as food, water, soil, and processed foods, including dairy products. In addition, Chronobacter sakazaki is known to cause meningitis and necrotizing enterocolitis. Because PIF is easily contaminated by Chronobacter sakazaki during improper storage, it is considered a major cause of infection in newborns with a weaker immune system than adults.

On the other hand, antibiotic resistance makes antibiotics no longer able to effectively inhibit pathogenic bacteria. In other words, bacteria change to become resistant to the chemicals in the drug. In this case, general infections or diseases can no longer be treated with general antibiotics, and resistant bacteria continue to survive and multiply due to antibiotics that are less effective, causing fatal damage. Antibiotic resistance is now considered one of the greatest threats to global health, but since there is no way to stop the development of resistant bacteria as long as antibiotics are used, the most effective way to slow the growth of resistant bacteria is not to use antibiotics, or even if necessary, the most effective way. should be used only as For this reason, the number of newly developed antibiotics is also rapidly decreasing in recent years, and therefore, a biocontrol agent capable of controlling pathogenic bacteria without the problem of antibiotic resistance is required.

Bacteriophages (phages) are viruses of bacteria that can kill and lyse infected bacteria. Bacteriophages have been used to treat a variety of bacterial diseases in humans and animals since their discovery in the early 20th century. The most important characteristic of bacteriophages is that they are highly specific to specific bacterial strains, unlike antibiotics that act universally against various pathogens. Thanks to these characteristics, bacteriophages do not kill beneficial bacteria in our body, but only pathogenic bacteria can be selectively killed. Consequently, the antibacterial properties of bacteriophages are suitable for applying bacteriophages as an alternative to antibiotics. In 2006, the US Food and Drug Administration (FOOD and Drug Administration, FDA) allowed the use of purified bacteriophages as food additives, and accordingly, the use of bacteriophages in many fields such as medicine and nanomaterials, including the food industry, is increasing. Therefore, as the demand for bacteriophages is increasing, additional studies are needed to efficiently produce bacteriophages.

Accordingly, the present inventors made efforts to develop a new mass production method to meet the high demand for bacteriophages, and as a result, introduced a two-stage self-cycling (TSSC) process, and cultured and propagated the host bacterium Chronobacter sakazaki. By measuring the parameters (pH, optical density, number of viable cells, number of colonies formed) according to It was confirmed that it can be obtained continuously. Therefore, the Chronobacter Sakazaki bacteriophage mass production method was stably and continuously by revealing that it can be usefully used as a method to obtain a high-concentration bacteriophage solution, thereby completing the present invention.

It is an object of the present invention to provide a method for mass production of Cronobacter sakazakii bacteriophages.

In order to achieve the above object, the present invention provides a method comprising: 1) a self-cycling fermentation (SCF) step of culturing bacteria to obtain a bacterial culture; And 2) a self-cycling infection (SCI) step to obtain a bacteriophage by culturing and collecting the infected bacteria after infecting the bacteria of the bacterial culture medium obtained in step 1) with the bacteriophage; As a production method, the self-circulating fermentation (SCF) step comprises the steps of: i) resuspending a single colony of a host bacterial strain in a medium and then culturing; ii) monitoring the pH while subculturing the culture solution of step i) in the first bioreactor; iii) when the pH is 5.5 to 6.0, obtaining a portion of the culture solution of step ii) and transferring it to a second bioreactor; and iv) replenishing the culture medium remaining in the first bioreactor among the culture medium of step ii) as a sub-step, wherein the autologous circulation infection (SCI) step is performed in A) under pH monitoring, in a second bioreactor Proliferating the bacteriophage while culturing after adding the bacteriophage to the bacteria in my bacterial culture; B) when the pH is 4.5 to 5.5, collecting and filtering the proliferated bacteriophage of step A) to obtain a filtrate; C) re-adding a portion of the filtrate obtained in step B) to a second bioreactor; And D) purifying the remaining filtrate of step B) to produce a bacteriophage; it provides a method for mass production of bacteriophages, including as a detailed step.

The invention also provides an apparatus for carrying out the method.

The mass production method of Cronobacter sakazakii bacteriophage of the present invention consists of a two-stage self-cycling (TSSC) process, and at the beginning of the process, a nutrient medium necessary for culturing Cronobacter sakazakii By adding and measuring the pH change of the culture medium, it is possible to set the optimal bacteriophage inoculation time and harvest time, so that it is possible to simply and continuously mass-produce high-concentration bacteriophages in comparison with the existing process, so the method It can be usefully used for mass production of Chronobacter sakazaki bacteriophage.

1 is a diagram showing an in vitro growth curve of Cronobacter sakazakii.
Figure 2 is a diagram showing a one-step growth curve (one-step growth curve) of the bacteriophage Φ CS01.
3 is a simplified schematic diagram of the modified two-stage self-cycling process of the present invention.
Figure 4 is a diagram showing the shape of the bacteriophage Φ CS01.
Figure 5 is a diagram showing the optimal initial infection conditions for the production of bacteriophage Φ CS01.
Figure 6 is a diagram showing the results of the thermal stability test of the bacteriophage Φ CS01.
7 is a diagram showing the results of the pH stability test of bacteriophage Φ CS01.
8 is a diagram showing the results of blue-white selection of E. coli ER2738.
9 is a diagram showing the results of agarose gel electrophoresis of Φ CS01 phage DNA cut with restriction enzymes.
FIG. 10 is a diagram showing a neighbor-joining phylogenetic tree of bacteriophage Φ CS01 based on a tape measure protein sequence.
11 is a diagram showing a neighbor-joining phylogenetic tree of bacteriophage Φ CS01 based on a tail fiber protein sequence.
12 is a diagram showing the correlation between pH, viable cell number, and OD in a Chronobacter sakazaki culture medium.
Figure 13 is a diagram showing the results of the bacterial induced test of bacteriophage Φ CS01 using Chronobacter Sakazaki.
14 is a diagram showing the correlation between the pH and the number of viable cells of the Chronobacter sakazaki culture medium pretreated with lysozyme buffer and sonication.
15 is a diagram showing the characteristics of the bacteriophage Φ CS01 induced by Chronobacter Sakazaki.
16 is a diagram showing the titer of a high titer phage solution obtained by repeating the TSSC process.
17 is a diagram showing the infection rate according to time of the freeze-dried bacteriophage Φ CS01.

Hereinafter, the present invention will be described in detail.

The present invention is a self-cycling fermentation (SCF) step of culturing bacteria to obtain a bacterial culture; And 2) a self-cycling infection (SCI) step to obtain a bacteriophage by culturing and collecting the infected bacteria after infecting the bacteria of the bacterial culture medium obtained in step 1) with the bacteriophage; As a production method, the self-circulating fermentation (SCF) step comprises the steps of: i) resuspending a single colony of a host bacterial strain in a medium and then culturing; ii) monitoring the pH while subculturing the culture solution of step i) in the first bioreactor; iii) when the pH is 5.5 to 6.0, obtaining a portion of the culture solution of step ii) and transferring it to a second bioreactor; and iv) replenishing the culture medium remaining in the first bioreactor among the culture medium of step ii) as a sub-step, wherein the autologous circulation infection (SCI) step is performed in A) under pH monitoring, in a second bioreactor Proliferating the bacteriophage while culturing after adding the bacteriophage to the bacteria in my bacterial culture; B) when the pH is 4.5 to 5.5, collecting and filtering the proliferated bacteriophage of step A) to obtain a filtrate; C) re-adding a portion of the filtrate obtained in step B) to a second bioreactor; And D) purifying the remaining filtrate of step B) to produce a bacteriophage; it provides a method for mass production of bacteriophages, including as a detailed step.

The bacterium may be Cronobacter sakazakii , but is not limited thereto.

In addition, the first bioreactor and the second bioreactor may be a jar type fermenter, but is not limited thereto.

In addition, the medium of detailed step i) of step 1) may be a liquid medium, but is not limited thereto.

In addition, the incubation temperature of detailed step ii) of step 1) may be 20 to 35 ℃, specifically may be set to 21 to 30 ℃, more specifically may be set to 22 to 26 ℃, most preferably It may be set to 24° C., but it is not limited thereto, and if there is a difference in the effect according to the temperature, it may be appropriately adjusted.

In addition, the incubation time of detailed step iii) of step 1) may be 2 to 6 hours, specifically 3 to 5 hours, and most preferably 4 hours, but is not limited thereto. If there is a difference in effect, it can be appropriately adjusted.

In addition, the subculture of sub-step ii) of step 1) may be when an early exponential phase is reached on the bacterial growth curve, but is not limited thereto.

The exponential phase may be the same as a logarithmic phase, log phase, logarithmic period, log period, proliferative phase, and logarithmic phase, but is not limited thereto.

In addition, the pH of detailed step iii) of step 1) may be 4 to 9, specifically, may be 4.5 to 6, and most preferably may be pH 5.8, but is not limited thereto. If there is a difference, it can be appropriately adjusted.

In addition, the volume ratio of a portion of the culture solution of sub-step iii) of step 1) and the culture solution remaining in the first bioreactor of sub-step iv) may be 9:1, but is not limited thereto.

In addition, the volume ratio of the culture medium remaining in the first bioreactor in detailed step iv) of step 1) and the supplemented medium may be 1:9, but is not limited thereto.

In addition, the bacteriophage of step 2) may be Φ CS01, but is not limited thereto.

In addition, the bacteriophage of detailed step A) of step 2) may be input to have 0.01 to 0.5 multiplicity of infection (Multiplicity of Infection, MOI), specifically 0.05 to 0.3 MOI may be input to have, most preferably It may be input to have an MOI of 0.1, but it is not limited thereto, and if there is a difference in the effect according to the MOI, it may be appropriately adjusted.

In addition, the pH of detailed step B) of step 2) may be 4 to 9, specifically pH 4.5 to 6, and most preferably may be pH 5.0, but is not limited thereto. If there is a difference, it can be appropriately adjusted.

In addition, the filtration of detailed step B) of step 2) may consist of a filter having a pore size of 0.2 to 0.7 μm, specifically, a filter having a pore size of 0.3 to 0.5 μm, and most preferably a filter having a pore size of 0.45 μm. However, the present invention is not limited thereto, and when there is a difference in the effect according to the pore size of the filter, it may be appropriately adjusted.

In addition, the purification of detailed step C) of step 2) may be performed with one or more selected from chloroform, sodium chloride (NaCl) and polyethylene glycol (PEG), but is not limited thereto.

In addition, the SCI step may be repeated 2 to 5 times in a row, specifically 3 to 4 times in a row, and most preferably may be repeated 3 times, but is not limited thereto, and the effect according to the number of repetitions If there is a difference in , it can be appropriately adjusted.

The present invention provides an apparatus for performing the mass production method of the bacteriophage.

The device may include, but is not limited to, two or more bioreactors.

In addition, the device may be independent of the culture space of the bacteria to be infected with the bacteriophage and the bacteria infected with the bacteriophage, but is not limited thereto.

In a specific embodiment of the present invention, the present inventors constitute a two-stage self-cycling (TSSC) process, and in order to further increase production efficiency, the host bacterium Cronobacter sakazakii In the culturing process Changes in various parameters of the culture medium (pH, optical density, number of viable cells) were measured, and parameters according to the propagation of bacteriophages (pH, optical density, number of colonies) were also measured.

As a result, it was possible to increase the continuity and efficiency of production by calculating the optimal inoculation time and harvest time of the bacteriophage, and it was possible to obtain a bacteriophage solution of a stable and continuously high concentration compared to the conventional process.

Hereinafter, the present invention will be described in detail by way of Examples and Experimental Examples.

However, the following Examples and Experimental Examples only illustrate the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

<Experimental Example 1> Confirmation of the in vitro growth curve of Chronobacter sakazaki

To analyze the growth over time of Chronobacter sakazaki, a growth curve was identified. First, the bacterium Chronobacter sakazaki NCTC 11467 ( C. sakazakii NCTC 11467) used in the present invention was obtained from the National Collection of Type Cultures (NCTC). _{In order to confirm the growth curve, the bacterial culture medium was adjusted to an optical density of 0.1 at 540 nm (OD 540} ) using a microplate reader (Multiskan FC, Thermo, USA), and after inoculation of 20 μl/ml host bacteria, It was grown in TSB broth (TSB broth, BD Bacto, USA) under microaerophilic conditions in a shaking incubator at 37° C. for 3 hours. The number of colony forming units (CFU) in the cultured cells was measured every 10 minutes.

As a result, in the growth curve of the confirmed Chronobacter sakazaki, The lag phase was maintained for about 30 minutes, and it was found to take 140 minutes to complete the exponential phase in which exponential growth takes place, after which it enters the stationary phase. was observed (Fig. 1).

<Experimental Example 2> Confirmation of one-step growth curve of bacteriophage Φ CS01

After being infected with the bacteriophage, a one-step proliferation curve was confirmed to confirm the proliferation of the bacteriophage over time. For the analysis of the one-step growth curve, 5 ml of the bacterial culture was centrifuged at 4,000 × g for 10 minutes in the logarithmic phase with an _{OD 540 value of 0.3, and the pellet was resuspended in 1 ml of TSB liquid medium.} . Then, the bacteriophage solution was added to the suspension at a multiplicity of infection (MOI) of 0.1, and the mixture was incubated at 37°C for 30 minutes, then centrifuged at 11,000 × g for 10 minutes, and the pellet was 10 Resuspended in ml TSB liquid medium. The titer of the bacteriophage (titer), the number of plaque formation per ml of bacteriophage solution (plaque forming unit, PFU) was measured at 10 minute intervals for 120 minutes using a double layer plaque assay.

As a result, it was observed that the latent period was maintained for about 60 minutes, and the exponential phase was 60 to 80 minutes in the one-step proliferation curve of the identified Φ CS01. It was confirmed that it took 80 minutes to complete release after initial infection with a burst size of 9.8 viral particles per host cell (PFU/infected cell) (FIG. 2).

<Example 1> Amplification process of bacteriophage

A single colony of the host bacterial strain, Cronobacter sakazakii , was resuspended in 5 ml of TSB liquid medium, and cultured at 100 rpm in a shaking incubator at 37°C. When the culture medium _{reached an early exponential phase with an OD 540} value of 0.3, 1 ml of the solution was sub-cultured with 100 ml of TSB liquid medium, and then the subcultured medium had an OD 540 value of _540. It was cultured at 100 rpm in a shaking incubator at 37°C until an early exponential phase of 0.3 was reached. After incubation, the bacteriophage was inoculated to have an MOI of 0.1, and cultured in a shaking incubator at 37° C. for 20 minutes.

<Example 2> Purification (purification) process of bacteriophage

After amplification of the bacteriophage, 1 ㎖ of chloroform and 1 mole of sodium chloride (NaCl) at a concentration of 1% (w/v) were added, and incubated for 30 minutes at 37° C. at 200 rpm in a shaking incubator. Then, the culture solution was centrifuged at 11,000 × g at 4°C for 30 minutes, and the supernatant was filtered using a syringe filter (syringe filter, vantec, USA) having a pore size of 0.45 μm, and solid polyethylene glycol ( polyethylene glycol, PEG 8000, Aldrich) was added to the suspension to a final concentration of 20% (w/v), and the mixture was cooled on ice overnight. Then, the lysate was centrifuged at 11,000×g for 100 min, and the supernatant was discarded. The pellet was resuspended in 5 ml of SM buffer, half the volume of chloroform was added, and then gently mixed. The mixed mixture was centrifuged at 3,000×g at 4°C for 15 minutes, and the supernatant was recovered using a syringe filter (syringe filter, Advantec, USA) with a pore size of 0.45㎛, filtered, and stored at 4°C in a refrigerator. did

<Example 3> Two-stage self-cycling (TSSC) process

The type of bioreactor used plays an important role in establishing infection and action conditions leading to optimal bacteriophage titers. In the meantime, the types of general bioreactors used for bacteriophage production are batch, continuous, two-stage continuous and two-stage self-cycling, and the present invention used a two-stage self-cycling (TSSC) process. The process consists of a self-cycling fermentation (SCF) step and a self-cycling infection (SCI) step, and the TSSC process of the present invention was performed by modifying the existing process (FIG. 3) .

<3-1> Self-cycling fermentation (SCF) step

In the SCF stage, after resuspending a single colony of the host bacterial strain Chronobacter sakazaki in 10 ml of TSB liquid medium, _{when the early exponential phase of OD 540} is 0.3 and pH 5.8 is reached incubated at 37°C until After incubation, 1 ml of the culture solution was sub-cultured in the first bioreactor at a concentration of 1% v/v of 10 ml in 1000 ml of the culture solution of the host culture. Then, the subcultured bacterial culture solution _{was incubated at 24° C. for 4 hours until an OD 540} value of 0.3 and an initial exponential phase of pH 5.8 were reached. After propagation of the host bacteria, 900 ml of culture medium was transferred to the second bioreactor and 100 ml of solution was left in the first bioreactor supplemented with 900 ml of fresh TSB broth.

<3-2> Self-cycling infection (SCI) stage

In the SCI step, the bacteriophage was cultured at 37° C. for 4 hours in the second bioreactor after inoculation so that the MOI was 0.1. When the pH of the lysate reached 5.0, it was collected and filtered through a bottle-top vacuum filter (Jet Biofil, USA) having a pore size of 0.45 μm. After filtration, 900 ml of the filtered solution was purified by the method of <Example 2> to make a high titer bacteriophage solution, and 100 ml of the filtered solution was added to the second bioreactor for repeated infection.

This process was repeated three times in succession.

In the TSSC process, in order to evenly rotate the SCF stage, the first stage, and the SCI stage, the second stage, the growth of Chronobacter sakazaki because the proliferation of the bacteriophage must be completed in the second stage until the host bacteria reaches the logarithmic stage in the SCF stage I needed to slow down. Accordingly, in the present invention, the temperature of the first bioreactor was adjusted to 24° C., unlike the culture method of the subcultured culture medium of Example 1, so that the host bacteria reached the initial log phase after 4 hours. Further experiments confirmed that when the host bacteria were cultured at 24°C, there was no difference from the host bacteria cultured at 37°C except for growth rates (data not shown). Through this control, the circulation between the first and second steps of the TSSC process could be made uniformly, and a bacteriophage solution of high concentration could be obtained up to three times on the basis of this.

<Experimental Example 3> Confirmation of the shape of the bacteriophage Φ CS01 using a transmission electron microscopy (TEM)

The structure of the bacteriophage Φ CS01 was observed using a transmission electron microscope (JEOLJEM-2100F FE-TEM, KBSI, Korea) at 200 kV. First, a high titer phage solution containing 108 PFU was stained with 2% w/v uranyl acetate. The phage particles were placed on a carbon-coated grid and immersed in distilled water containing a drop of uranyl acetate at a concentration of 2% w/v. Then, 200-mesh grids (200-mesh grids, Gatan, USA) were coated with a collodion film prepared with 2% collodion in amyl acetate, and carbon film pieces with phage particles. film fragments) were used to absorb. After air drying for 10 minutes, TEM was applied to the grid. Images of the negatively stained phage particles were taken using a one-view camera (Gatan, USA) at 100,000× and 150,000× magnification.

As a result, the spherical head (Spherical head) with icosahedral symmetry of the bacteriophage Φ CS01 was 65.74 nm, and the length of the rigid tail was 89.75 nm. In addition, Φ CS01 was observed to have a non-contracted tail and a contracted tail, and a sheath-like structure in the tail (Fig. 4). Due to its morphological characteristics, phage CS01 was presumed to belong to the family Myoviridae.

<Experimental Example 4> Confirmation of optimal infection conditions for bacteriophage Φ CS01

The productivity of the bacteriophage solution according to the infection conditions was confirmed. For a typical infective range _{, host cultures at an OD 540} value of 0.4 (cell concentration of about 5.0×108 CFU/ml) for infection loads (0.1 to 5% v/v in 10 ml culture) 0.5 ml), and has a range of ^{1.0×10 −4} to 1.0×10 ⁻¹ with respect to the initial MOI. The titer of the bacteriophage (titer) was measured using a double-layer plaque assay for PFU per ml of the bacteriophage solution.

As a result, the optimal titer of the bacteriophage Φ CS01 under the given treatment conditions was obtained from an initial MOI between ^{1.0×10 −1} and 1.0×10 ^{−2 with an infection load of 1% ( FIG. 5 ).} After cell lysis, an infection load of 1% was chosen with an initial MOI of ^{1.0×10 −1 to minimize cell debris.} These initial infection conditions were used to operate the bioreactor for optimal bacteriophage production.

<Experimental Example 5> Confirmation of thermal stability of bacteriophage Φ CS01

The thermal stability of the bacteriophage solution according to the temperature conditions was confirmed. The thermal stability of the bacteriophage Φ CS01 was evaluated by exposure to various temperature ranges for 1 hour.

As a result, the bacteriophage Φ CS01 was almost maintained when cultured at 4 ° C to 37 ° C. Infectivity activity (infection activity) was almost maintained, and when cultured at 50 ° C to 60 ° C, the infectivity was slightly reduced. However, at 70° C., it was confirmed that a single plaque was insufficient ( FIG. 6 ).

<Experimental Example 6> Confirmation of pH stability of bacteriophage Φ CS01

The pH stability and optimum pH of the bacteriophage solution according to the pH change were confirmed. The pH stability of the bacteriophage Φ CS01 was evaluated by exposure to various pH ranges for 1 hour.

As a result, almost no decrease was observed in the infectious phage after incubation at pH 4 to 9 for 1 hour. The number of bacteriophages was shown to be reduced at pH 10-11, but no active infectious phages were detected at pH 3 and 12. As a result, the pH range 4 to 9 was confirmed to be optimal for bacteriophage stability (FIG. 7).

<Experimental Example 7> Screening through bio-panning method

In order to broaden the host range of the bacteriophage Φ CS01, screening was performed to confirm the peptide sequence with high affinity from the bacteriophage library by the biopanning method.

<7-1> Immobilization of target pathogen Chronobacter sakazaki

Heat-inactivated Chronobacter sakazaki suspension incubated at 65° C. for 1 hour was mixed with carbonate buffer (sodium bicarbonate (NaHCO ₃ ) 35 mM, sodium carbonate (Na ₂ CO _{3 )} 15 mM (pH=9.80)) and adjusted to an optical density of 0.5 at 540 nm corresponding to about 109 CFU per ml. 1.5 ml of bacterial suspension was filled in a 60 mm × 15 mm cell culture dish (Cell Culture Dishes, Falcon®, USA), and cultured overnight in a bacterial incubator at 37°C. The cell culture dish was blocked at 4° C. for 1 hour with a blocking buffer, washed 6 times with TBST buffer to prepare a coated plate, and the target pathogen, Chronobacter sakazaki, was fixed ( immobilization).

<7-2> Selection of phage displayed peptides using the biopanning method

The entire bacteriophage library (library) was diluted 100-fold in 1 ml of TSBT buffer, and then pipetted onto a plate coated with bacteria, gently shaken at room temperature for 1 hour. Then, the plate was poured on a clean paper towel to discard the non-binding phages, and the plate was washed 10 times with TBST buffer. The bound phage was eluted with 1 ml of an elution buffer (glycine-hydrogen chloride (Glycine-HCI) 0.2M (pH=2.2)) and gently mixed for 20 minutes at room temperature. The mixed eluate was transferred to a microcentrifuge tube (microcentrifuge tube, Axygen, USA) with a pipette, neutralized with 150 μl of neutralization buffer, and stored at 4°C.

<7-3> Amplification of the selected phage

The stored phage lysate was used for amplification by infecting E. coli ER2738 host bacteria. After growth for 4.5 hours at 37°C with vigorous shaking, bacterial cell debris was removed by centrifugation, and 1/6 volume of a solution (20% w/v polyethylene glycol-8,000 (polyethylene flycol-8,000) and 2.5 M sodium chloride (NaCl)) was added to precipitate the phages in the supernatant overnight at 4°C. Then, the precipitate was centrifuged, the pellet was resuspended in 1 ml of TSB buffer, and 100 μl of the amplified phage eluate was titrated to measure the phage concentration.

The remainder of the eluate was amplified with E. coli ER2738 cultured in LB medium. A 250 ml Erlenmeyer flask containing E. coli ER2738 was inoculated with 20 ml of LB medium. Incubation was performed with vigorous shaking at 37°C, and the 20 ml culture was carefully monitored to ensure that the OD ₆₂₀ value did not exceed the initial logarithmic phase of 0.01 to 0.05. The remaining eluate was amplified by adding the eluate to the 20 ml culture solution of E. coli ER2738. After incubation for 4.5 hours with vigorous shaking at 37°C, centrifugation at 12,000×g for 10 minutes to remove bacterial cell debris, phages were precipitated in the supernatant by adding 1/6 volume of PEG solution, and overnight at 4°C. kept.

The precipitated PEG was spun at 12,000×g for 15 min at 4° C., the supernatant was discarded, the tube was briefly rotated and the residual supernatant was removed with a pipette. The pellet was suspended in 1 ml of TBS, and the suspension was transferred to a microcentrifuge tube, and then spun at 12,000×g for 5 minutes at 4° C. to pellet residual cells. The supernatant was transferred to a sterile microcentrifuge tube and precipitated by adding 1/6 volume of PEG solution.

The precipitated PEG was incubated on ice for 15 to 60 minutes and centrifuged at 12,000×g for 10 minutes at 4°C. After discarding the supernatant and removing the residual supernatant with a micropipette, centrifuged for 1 minute at 12,000 × g in a TBS centrifuge for pelleting to suspend the 200 μL pellet, and transfer the supernatant to a new tube.

<7-4> Perform blue-white selection

For phage titration, a single colony of E. coli ER2738 was inoculated into 10 ml of LB liquid medium, and cultured at 37° C. in a shaking incubator at 250 rpm until the mid- _{log phase with an OD 620 value of 0.5.} After dispensing 3 ml of the equilibrated agarose tower into a sterile culture tube and putting it in a water bath at 45° C., the phage solution was serially diluted with a 10-fold dilution in LB liquid medium. When the culture of E. coli ER2738 reached the meta-log phase, 200 µL of the culture was dispensed into each microcentrifuge tube containing the diluted phage solution. 10 μL of each dilution was aliquoted into a separate tube, mixed and incubated for 5 minutes at room temperature.

One at a time, infected cells were transferred to culture tubes containing agarose warmed to 45° C. and immediately poured into preheated LB/IPTG/X-gal plates. By tilting the plate, the upper agarose gel was spread evenly. After cooling for 5 minutes, the plates were incubated overnight at 37°C. The next day, plaques on the plate were counted and converted to PFU per 10 μL. Since the library cloning vector M13KE carries the lacZα gene, it was confirmed that the bacteriophage plaques appeared blue in the medium containing X-gal and IPTG due to α-complementation (Fig. 8).

<Experimental Example 8> Confirmation of the host range of bacteriophage Φ CS01

The host range spectrum of the bacteriophage was confirmed using different bacterial strains (Table 1). A spot lysis assay was performed according to the protocol. Each of the 10 different host bacteria was inoculated into 5 ml of soft 0.75% agar and poured onto a solidified 1.5% agar plate. Before the soft agar was hardened, 20 μl of the phage solution was spread on a top agar plate and incubated at 37° C. for 12 hours. Susceptibility to the bacteriophage Φ CS01 was confirmed by measurement of plaque formation and the appearance of lysis zones after inoculation. According to the degree of sharpness, the observed results were divided into two categories: (+) distinct plaques and (-) no plaques. Enterobacter (Enterobacter) and other strains were obtained from the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center (Korean Collection for Type Cultures, KCTC ) or ATCC (American Type Culture Collection) for the host range test (host-range test).

As a result, it was confirmed that only Chronobacter sakazaki among ten different host bacteria had sensitivity to Φ CS01.

<Experimental Example 9> Confirmation of restriction enzyme that degrades bacteriophage Φ CS01 DNA

For molecular characterization of bacteriophage, restriction enzymes for cutting bacteriophage DNA were identified. Bacteriophage DNA samples were digested with restriction enzymes EcoRI, EcoRV, HindIII, BamHI, xhoI and SalI.

As a result, it was confirmed that Chronobacter Sakazaki bacteriophage Φ CS01 is sensitive to restriction enzymes EcoRI, EcoRV, HindⅢ, BamHI and SalI, but not to XhoI (FIG. 9).

<Experimental Example 10> Confirmation of evolutionary tree of bacteriophage Φ CS01

The classification of adjacent bacteriophages was confirmed by analyzing the Neighbor-joining phylogenetic tree of Chronobacter bacteriophage. The evolutionary history of bacteriophages is based on the isolated phage tape measure protein (TMP) sequence ( FIG. 10 ), tail fiber protein sequence ( FIG. 11 ) and 19 additional phages infected with different pathogens. It was inferred using the neighbor-joining method. The percentage of replicate trees that are associated taxa that have been grouped together in more than 1000 bootstrap tests is displayed next to the branch. Trees are drawn to scale with branch lengths in units equal to the evolutionary distance used to infer the phylogenetic tree. Evolutionary analysis was performed using the NCBI BLAST neighbor joining clustering method and Mega X software (http://www.megasoftware.net).

As a result, the maximum sequence difference was 0.75. The phylogenetic classification revealed that the TMP and tail fiber protein of the bacteriophage Φ CS01 were closest to the classification of the Cronobacter phage, followed by the classification of the Escherichia phage and it was confirmed to be most similar to the classification of the phage.

<Experimental Example 11> Chronobacter sakazaki Confirmation of relationship according to changes in pH, viable cell counts and OD in culture medium

By measuring the changes in the variable parameters (pH, viable cell number, and OD) according to the growth of Chronobacter sakazaki, the productivity according to the pH change was confirmed. The host bacterium Chronobacter sakazaki _{was adjusted to an absorbance of 0.1 at 540 nm (OD 540} ), and grown in TSB broth in a shaking incubator at 37° C. for 3 hours.

As a result of observing the change in the number of viable cells and OD in the bacterial culture, it was generally found that the number of viable cells and the OD were directly proportional, and the pH and the OD were inversely proportional (Fig. 12(A), 12(B)). During incubation, the pH of the culture medium increased from 6.4 to 4.5 and the OD increased from 0.05 to 0.62. Through this result, it was confirmed that the bacterial strain was in the log phase when the pH of the culture medium was about 5.8. In order to support these results, as a result of observing the correlation between the pH of the culture and the number of viable cells, the pH of the culture and the number of viable cells were inversely proportional. In addition, it was confirmed that the Chronobacter sakazaki culture was in the log phase when the pH of the culture medium was about 5.8 (FIG. 12(C)).

<Experimental Example 12> Confirmation of relationship according to changes in pH, viable cell counts, and OD in the bacterial challenge test of bacteriophage Φ CS01 using Chronobacter sakazaki

Changes in variable parameters (pH, viable cell number, and OD) according to the proliferation of Φ CS01 were measured to determine the reason for the change in pH. In order to observe the effect of the phage ΦCS01 on the growth of Chronobacter sakazaki, it was incubated at 37° C. for 180 minutes.

As a result, a difference was confirmed between the experimental group (cultures infected with phage ΦCS01) and the control group (cultures infected with non-phage ΦCS01). _{The difference between the number of viable cells and OD 540} of the experimental group and the control group appeared in the late log phase (FIGS. 13(A), 13(B)). In addition, the difference between the pH of the experimental group and the control group was also clearly observed in the late log phase (Fig. 13(C)). Through this result, it was confirmed that the lysis activity of the phage is one of the reasons for the pH rebound of the culture medium.

<Experimental Example 13> Confirmation of relationship according to changes in pH and viable cell number of Chronobacter sakazaki culture medium pretreated with lysozyme buffer and sonication

Changes in variable parameters (pH and number of viable cells) according to the growth of Chronobacter sakazaki were measured to determine the reason for the change in pH. Chronobacter sakazaki was cultured in BHI medium in a 50 ml conical tube at 37° C. for 2 hours at 250 rpm in a shaking incubator until reaching the late log phase. The samples were then centrifuged and the STET buffer (Tris-HCl 10 mM, pH=8.0, sodium chloride (NaCl) 0.1 M, EDTA 1 mM, and TRITON X-100 5% w/v), and the prepared lysozyme solution (10 mg/mL in 10 mM Tris-HCl, pH=8.0) was added. Thereafter, the pH of the solution was adjusted by adding a hydrogen chloride (HCl) solution to the pH before addition of the STET buffer and lysozyme solution, the solution was sonicated for 30 minutes, and transferred to a 50 ml conical tube at 37° C. for 30 minutes After incubation for a while, measurements were taken every 10 minutes using an Ag/AgCl electrode (Beckman Coulter Inc., Sharon Hill PA).

As a result, in general, there was an inverse proportion between the number of viable cells and the pH (FIG. 14). On the other hand, it was found that the pH of the solution rebounded when ultrasonication was performed after treatment with lysozyme solution and STET buffer. Through these results, it was confirmed that bacterial cell lysis is one of the reasons for the pH rise of the culture medium.

<Experimental Example 14> Confirmation of characteristics of bacteriophage Φ CS01 induced by Chronobacter Sakazaki

Changes in parameters (pH, viable cell number, and OD) according to the proliferation of Φ CS01 were measured to confirm the reason for the change in pH. The host bacterium Chronobacter sakazaki _{was adjusted to 0.1 absorbance at 540 nm (OD 540} ), and grown for 90 minutes in TSB medium in a shaking incubator at 37 ° C. Bacteriophage Φ CS01 induced bacterial culture when _{OD 540 was 0.3.} .

As a result, it was confirmed that the phage induces bacterial cell lysis which causes the release of intracellular substances of the culture medium, and acts as a buffer solution in the culture medium to cause the pH to rise. To support these results, a correlation between the pH of the culture medium and the number of viable cells was observed (FIG. 15). The pH of the culture medium was decreased from 5.61 to 4.65 until 60 minutes after induction of bacterial culture, and then slowly rebounded to 5.08. On the other hand, it was confirmed that the titer of the phage slightly increased up to 30 minutes and then increased rapidly (Fig. 15(A). On the other hand, when the pH rebounded after the culture induced phage Φ CS01, the pH and the number of viable cells It was confirmed that there was an inverse proportion between (Fig. 13(B)).As the number of viable cells decreased due to the phage lysis effect, it was observed that the pH of the culture rebounded, and in order to support this result, viable cells of Chronobacter sakazaki The correlation between the number and titer of phage Φ CS01 confirmed that as the concentration of phage Φ CS01 increased, the number of viable cells in the culture medium decreased inversely (Fig. 15(C)). However, no significant change in titer and pH was measured after 4 hours, so the optimal incubation time of the present invention was confirmed to be 4 hours.

<Experimental Example 15> Confirmation of high titer phage solution obtained by TSSC process

Through the correlation graph and data for the number of viable cells (CFU / ml), the bacteriophage titer (PFU / ml) and the pH of the culture medium of <Experimental Example 11> to <Experimental Example 14>, the production efficiency of bacteriophages can be increased The inoculation and harvest time of the bacteriophage could be confirmed as a change in pH instead of the passage of time.

As a result, the concentration of the bacteriophage solution was increased compared to the conventional method in a stable and continuous process, and the yield of Φ CS01 obtained according to the process of <Example 3> was about 24 times higher than that of the conventional flask culture method. was done (FIG. 16). By simply adding a nutrient medium at the beginning of the process without adding a phage solution or bacterial culture medium, the bacteria to be infected with the bacteriophage and the bacteria infected with the bacteriophage have an independent culture environment. Continuously high as a mass production method of Chronobacter sakazaki bacteriophage It was confirmed that a titer phage lysate could be stably obtained.

<Experimental Example 16> Confirmation of stability of bacteriophage Φ CS01 after lyophilization

After freeze-drying the bacteriophage Φ CS01 to prevent infection caused by Chronobacter sakazaki, which survives even in a dry environment, the infection rate was confirmed. Among many cryoprotectants, the most used is sugar, and in particular, 0.5 M sucrose is known as the most effective stabilizer. Thus, phage Φ CS01 was freeze-dried in a stabilizer (0.5 M sucrose) and stored at 4°C, then suspended in sterile distilled water in the same volume as before freeze-drying, and a double layer to measure infectivity suspended solutions. A double layer plaque assay was performed.

As a result, a decrease in the infection rate of about 1 log scale was observed immediately after lyophilization, but it was confirmed that the infection rate was maintained for 9 weeks without a decrease thereafter (FIG. 17).

Claims (19)

1) a self-cycling fermentation (SCF) step of culturing bacteria to obtain a bacterial culture; and
2) After infecting the bacteria of the bacterial culture medium obtained in step 1) with the bacteriophage, culturing and collecting the infected bacteria to obtain a bacteriophage self-cycling infection (SCI) step; mass production of bacteriophages, including As a method,
The self-circulating fermentation (SCF) step is
i) resuspending a single colony of the host bacterial strain in a medium and then culturing;
ii) monitoring the pH while subculturing the culture solution of step i) in the first bioreactor;
iii) when the pH is 5.5 to 6.0, obtaining a portion of the culture solution of step ii) and transferring it to a second bioreactor; and
iv) replenishing the culture medium remaining in the first bioreactor among the culture medium of step ii) as a detailed step;
The autologous circulating infection (SCI) step is
A) under pH monitoring, adding the bacteriophage to the bacteria in the bacterial culture solution in the second bioreactor and then culturing the bacteriophage to multiply;
B) when the pH is 4.5 to 5.5, collecting and filtering the proliferated bacteriophage of step A) to obtain a filtrate;
C) re-adding a portion of the filtrate obtained in step B) to a second bioreactor; and
D) purifying the remaining filtrate of step B) to produce a bacteriophage;
The bacterium of step 1) is Cronobacter sakazakii ,
The bacteriophage of step 2) is Φ CS01, the mass production method of the bacteriophage.
delete The method according to claim 1, characterized in that the first bioreactor and the second bioreactor are jar type fermentors.
According to claim 1, wherein the medium of the detailed step i) of step 1) is a liquid medium, characterized in that, bacteriophage mass production method.
According to claim 1, wherein the incubation temperature of detailed step ii) of step 1) is characterized in that 20 to 35 ℃, mass production of bacteriophage.
According to claim 1, wherein the incubation time of detailed step iii) of step 1) is characterized in that 2 to 6 hours, the mass production method of bacteriophage.
The method of claim 1, wherein the sub-culture of sub-step ii) of step 1) is characterized in that when it reaches an early exponential phase on the bacterial growth curve, the mass production method of bacteriophages.
According to claim 1, wherein the pH of the detailed step iii) of step 1) is characterized in that 5.8, the mass production method of bacteriophage.
According to claim 1, wherein the volume ratio of a part of the culture solution of sub-step iii) of step 1) and the culture solution remaining in the first bioreactor of sub-step iv) is 9:1, the mass production method of bacteriophages.
According to claim 1, wherein the volume ratio of the culture medium to be supplemented with the culture medium remaining in the first bioreactor in detailed step iv) of step 1) is 1:9, characterized in that, mass production method of bacteriophage.
delete According to claim 1, wherein the bacteriophage of detailed step A) of step 2) is 0.01 to 0.5 multiplicity of infection (Multiplicity of Infection, MOI) characterized in that the input to have, mass production of bacteriophage.
According to claim 1, wherein the pH of the detailed step B) of step 2) is 5.0, characterized in that, bacteriophage mass production method.
According to claim 1, wherein the filtration of the detailed step B) of step 2) is characterized in that consisting of a filter having a pore size of 0.2 to 0.7 μm, the mass production method of bacteriophages.
According to claim 1, wherein the purification of detailed step C) of step 2) is chloroform (chloroform), sodium chloride (NaCl) and polyethylene glycol (polyethylene glycol, PEG) characterized in that carried out with one or more selected from, bacteriophage of mass production method.
According to claim 1, wherein the SCI step is characterized in that repeated 2 to 5 times in a row, the mass production method of bacteriophage.
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