Salmonella bacteriophage nanoemulsion preparation as well as preparation method and application thereof

Abstract

The invention discloses a salmonella bacteriophage nano-emulsion preparation as well as a preparation method and application thereof. The salmonella bacteriophage nano-emulsion preparation is mainly prepared from the following active ingredients in percentage by mass: 5 to 15 percent of salmonella bacteriophage suspension, 84 to 94 percent of edible oil, 0.5 to 3.0 percent of Pickering stabilizer dispersion liquid and 0 to 2.0 percent of protective agent. The invention also discloses a preparation method of the salmonella bacteriophage nano-emulsion preparation and application of the salmonella bacteriophage nano-emulsion preparation in bacteriostasis and preservation of meat products. The salmonella bacteriophage nano-emulsion preparation disclosed by the invention is more essential in stable mechanism, more stable in system, more beneficial to bacteriophage activity maintenance, better in food applicability, safe, friendly, more durable in bacteriostatic action and more suitable for fresh keeping of meat products when being applied.

Description

Technical Field

[0001] This invention belongs to the field of food preservation and antibacterial delivery technology, specifically relating to a Salmonella phage nanoemulsion formulation, its preparation method, and its application. Background Technology

[0002] Meat products are susceptible to microbial contamination during processing, transportation, and storage. Bacteriophages, as biological antimicrobial agents, offer advantages such as strong targeting and low residue risk; however, their activity in emulsion systems can decrease due to factors like interfacial shearing, oxidation, and storage stratification. Existing phage emulsion systems often employ surfactants such as Tween / Span / phospholipids and vegetable oils to construct droplet structures. While this improves dispersibility, it may still suffer from insufficient interfacial film strength, easy aggregation and stratification during long storage, and fluctuating activity levels. Therefore, there is a need for an emulsion carrier system that is more stable in food systems, can improve phage activity retention, and is more suitable for coating meat products. Summary of the Invention

[0003] The purpose of this invention is to provide a Salmonella phage nanoemulsion formulation.

[0004] The present invention also aims to provide a method for preparing the above-mentioned Salmonella phage nanoemulsion formulation.

[0005] The final objective of this invention is to provide the application of the above-mentioned Salmonella phage nanoemulsion formulation in the antibacterial and preservation of meat products.

[0006] The first objective of the present invention can be achieved by the following technical solution: a Salmonella phage nanoemulsion formulation, mainly composed of the following effective ingredients in weight percentage: 5%~15% Salmonella phage suspension, 84%~94% edible oil, 0.5%~3.0% Pickering stabilizer dispersion, and 0~2.0% protectant.

[0007] In the Salmonella phage nanoemulsion formulation of the present invention:

[0008] Preferably, the Salmonella phage suspension is prepared by activating Salmonella phage, dissolving and diluting it with PBS buffer solution, and the phage titer in the Salmonella phage suspension is 10. 6 ~10 9 PFU / mL.

[0009] The Salmonella phage described in this invention can be any publicly available Salmonella phage.

[0010] Preferably, the Salmonella phage described in this invention is recommended, but not limited to, the Salmonella phage with accession number CCTCC NO.M2021765.

[0011] Preferably, the PBS buffer solution of the present invention is food-grade phosphate buffer (PBS), whose components are sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, etc., and meets the requirements of GB 2760 or FDA food contact materials.

[0012] Preferably, the edible oil of the present invention includes, but is not limited to, one or a mixture of two or more of soybean oil, corn oil, and medium-chain triglycerides.

[0013] Preferably, the Pickering stabilizer of the present invention is one or a mixture of two or more of nanocellulose, modified starch granules, and protein-polysaccharide composite granules.

[0014] The nanocellulose refers to food-grade cellulose nanocrystals (CNC) or microfibrillated cellulose (MFC); the modified starch granules refer to common modified starches such as food-grade sodium octenyl succinate starch and acetylated distarch phosphate; and the protein-polysaccharide composite granules refer to edible composite granules formed by food-grade proteins (such as whey protein and soy protein isolate) and food-grade polysaccharides (such as pectin and gum arabic) through known methods such as electrostatic compounding and heat treatment.

[0015] The Pickering stabilizer described in this invention is an edible solid particle that can be adsorbed at the oil-water interface to form a particle film to stabilize nanoemulsion droplets.

[0016] Preferably, the protective agent of the present invention is one or a mixture of two or more of gelatin, whey protein, trehalose and sucrose.

[0017] Furthermore, the mass percentage of the protective agent described in this invention is 0.01% to 2.0%, more preferably 0.5% to 1.5%.

[0018] Preferably, the average particle size of the Salmonella phage nanoemulsion formulation of the present invention is 100-230 nm.

[0019] The edible oil, Pickering stabilizer, protectant, and PBS buffer solution described in this invention all refer to food-grade or food additive-grade products that comply with the relevant regulations of my country's National Food Safety Standard or GB 2760 Standard for the Use of Food Additives. These raw materials are all commercially available products known in the art and can be obtained through conventional commercial channels.

[0020] The Salmonella phage nanoemulsion formulation of this invention is free of Tween-type and Span-type surfactants; and utilizes a solid particle interface film to achieve droplet stabilization.

[0021] The Salmonella phage nanoemulsion formulation of this invention is a solid particle stabilized (Pickering) Salmonella phage nanoemulsion formulation. By forming a stable particle film at the oil-water interface through edible solid particles, the influence of traditional surfactants on phage activity is reduced or avoided, thereby improving the stability of emulsion droplets and maintaining phage activity, and can be used for preservation and antibacterial treatment of meat products.

[0022] The second objective of this invention can be achieved through the following technical solution: the preparation method of the above-mentioned Salmonella phage nanoemulsion formulation includes the following steps:

[0023] (1) After activating Salmonella phage, dissolve and dilute it with PBS buffer solution to prepare Salmonella phage suspension;

[0024] (2) Add Pickering stabilizer to aqueous buffer solution and disperse it under high-speed stirring to form a uniform particle Pickering stabilizer dispersion;

[0025] (3) Add the edible oil to the Pickering stabilizer dispersion obtained in step (2) in a continuous or partial manner, and pre-emulsify it under high-speed shear conditions to obtain a crude emulsion;

[0026] (4) The crude emulsion obtained in step (3) is subjected to high-pressure homogenization or ultrasonic treatment to prepare nanoemulsion;

[0027] (5) Add the Salmonella phage suspension obtained in step (1) to the nanoemulsion obtained in step (4) and mix under low shear conditions. Then add a protectant and stir to adjust the pH to 6.4-6.8 to obtain the Salmonella phage nanoemulsion preparation.

[0028] In the above preparation method of Salmonella phage nanoemulsion formulation:

[0029] Preferably, the aqueous buffer solution in step (2) is food-grade phosphate buffer (PBS).

[0030] Preferably, the high-speed stirring conditions in step (2) are dispersion at a high speed of 3000 to 12000 rpm for 10 to 20 minutes, more preferably 15 minutes.

[0031] Preferably, the Pickering stabilizer in the Pickering stabilizer dispersion in step (2) has a mass percentage content of 0.5-3.0%.

[0032] Preferably, the high-speed shearing conditions in step (3) are shearing at a speed of 5000 to 20000 rpm for 10 to 15 minutes.

[0033] Preferably, the pressure of the high-pressure homogenization in step (4) is 10-80 MPa, and the number of homogenization cycles is 5-8.

[0034] Preferably, the ultrasonic treatment in step (4) has a power of 200-1200W and a treatment time of 15-20min.

[0035] Preferably, the temperature during the high-pressure homogenization or ultrasonic treatment in step (4) is not higher than 25°C.

[0036] Preferably, the rotational speed of the low shear condition in step (5) is 100-800 rpm and the time is 10-30 min.

[0037] The method for preparing Salmonella phage nanoemulsion preparations provided by this invention improves the retention of phage activity through a process of "first forming emulsion and then adding phage, controlling temperature throughout the process, and adjusting pH with a buffer system".

[0038] The last objective of the present invention can be achieved by the following technical solution: the application of the above-mentioned Salmonella phage nanoemulsion preparation in the antibacterial and preservation of meat products.

[0039] More preferably, the meat product is a chicken product.

[0040] Compared with the prior art, the present invention has the following advantages:

[0041] (1) More fundamental stabilization mechanism and more stable system: This invention uses edible solid particles to form a dense particle film (Pickering interface film) at the oil-water interface to stabilize the emulsion droplets. Compared with the traditional emulsification system that relies on surfactants such as Tween / Span, it is less prone to aggregation, layering and demulsification, has higher storage stability, and is easier to form a uniform coating layer on the surface of meat products.

[0042] (2) More conducive to the preservation of phage activity: The present invention can reduce the impact of high shear and temperature rise on phage by combining measures such as "physical shielding effect of solid particle interface membrane + optimized temperature control in process, adding phage after emulsification and low shear, and pH 6.4-6.8 buffer adjustment", thereby improving the survival rate / potency retention rate of phage in preparation, storage and use, and thus improving the sustainability of actual antibacterial effect;

[0043] (3) Better food applicability and safety: The oil phase used is an edible oil system, and the Pickering stabilizer is an edible material such as nanocellulose, modified starch particles, and protein-polysaccharide composite particles. It is preferred to be free of Tween / Span surfactants, which is more in line with the use requirements of food contact scenarios, with lower residue risk and higher consumer acceptance.

[0044] (4) The antibacterial effect is longer and the application is more suitable for meat product preservation: Nano-emulsion droplets have better adhesion and spread on the surface of meat products. The particle film structure can provide a certain "continuous effect" characteristic, so that the bacteriophage can maintain an effective concentration in the high-risk surface environment of the target bacteria, which is more suitable for industrial preservation processes such as spraying, impregnation, and coating. Attached Figure Description

[0045] Figure 1 Comparative photographs of the physical states of the Salmonella phage nanoemulsion formulation prepared in Example 2 and the conventional formulation prepared in Comparative Example 1.

[0046] Figure 2 The release capacity curves of the Salmonella phage nanoemulsion preparation prepared in Example 2 and the conventional preparation prepared in Comparative Example 1 are shown. Detailed Implementation

[0047] The present invention will be further described below through specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0048] All raw materials used in the following embodiments and comparative examples of this invention, including but not limited to edible oils, Pickering stabilizers (nanocellulose, modified starch granules, protein-polysaccharide composite granules, etc.), protective agents (gelatin, whey protein, trehalose, sucrose, etc.), phosphate-buffered saline (PBS), and pH adjusters, are food-grade or commercially available products that meet the requirements for food contact materials. The protein-polysaccharide composite granules can be prepared from corresponding food-grade proteins and polysaccharides according to known methods.

[0049] In addition to using commercially available or publicly available Salmonella phages, Salmonella phages can also be obtained through screening using methods known in the art, such as those described in the following literature:

[0050] Anjay ,Ashok K ,Abhishek , et al.Isolation and characterization ofSalmonella phages and phage cocktail mediated biocontrol of Salmonellaenterica serovar Typhimurium in chicken meat[J].LWT,2022,155DOI:10.1016 / J.LWT.2021.112957.

[0051] Example 1

[0052] The Salmonella phage nanoemulsion formulation provided in this embodiment is mainly composed of the following active ingredients in weight percentage: potency 3.63 × 10 8 The composition consists of 5% PFU / mL Salmonella phage suspension, 92.5% corn oil, 1.0% modified starch granule dispersion, 0.8% gelatin, and 0.7% sucrose.

[0053] The Salmonella phage is the Salmonella phage with accession number CCTCC NO.M2021765.

[0054] The preparation method of this Salmonella phage nanoemulsion formulation includes the following steps:

[0055] (1) After activating Salmonella phage, it was dissolved and diluted with PBS buffer to prepare a titer of 3.63 × 10⁻⁶. 8 PFU / mL Salmonella phage suspension;

[0056] (2) Add 0.01g of modified starch granules to 0.99g of aqueous buffer (PBS) and disperse for 15 min under high-speed stirring at 6000rpm to form a uniform granular modified starch granule dispersion. The mass percentage of modified starch granules in the modified starch granule dispersion is 1%.

[0057] (3) Add 92.5g of corn oil to the 1g modified starch particle dispersion obtained in step (2) in portions, and pre-emulsify it by high-speed shearing at 12000rpm for 12min to obtain a crude emulsion;

[0058] (4) The crude emulsion obtained in step (3) was placed in an ice-water bath to keep the temperature below 20°C and subjected to high-pressure homogenization treatment at 40 MPa for 7 times to prepare nanoemulsion;

[0059] (5) Add 5g of the Salmonella phage suspension prepared in step (1) to the nanoemulsion obtained in step (4), mix well for 15min under low shear conditions at 200rpm, then add 0.8g of gelatin and 0.7g of sucrose, stir for 30min; then adjust the pH to 6.6 with a small amount of 0.5M food-grade sodium bicarbonate solution to obtain the Salmonella phage nanoemulsion preparation.

[0060] The average particle size of the prepared Salmonella phage nanoemulsion formulation was determined by the Malvern Zetasizer Nano ZS method, and the average particle size was 228 nm.

[0061] Example 2

[0062] The Salmonella phage nanoemulsion formulation provided in this embodiment is mainly composed of the following active ingredients in weight percentage: potency 5.6 × 10⁻⁶. 8 The composition consists of 15% PFU / mL Salmonella phage suspension, 84% soybean oil, 0.5% nanocellulose dispersion, 0.3% whey protein, and 0.2% trehalose.

[0063] The Salmonella phage is the Salmonella phage with accession number CCTCC NO.M2021765.

[0064] The preparation method of this Salmonella phage nanoemulsion formulation includes the following steps:

[0065] (1) After activating Salmonella phage, it was dissolved and diluted with PBS buffer to prepare a titer of 5.6 × 10⁻⁶. 8 PFU / mL Salmonella phage suspension;

[0066] (2) Add 0.005 g of nanocellulose to 0.995 g of aqueous buffer (PBS) and disperse it for 15 min under high-speed stirring at 8000 rpm to form a uniform nanocellulose dispersion. The mass percentage of nanocellulose in the nanocellulose dispersion is 0.5%.

[0067] (3) Add 84g of soybean oil to the 0.5g nanocellulose dispersion obtained in step (2) in a continuous manner, and pre-emulsify it by high-speed shearing at 10000rpm for 15min to obtain a crude emulsion;

[0068] (4) The crude emulsion obtained in step (3) was subjected to high-pressure homogenization treatment at room temperature (25°C) and pressure of 60 MPa for 6 times to prepare nanoemulsion;

[0069] (5) Add 15g of the Salmonella phage suspension prepared in step (1) to the nanoemulsion obtained in step (4), mix well for 20min under low shear conditions at 500rpm, then add 0.3g of whey protein and 0.2g of trehalose, stir for 15min; then adjust the pH to 6.5 with a small amount of 0.1M citric acid solution to obtain the Salmonella phage nanoemulsion preparation.

[0070] The average particle size of the prepared Salmonella phage nanoemulsion formulation was determined by the Malvern Zetasizer Nano ZS method, and the average particle size was 144 nm.

[0071] Example 3

[0072] The Salmonella phage nanoemulsion formulation provided in this embodiment is mainly composed of the following active ingredients in weight percentage: potency 3.5 × 10 8 The mixture contains 10% PFU / mL Salmonella phage suspension, 88.2% medium-chain triglycerides (MCT), 1.0% protein-polysaccharide complex particle dispersion, 0.5% whey protein, and 0.3% trehalose.

[0073] Salmonella phages were obtained by screening according to the method described in (Anjay, 2022).

[0074] The preparation method of this Salmonella phage nanoemulsion formulation includes the following steps:

[0075] (1) After activating Salmonella phage, dissolve and dilute it with PBS buffer to prepare a titer of 3.5 × 10⁻⁶. 8 PFU / mL Salmonella phage suspension;

[0076] (2) Add 0.02g of protein-polysaccharide composite particles to 0.98g of aqueous buffer (PBS) and disperse for 15min under high-speed stirring at 10000rpm to form a uniform protein-polysaccharide composite particle dispersion. The mass percentage of protein-polysaccharide composite particles in the protein-polysaccharide composite particle dispersion is 2%.

[0077] (3) 88.2g of medium-chain triglycerides were added to the 1.0g protein-polysaccharide composite particle dispersion obtained in step (2) in a continuous manner, and pre-emulsification was carried out by high-speed shearing at 8000 rpm for 10 min to obtain a crude emulsion;

[0078] (4) The crude emulsion obtained in step (3) was treated in a water bath at 20°C for 18 minutes with an ultrasonic cell disruptor (power 600 W, working for 2 seconds, intermittent for 3 seconds) to prepare nanoemulsion;

[0079] (5) Add 10g of the Salmonella phage suspension prepared in step (1) to the nanoemulsion obtained in step (4), mix at 600 rpm for 30 min under low shear conditions, then add 0.5g of whey protein and 0.3g of trehalose, stir for 10 min; then adjust the pH to 6.7 with a small amount of 0.1M citric acid solution to obtain the Salmonella phage nanoemulsion preparation.

[0080] The average particle size of the prepared Salmonella phage nanoemulsion formulation was determined by the Malvern Zetasizer Nano ZS method, and the average particle size was 201 nm.

[0081] Comparative Example 1

[0082] The difference from Example 2 is that Pickering stabilizer is not used, but food-grade Tween 80 is used as emulsifier. The other conditions are kept as consistent as possible with Example 2 to compare the stability of the emulsifiable concentrate and the difference in phage activity. The average particle size is determined by dynamic light scattering method (Malvern Zetasizer Nano ZS), and the average particle size of the obtained formulation is 326 nm.

[0083] Comparative Example 2

[0084] The difference from Example 2 is that no stabilizer (Pickering stabilizer or surfactant) was added; in the preparation method, soybean oil and PBS were directly mixed and then subjected to high-speed shearing and homogenization. The resulting emulsion showed severe oil-water separation after homogenization and could not form a stable nanoemulsion, so no further characterization was performed.

[0085] Comparative Example 3

[0086] The difference from Example 2 is that the amount of nanocellulose dispersion added is only 0.01%; the preparation method is the same as in Example 2.

[0087] The average particle size was determined by dynamic light scattering method (Malvern Zetasizer Nano ZS), and the average particle size of the obtained formulation was 302 nm. After standing for 3 days, obvious stratification appeared.

[0088] Comparative Example 4

[0089] The difference from Example 2 is that the amount of nanocellulose dispersion added is too high, at 6.0%. The preparation method is the same as in Example 2; the average particle size is determined by dynamic light scattering method (Malvern Zetasizer Nano ZS), and the average particle size of the obtained formulation is 264 nm. However, the viscosity of the system is too high, the fluidity is poor, and it is not conducive to spraying application.

[0090] Comparative Example 5

[0091] The difference from Example 2 lies in the change of process sequence, where the phage is added to the aqueous phase before emulsification. Specifically, a 15% phage suspension is first mixed with PBS and nanocellulose dispersion as the aqueous phase, then subjected to high-speed shearing and high-pressure homogenization with soybean oil. Subsequent steps for adding the protective agent and adjusting the pH are the same. The average particle size was determined using Malvern Zetasizer Nano ZS, and the resulting formulation had an average particle size of 274 nm. However, the phage titer immediately decreased by approximately two orders of magnitude after preparation, indicating that this process severely damaged the phage activity.

[0092] Example 4

[0093] 1. Choosing cooking oil

[0094] Taking Example 1 as an example, different edible oil phases were selected: soybean oil, corn oil, sunflower seed oil and medium chain triglyceride oil (MCT), with the same amount. The effects of different oil phases on the stability of emulsifiable concentrate and the maintenance of phage activity were analyzed (the phage was added in the form of Salmonella phage suspension, and the amount added accounted for 5% of the total mass of the system). The results are shown in Table 1.

[0095] Table 1. Effects of different oils on the stability and phage activity retention of emulsifiable concentrate formulations

[0096]

[0097] As shown in Table 1, the phage titers, from highest to lowest, are soybean oil > medium-chain triglyceride (MCT) oil > corn oil > sunflower oil. Therefore, soybean oil was selected as the best solvent.

[0098] 2. Screening of Pickering Stabilizers

[0099] Taking Example 1 as an example, various Pickering stabilizers were selected: 0.01g of nanocellulose, 0.01g of modified starch particles, or 0.01g of protein-polysaccharide composite particles were added to 0.99g of aqueous buffer solution and dispersed under high-speed stirring to form a uniform particle Pickering stabilizer dispersion. The prepared particle Pickering stabilizer dispersions were then used with soybean oil at mass percentages of 0.05%, 0.1%, 0.5%, 1.0%, and 3.0% (accounting for the entire nanoemulsion system) to construct Pickering nanoemulsion systems. The results are shown in Table 2.

[0100] Table 2. Effect of the mass percentage of Pickering stabilizer dispersion in the entire nanoemulsion system on the stability and dispersibility of the emulsion formulation.

[0101]

[0102] As can be seen from Table 2, among the three Pickering stabilizers—nanocellulose, modified starch granules, and protein-polysaccharide composite granules—nanocellulose has the best effect, achieving superior stability and dispersibility within the range of 0.5%-1.0%. Modified starch granules and protein-polysaccharide composite granules require relatively higher concentrations (≥1.0%) to achieve the stabilizing effect.

[0103] 3. Particle size analysis

[0104] The average particle size was determined using the dynamic light scattering method (Malvern Zetasizer Nano ZS).

[0105] Table 3 Particle Size Analysis Table

[0106]

[0107] As can be seen from Table 3, the average particle size of the Pickering nanoemulsions prepared in Examples 1-3 of this invention is between 100-230 nm, and the distribution is uniform. In comparison, the particle size of Comparative Example 1 (conventional Tween 80 emulsification) is larger (326 nm); the particle size of Comparative Example 3 (insufficient stabilizer) is the largest and the distribution is wide (302 nm); the particle size of Comparative Example 4 (excessive stabilizer) is also increased (264 nm) due to the excessive viscosity of the system. The appropriate Pickering stabilizer and its concentration are the key to obtaining small-sized, stable nanoemulsions.

[0108] 4. Physical state of Salmonella phage nanoemulsion formulation

[0109] Comparative photos of the Salmonella phage nanoemulsion formulations prepared in Example 2 and Comparative Example 1 after standing for 12 days are shown below. Figure 1 As shown.

[0110] from Figure 1 As can be seen, the sample of Example 2 (Pickering stable) remained a homogeneous emulsion with no significant changes; while the sample of Comparative Example 1 (Tween 80 stable) showed severe oil phase floating and water phase settling, completely demulsifying and separating. This image visually demonstrates the significant advantage of the Pickering stabilization mechanism in improving the long-term storage stability of emulsions.

[0111] 5. Long-term storage stability test

[0112] The emulsifiable concentrates prepared in Examples 1-3 and Comparative Examples 1-5 were subjected to quality testing, including phage activity (potency retention), pH value, antibacterial activity, and stability. The results are shown in Tables 4-6. Storage conditions were 25°C at room temperature. Because Comparative Example 2 could not form a stable emulsion, phage activity (potency retention), pH value, antibacterial activity, and stability were not tested.

[0113] Antimicrobial activity was evaluated according to the principles of the agar diffusion method (i.e., the Oxford cup method) recommended by the Clinical Laboratory Standards Institute (CLSI).

[0114] Colony growth inhibition rate (%) = [(sample inhibition zone diameter - Oxford cup outer diameter) / (positive control inhibition zone diameter - Oxford cup outer diameter)] × 100%.

[0115] Note: Positive controls are prepared using free phage stock solution with known titers.

[0116] Table 4. Phage activity and pH stability

[0117]

[0118] As shown in Table 4, the phage titer of each embodiment of the present invention (using Pickering stabilization and optimization process) decreased slowly, and the pH remained stable. Comparative Example 1 (Tween 80) showed the fastest titer decrease and a significant pH reduction, possibly related to the degradation of traditional emulsifiers or insufficient protection of the phage. Comparative Example 3 (insufficient stabilizer) also exhibited poor titer retention. Comparative Example 5 (improper process) showed a significant initial titer loss, and while Comparative Example 4 maintained good phage titer, the system was too viscous, resulting in consistently poor dispersibility (flowability).

[0119] Table 5. Inhibitory effect of Salmonella phage Pickering nanoemulsion on Salmonella.

[0120]

[0121] As shown in Table 5, the nanoemulsions of the present invention exhibited a smoother and higher antibacterial rate retention curve during storage. The antibacterial rates of Comparative Examples 1 and 3 decreased significantly with a rapid decline in potency. Comparative Example 5 showed consistently poor antibacterial effect due to severe damage to its activity.

[0122] The release capacity curves of the Salmonella phage nanoemulsion formulation prepared in Example 2 and the conventional formulation prepared in Comparative Example 1 are shown in the figure below. Figure 2 As shown.

[0123] from Figure 2 As can be seen, the phages in the nanoemulsion exhibit a slow but continuous release over 8 hours, with a cumulative release rate of approximately 75% after 8 hours, reaching a plateau (approximately 94%) after 24 hours. This indicates that the Pickering nanoemulsion structure of the present invention has a certain encapsulation and protection effect on the phages and can achieve a certain degree of sustained release, which is beneficial for prolonging the antibacterial effect time.

[0124] Table 6 Evaluation of the physical stability and dispersibility of nanoemulsions

[0125]

[0126] As shown in Table 6, the Pickering nanoemulsions of this invention (Examples 1-3) exhibit excellent physical stability and good redispersibility during a 12-day storage period. Comparative Example 1 (Tween 80) and Comparative Example 3 (insufficient stabilizer) showed severe stratification within 6-12 days, indicating poor stability. Comparative Example 4 (excessive stabilizer), while showing good stability, suffered from consistently poor viscosity and dispersibility (flowability), hindering its application. Comparative Example 5 (improper process) also showed stratification in the later stages of storage. This further demonstrates the comprehensive advantages of this invention in terms of the type, concentration, and preparation process of the Pickering stabilizer.

[0127] Therefore, this invention solves the interfacial film strength problem through Pickering particle stabilizers. Pickering solid particles can adsorb onto the oil-water interface to form a particle film to stabilize nano-emulsion droplets. Optimized processes, such as adding phages after emulsification, temperature control throughout the process, and pH adjustment using a buffer system, can improve phage activity and stability, thereby solving the problem of phage activity fluctuations. Furthermore, in Example 2, the phage titer increased from 5.6 × 10⁻⁶ within 12 days. 8 Reduced to 4.53×10 8 The PFU / mL decreased only slightly; Comparative Example 1 (Tween 80) decreased from 1.45 × 10⁻⁶ PFU / mL over 12 days. 7 Reduced to 5.85×10 4 The PFU / mL level decreased drastically; in Comparative Example 5 (incorrect process sequence), the activity immediately decreased by two orders of magnitude.

[0128] Example 5

[0129] 1. Experimental Methods

[0130] 1.1. Sample preparation: Fresh chicken breast was purchased from Panlong Market in Haizhu District, Guangzhou City, Guangdong Province, and aseptically cut into pieces of about 10g each (5cm×5cm×1cm).

[0131] 1.2. Contamination: Soak the meat slices in 10... 4 Immerse the surface in a Salmonella bacterial solution at CFU / mL for 1 minute, then remove and drain to ensure an initial bacterial load of approximately 10. 3 CFU / cm².

[0132] 1.3. Processing Group:

[0133] Group A (Invention): Spraying the nanoemulsion prepared in Example 2, at a dosage of 0.2 mL / cm³. 2 ;

[0134] Group B (free phage): Spray with a free phage suspension of the same total amount as Group A phage (diluted with PBS to the same titer);

[0135] Group C (blank nanoemulsion): Spraying blank Pickering nanoemulsion without bacteriophage (same process as in Example 2, but without adding bacteriophage);

[0136] Group D (blank control): Sprayed with an equal volume of sterile PBS.

[0137] 1.4. Storage and Testing: Place all processed meat slices on a sterile tray, lightly cover with plastic wrap, and store in a refrigerator at 4°C. Take samples on days 0, 3, 7, and 10.

[0138] 1.5. Colony Count: Take 1 g of meat sample (including surface), add 9 mL of sterile physiological saline, homogenize, perform serial dilutions, and determine the total number of Salmonella (CFU / g) in the sample using the plate count method (PCA medium).

[0139] 1.6. Sensory evaluation: Five trained evaluators will score the color and odor of the meat samples on a 5-point scale (1 point: severe browning and putrid odor; 5 points: bright red and fresh meat odor).

[0140] 2. Experimental Results

[0141] Table 7. Changes in Salmonella count on chicken surface during storage at 4℃ (logCFU / g)

[0142]

[0143] Table 8. Sensory scores of chicken during storage at 4℃ (average scores for color and odor)

[0144]

[0145] 3. Conclusion

[0146] Experimental results show that:

[0147] 3.1 Antibacterial Effect: As shown in Table 7, Group A (the nanoemulsion of this invention) exhibited the most significant inhibitory effect on the growth of Salmonella on the surface of chicken throughout the entire 10-day refrigeration period. By day 10, its colony count was approximately four orders of magnitude lower than Group D (control) and approximately two orders of magnitude lower than Group B (free bacteriophages). This demonstrates that the Pickering nanoemulsion carrier not only effectively protects bacteriophage activity but also significantly prolongs the antibacterial time by enhancing its adhesion to the meat surface and its potential sustained-release effect.

[0148] 3.2 Preservation effect: As can be seen from Table 8, consistent with the antibacterial effect, the sensory quality (color and odor) of the chicken in Group A was best preserved, significantly delaying the occurrence of spoilage.

[0149] 3.3 Carrier Role: As shown in Tables 7-8, group C (blank nanoemulsion) exhibited certain antibacterial and preservative effects compared to group D (control). This may be due to the physical isolation effect of the nanoemulsion membrane on the meat surface and certain characteristics of the Pickering particles themselves, but the effect was far less than that of group A, which was loaded with bacteriophages. This demonstrates that bacteriophages are the main active antibacterial component, while nanoemulsions are excellent delivery and protective carriers.

[0150] Other meats, such as pork and beef, can achieve similar results.

[0151] In summary, the Salmonella phage Pickering nanoemulsion formulation of the present invention can be effectively applied to the preservation of meat products such as chicken, significantly inhibiting the growth of specific pathogenic bacteria and extending shelf life.

[0152] The above examples illustrate specific embodiments of the present invention. It is important to note that these specific embodiments are only for further explanation and do not constitute a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the present invention still fall within the scope of protection of the present invention.

Claims

1. A Salmonella phage nanoemulsion formulation, characterized in that, It is mainly composed of the following active ingredients by mass percentage: Salmonella phage suspension 5%~15%, edible oil 84%~94%, Pickering stabilizer dispersion 0.5%~3.0%, and protectant 0~2.0%.

2. The Salmonella phage nanoemulsion formulation according to claim 1, characterized in that, The Salmonella phage suspension was prepared by activating Salmonella phages, dissolving and diluting them with PBS buffer solution, and the phage titer in the suspension was 10. 6 ~10 9 PFU / mL.

3. The Salmonella phage nanoemulsion formulation according to claim 1, characterized in that, The edible oil is one or a mixture of two or more of soybean oil, corn oil, and medium-chain triglycerides; the Pickering stabilizer is one or a mixture of two or more of nanocellulose, modified starch granules, and protein-polysaccharide composite granules; and the protective agent is one or a mixture of two or more of gelatin, whey protein, trehalose, and sucrose.

4. The Salmonella phage nanoemulsion formulation according to claim 1, characterized in that, The average particle size of the Salmonella phage nanoemulsion formulation is 100–230 nm.

5. The method for preparing the Salmonella phage nanoemulsion formulation according to any one of claims 1-4, characterized in that, Includes the following steps: (1) After activating Salmonella phage, dissolve and dilute it with PBS buffer solution to prepare Salmonella phage suspension; (2) Add Pickering stabilizer to aqueous buffer solution and disperse it under high-speed stirring to form a uniform particle Pickering stabilizer dispersion; (3) Add the edible oil to the Pickering stabilizer dispersion obtained in step (2) in a continuous or partial manner, and pre-emulsify it under high-speed shear conditions to obtain a crude emulsion; (4) The crude emulsion obtained in step (3) is subjected to high-pressure homogenization or ultrasonic treatment to prepare nanoemulsion; (5) Add the Salmonella phage suspension obtained in step (1) to the nanoemulsion obtained in step (4) and mix under low shear conditions. Then add a protectant and stir to adjust the pH to 6.4-6.8 to obtain the Salmonella phage nanoemulsion preparation.

6. The method for preparing the Salmonella phage nanoemulsion formulation according to claim 5, characterized in that, The aqueous buffer solution in step (2) is food-grade phosphate buffer PBS; the high-speed stirring conditions are dispersion at a speed of 3000-12000 rpm for 10-20 min; the mass percentage of Pickering stabilizer in the Pickering stabilizer dispersion is 0.5-3.0%.

7. The method for preparing the Salmonella phage nanoemulsion formulation according to claim 5, characterized in that, The high-speed shearing conditions mentioned in step (3) are shearing at a speed of 5000 to 20000 rpm for 10 to 15 minutes.

8. The method for preparing the Salmonella phage nanoemulsion formulation according to claim 5, characterized in that, The pressure of the high-pressure homogenization in step (4) is 10-80 MPa, and the number of homogenizations is 5-8; the power of the ultrasonic treatment is 200-1200 W, and the treatment time is 15-20 min; the temperature during the high-pressure homogenization or ultrasonic treatment is not higher than 25℃.

9. The method for preparing the Salmonella phage nanoemulsion formulation according to claim 5, characterized in that, The low shear conditions described in step (5) involve a rotation speed of 100–800 rpm and a time of 10–30 min.

10. The application of the Salmonella phage nanoemulsion formulation according to any one of claims 1-4 in the antibacterial and preservation of meat products.

Images