Salmonella phage Y1 with wide lysis spectrum and preparation method of low-endotoxin preparation of salmonella phage Y1
By preparing the wide-cleavage spectrum Salmonella phage Y1 and its low endotoxin preparation, using chitosan to remove endotoxins in the phage preparation, the problem of high cost and limited effect of removing endotoxins in the prior art is solved, and low-cost and effective endotoxin removal and phage activity maintenance are achieved, which is suitable for industrial production and animal applications.
Patent Information
- Application Number
- CN202510638138.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-19
- Publication Date
- 2025-08-15
AI Technical Summary
The prior art lacks methods for low-endotoxin bacteriophage preparations that are widely adaptable and industrially produced. The existing methods for removing endotoxins are costly, limited in effect or residual problems.
The preparation method of Salmonella phage Y1 and its low endotoxin preparation was adopted by adding chitosan solution to the phage suspension, mixing evenly, and precipitating at room temperature overnight, and the endotoxins were removed by using the biocompatibility and antibacteriality of chitosan.
It has achieved low-cost and effective removal of endotoxins in bacteriophage preparations, maintained phage activity, is suitable for industrial production, and has no adverse effects on animals, and has broad market prospects.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a preparation method of a Salmonella phage Y1 with a broad lysis spectrum and a low-endotoxin preparation thereof. Background Art
[0002] Phage therapy is a method of treating bacterial infections using bacteriophages. With the growing problem of antibiotic resistance, phage therapy has regained attention as an alternative or supplement to antibiotic treatment and holds great promise. However, when phages specifically lyse host bacteria, they release large amounts of lipopolysaccharide (LPS), which acts as endotoxins and can induce systemic inflammatory response syndrome (SIRS), multiple organ failure syndrome (MOFS), and sepsis in humans or animals, potentially endangering their lives. Therefore, endotoxin removal is a crucial step in the production process.
[0003] Currently, there are many methods for removing endotoxins, but there is still no method that is widely applicable and can be industrially produced to produce low-endotoxin phage preparations. For example, ion exchange chromatography, which exploits the charge difference between endotoxins and proteins, and ultrafiltration technology using membrane filtration can effectively remove endotoxins, but are not suitable for removing endotoxins from phage. Gel filtration, which separates by molecular size, can also help purify protein products, but its endotoxin removal effect is limited and the gel cost is relatively high. Using adsorbents such as activated carbon to remove endotoxins is low-cost and has a large processing capacity. However, activated carbon has poor selectivity and easily adsorbs active ingredients, making residual activated carbon in the purified solution difficult to remove. Distillation and affinity chromatography have good removal effects, but the removal cost is high. Using detergents such as Triton X-110 is simple, efficient, and inexpensive, but there is the problem of detergent residue. Summary of the Invention
[0004] The present invention aims to overcome the shortcomings of the prior art by providing a broad-spectrum Salmonella phage Y1 and a method for preparing a low-endotoxin preparation thereof. The present invention aims to develop a broad-spectrum Salmonella phage Y1 and a method for removing endotoxins from phage preparations using chitosan. This method is simple, effective, low-cost, and suitable for industrial production. To achieve the above objectives, the present invention provides the following technical solutions:
[0005] The invention provides a broad lysis spectrum Salmonella phage (Salmonella phage) Y1, whose preservation number is CCTCC NO: M 20242911.
[0006] The phage is a broad-spectrum Salmonella phage, named Salmonella phage Y1. On December 26, 2024, the Salmonella phage Y1 was deposited with the China Center for Type Culture Collection at Wuhan University in Wuhan, Hubei Province, with a deposit number of CCTCC NO: M 20242911 and a deposit date of December 26, 2024. The Salmonella phage Y1 can lyse 14 different strains of Salmonella.
[0007] The present invention also provides a use of the above-mentioned Salmonella phage Y1 in preparing a low-endotoxin phage preparation.
[0008] The present invention also provides a method for preparing a low-endotoxin phage preparation, comprising the following steps:
[0009] 1) Activate the phage to obtain activated phage
[0010] 2) Cultivate the activated phage to obtain a titer of not less than 1×10 9 PFU / mL of phage suspension;
[0011] 3) Add chitosan solution to the phage suspension, mix well, and precipitate overnight at room temperature to obtain a low-endotoxin phage preparation.
[0012] Furthermore, in step 1), the bacteriophage is Salmonella phage Y1.
[0013] Furthermore, in step 1), the culture medium for activation and culture is LB culture medium.
[0014] Furthermore, in step 3), the final concentration of the chitosan solution in the low endotoxin phage preparation is 0.05-0.5%.
[0015] Furthermore, in the low-endotoxin phage preparation, the final concentration of chitosan is 0.05.
[0016] Furthermore, in step 3), the endotoxin content in the phage preparation is 500-1000 EU / mL.
[0017] The present invention also provides a use of the low-endotoxin phage preparation prepared by the above method in preparing a medicine for killing pathogenic Salmonella in a breeding environment or in an animal body.
[0018] Furthermore, the Salmonella is 14 different species of Salmonella, namely, any one of Salmonella 2017056, Salmonella 2018002, Salmonella 2018004, Salmonella Enteritidis 2018015, Salmonella 2018016, Salmonella 2018030, Salmonella 2018031, Salmonella 2018059, Salmonella 2018064, Salmonella 2018107, Salmonella 2018108, Salmonella 2018109, Salmonella Enteritidis 2019052, Salmonella 180414, and Salmonella 6087.
[0019] As a preferred option, the host bacteria used for phage Y1 is Salmonella 2018016.
[0020] Principle of the present invention:
[0021] Chitosan, also known as deacetylated chitosan, has been widely used as a drug and vaccine delivery vehicle due to its non-toxic, antibacterial, immunopotentiating, and biocompatible properties. This method utilizes chitosan to remove endotoxins from phage preparations. This method is simple, effective, low-cost, and suitable for industrial production.
[0022] Beneficial effects of the present invention:
[0023] 1. Salmonella phage Y1 has strong lysis ability and can be used to prepare drugs for the prevention and treatment of Salmonella.
[0024] 2. Low cost, simple equipment, simple operating procedures, stable effect, and easy to scale industrial production.
[0025] 3. The endotoxin removal effect is good and the effect on phage activity is small.
[0026] 4. Economic effect: low cost and wide market:
[0027] Low cost: It only costs more than 46.2 yuan to produce each liter of low-endotoxin phage preparation, with an average of 0.0462 yuan / mL.
[0028] Material name Price / Specifications Dosage cost Tryptone 325 yuan / 500g 10g 6.5 yuan yeast extract 140 yuan / 500g 5g 1.4 yuan Sodium chloride 15 yuan / 500g 10g 0.3 yuan Chitosan 100 yuan / 25g 0.5g 2 yuan EP tube, gun head, etc. 0.04 yuan / piece 10 0.4 yuan 0.22μm filter membrane 3 yuan / piece 10 30 yuan 50mL centrifuge tube 0.56 yuan / piece 10 5.6 yuan total 46.2 yuan
[0029] Wide market:
[0030] This patent enables the mass removal of endotoxins from phage preparations, significantly accelerating the pace of phage application. This will enable the formation of a complete phage therapy industry chain. Its economic value is immeasurable, and its social impact is enormous.
[0031] Specifically, the advantages of the present invention over the prior art are shown in the table below:
[0032] BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Figure 1 This is a picture of Salmonella phage Y1 plaque;
[0034] Figure 2 This is the optimal multiplicity of infection graph for Salmonella phage Y1;
[0035] Figure 3 Schematic diagram of the titer of phage Y1 after treatment with different concentrations of chitosan;
[0036] Figure 4 This figure shows the effectiveness of low-endotoxin preparations in killing Salmonella in chickens on farms. DETAILED DESCRIPTION
[0037] The present invention is further described in detail below with reference to specific embodiments so that those skilled in the art can understand.
[0038] Example 1 Screening of Salmonella phage Y1
[0039] 1. Isolation of bacteriophage
[0040] Wastewater was collected from a chicken farm in Hubei Province, centrifuged at 10,000 rpm for 5 min, and the supernatant was filtered and sterilized using a 0.22 μm filter membrane and stored at 4°C for later use.
[0041] To each 50 mL centrifuge tube, add 25% of Salmonella 2018016, 25% of the sterilized supernatant, and 50% of 2× LB medium. Mix thoroughly and incubate at 37°C for 12–14 hours. The next day, centrifuge the culture at 10,000 rpm for 5 minutes, and sterilize the supernatant by filtration through a 0.22 μm filter to obtain the phage enrichment solution.
[0042] The phages were separated by double-layer plate method: 0.1 mL of the phage enrichment solution was diluted 10 times in a row, and 10 -2 , 10 -4 , 10 -6 Add 0.1 mL of the diluent to 0.1 mL of the host bacteria in the logarithmic phase, and then add 3 mL of 0.6% semi-solid LB medium at approximately 45°C. Spread evenly on pre-prepared 1.2% LB solid medium, incubate at 37°C for 12-14 hours, and observe plaque formation.
[0043] 2. Morphological Identification of Phage
[0044] Pick a single clear plaque without halo, uniform in size and neat in edge, and place it in 1 mL of SM solution. Purify the isolated phage using the double-layer plate method. Repeat the experiment 3 times until each plaque is uniform in size. The purification results are as follows: Figure 1 As shown, the diameter of the plaque is about 0.4 cm, clear and bright. This phage is named Y1 and stored at 4℃ for future use.
[0045] The phage is a broad-spectrum Salmonella phage, named Salmonella phage Y1. On December 26, 2024, the Salmonella phage Y1 was deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan City, Hubei Province, with a deposit number of CCTCC NO: M 20242911 and a deposit date of December 26, 2024.
[0046] Example 2 Determination of Salmonella phage Y1
[0047] 1. Determination of the optimal multiplicity of infection (MOI)
[0048] Adjust the concentration of host bacteria 2018016 in the logarithmic phase to 1×10 6 CFU / mL, the phage stored at 4°C in Example 2 was added at a multiplicity of infection of 100, 10, 1, 0.1, and 0.01, respectively, and LB liquid medium was added to make the total volume of the culture system the same. After incubation at 37°C for 5 h, the cells were centrifuged at 10,000 rpm for 10 min, the supernatant was collected and diluted to an appropriate concentration, and the titer was determined by the double-layer method.
[0049] The results are as follows Figure 2 As shown: The optimal MOI of Salmonella phage Y1 is 1.
[0050] 2. Host spectrum identification
[0051] 0.1 ml of overnight culture of 21 Salmonella strains was added to 4 ml of LB semi-solid medium at about 50°C and evenly spread on the pre-prepared LB solid medium. Then each plate was divided into two areas, and 10 μL was taken from one area and the titer was adjusted to 1×10 8 PFU / ml, the phage stored at 4°C in Example 1 was added dropwise to the surface, and LB without bacteria was added dropwise to another area as a control. After the droplet dried, it was inverted and cultured at 37°C for 12 h, and the results were observed.
[0052] The results are shown in Table 1: the Salmonella phage Y1 can lyse 14 different strains of Salmonella, and the lysis rate of Salmonella phage Y1 reaches 66.6%.
[0053] Table 1 Spotting results of Salmonella phage Y1
[0054]
[0055]
[0056] Note: "+" indicates the degree of bacterial lysis by the phage. The more "+" there are, the higher the degree of lysis. "-" indicates that the phage has no ability to lyse bacteria.
[0057] Example 3 Preparation of Low-Endotoxin Phage Preparation 1
[0058] A method for preparing a low-endotoxin phage preparation 1 comprises the following steps:
[0059] 1) Activating Salmonella phage Y1 to obtain activated Salmonella phage Y1
[0060] 2) Cultivate the activated Salmonella phage Y1 to obtain a titer of not less than 1×10 9 PFU / mL of Salmonella phage Y1 suspension;
[0061] 3) Adding chitosan solution to an equal volume of an equivalent titer Salmonella phage Y1 suspension, mixing well, and allowing to precipitate overnight at room temperature to obtain a low-endotoxin phage preparation 1, wherein the final chitosan concentration in the low-endotoxin phage preparation 1 is 0.5%.
[0062] Example 4 Preparation of Low-Endotoxin Phage Preparation 2
[0063] The preparation method of phage preparation 2 prepared in this example is basically the same as that of Example 2, except that:
[0064] In low-endotoxin phage preparation 2, the final chitosan concentration was 0.5%.
[0065] Comparative Example 1 Preparation of phage preparation D1
[0066] The phage preparation D1 prepared in this example was prepared in the same manner as in Example 2, except that:
[0067] In the phage preparation D1, the final chitosan concentration was 0.005%.
[0068] Comparative Example 2 Preparation of phage preparation D2
[0069] The phage preparation D2 prepared in this example was prepared in the same manner as in Example 2, except that:
[0070] In phage preparation D2, the final chitosan concentration was 0.0005%.
[0071] Comparative Example 3 Phage Preparation D3
[0072] Phage preparation D3 is the Salmonella phage Y1 suspension prepared in the first two steps of the method of Example 2.
[0073] 1. Effect of different chitosan concentrations in phage preparations on endotoxin content
[0074] The endotoxin in phage preparations was semi-quantitatively detected using the Limulus amebocyte lysate gel method. The endotoxin removal effects of different chitosan concentrations on phage preparations 1-2 and phage preparations D1-D3 are shown in Table 2.
[0075] As shown in Table 2, when the chitosan concentration was 0.5% and 0.05%, the endotoxin removal effect was the best, and compared with the group without chitosan addition, the endotoxin decreased by at least 50 times.
[0076] Table 2 Effects of different concentrations of chitosan on endotoxin
[0077] Phage preparations Chitosan concentration EU / mL Phage preparation 1 0.5％ 1000 Phage preparation 2 0.05％ 1000 Phage preparation D1 0.005％ 25000 Phage preparation D2 0.0005％ 25000～50000 Phage preparation D3 No chitosan 50000～100000
[0078] 2. Effects of chitosan on bacteriophage
[0079] The titer of the phage preparation (ie, the phage suspension treated with different concentrations of chitosan) was determined using the double-layer plate method described in Example 1.
[0080] The results are as follows Figure 3 As shown, phage titers in phage preparations 1 and 2 decreased slightly after treatment with 0.5% and 0.05% chitosan, but remained very high. Phage titers in phage preparations D1 and D2 remained virtually unchanged after treatment with 0.005% and 0.0005% chitosan. Therefore, chitosan within this concentration range (0.05% to 0.5%) has minimal effect on phage Y1 activity. Considering both cost and endotoxin removal efficacy, the optimal concentration for phage preparation 1 is 0.05%.
[0081] III. Safety Assessment of Low-Endotoxin Phage Preparation 1 Prepared in Example 2
[0082] Twelve one-day-old broiler chickens weighing approximately 0.13 kg were randomly divided into two groups: a control group and a phage-treated group, with 6 chickens in each group. The groups were as follows:
[0083] Phage treatment group: low endotoxin phage preparation 1 was mixed with water at a volume ratio of 1:1000;
[0084] The control group did not receive any treatment, and except for drinking water, the other feeding conditions were the same as those of the phage-treated group.
[0085] The body temperature, weight and remaining feed of the chickens were measured daily, and the weight and feed-to-meat ratio of each group of chickens were calculated for 7 consecutive days.
[0086] The results are shown in Table 3. There was no significant difference between the phage-treated group and the control group in three safety indicators, namely body temperature, body weight and feed-to-meat ratio, indicating that the low endotoxin phage preparation 1 treated with 0.05% chitosan had no adverse effects on chickens.
[0087] Table 3 Safety evaluation of low endotoxin phage preparations
[0088]
[0089] IV. Therapeutic Application of the Low-endotoxin Phage Preparation 1 Prepared in Example 2 in Chicken Farms
[0090] In a young chicken farm in Shandong, an 8-day-old chicken house was randomly selected and bacteriophage preparation 1 was added to the drinking water to make the drinking water contain 1×10 7 PFU / mL, concentrated for 1 hour, and continued for 3 consecutive days. Anal swabs were collected from 20 chickens randomly selected before drinking the phage, on the first day after drinking the phage for 3 days, and on the third day after drinking the phage for 3 days. Each anal swab was diluted with 1 mL of PBS, and the dilutions were tested for Salmonella using fluorescent quantitative PCR.
[0091] The results are as follows Figure 4 As shown, before drinking the phages, the detection rate of Salmonella was 42.9%. After drinking the phages for three consecutive days, no Salmonella was detected on the first day. After drinking the phages for three consecutive days, the detection rate of Salmonella was 23.8% on the third day. These results indicate that low-endotoxin formulation 1 is highly effective in killing Salmonella in chickens on farms.
[0092] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without inventiveness, and these embodiments all fall within the scope of protection of the present invention.
Claims
1. A broad lysis spectrum Salmonella phage Y1, whose deposit number is CCTCC NO: M20242911.
2. Use of the Salmonella phage Y1 according to claim 1 in preparing a low-endotoxin phage preparation.
3. A method for preparing a low-endotoxin phage preparation, characterized in that: The following steps are included: 1) Activate the phage to obtain activated phage 2) Cultivate the activated phage to obtain a titer of not less than 1×10 9 PFU / mL of phage suspension; 3) Add chitosan solution to the phage suspension, mix well, and precipitate overnight at room temperature to obtain a low-endotoxin phage preparation.
4. The preparation method according to claim 3, wherein: In the step 1), the bacteriophage is Salmonella phage Y1.
5. The preparation method according to claim 3 or 4, characterized in that: In the step 1), the culture medium for activation and culture is LB culture medium.
6. The preparation method according to claim 3, wherein: In the step 3), the final concentration of the chitosan solution in the low-endotoxin phage preparation is 0.05-0.5%.
7. The preparation method according to claim 3 or 6, characterized in that: In the low-endotoxin phage preparation, the final concentration of chitosan is 0.
05.
8. The preparation method according to claim 3, 5 or 6, characterized in that: In step 3), the endotoxin content in the phage preparation is 500 to 1000 EU / mL.
9. Use of the low-endotoxin phage preparation prepared by the method of claim 3 in preparing a drug for killing pathogenic Salmonella in aquaculture environments or in animals.
10. The use according to claim 8, characterized in that: The Salmonella are 14 different species of Salmonella, namely, any one of Salmonella 2017056, Salmonella 2018002, Salmonella 2018004, Salmonella Enteritidis 2018015, Salmonella 2018016, Salmonella 2018030, Salmonella 2018031, Salmonella 2018059, Salmonella 2018064, Salmonella 2018107, Salmonella 2018108, Salmonella 2018109, Salmonella Enteritidis 2019052, Salmonella 180414, and Salmonella 6087.